designing of new age dairy foods_2015

LECTURE COMPENDIUM-cum-TRAINING MANUAL

OF

NATIONAL TRAINING PROGRAMME

ON

DESIGNING NEW AGE DAIRY FOODS

NOVEMBER 28 – DECEMBER 18, 2015

CENTRE OF ADVANCED FACULTY TRAINING

(Dairy Processing)

DAIRY TECHNOLOGY DIVISION

ICAR- NATIONAL DAIRY RESEARCH INSTITUTE (DEEMED UNIVERSITY)

KARNAL – 132 001, (HARYANA) INDIA

Course AdvisorsCourse AdvisorsCourse AdvisorsCourse Advisors

Dr. Latha SabikhiDr. Latha SabikhiDr. Latha SabikhiDr. Latha Sabikhi Director, Centre of Advanced Faculty Training in Dairy Processing and

Head, Dairy Technology Division &

Dr. Ashish Kumar SinghDr. Ashish Kumar SinghDr. Ashish Kumar SinghDr. Ashish Kumar Singh Principal Scientist & Acting Head, Dairy Engineering Division

Course DirectorCourse DirectorCourse DirectorCourse Director Mr. Sathish Kumar, M.H.Mr. Sathish Kumar, M.H.Mr. Sathish Kumar, M.H.Mr. Sathish Kumar, M.H.

Scientist, Dairy Technology Division

Course CoordinatorCourse CoordinatorCourse CoordinatorCourse Coordinator Dr. Shaik Abdul HussainDr. Shaik Abdul HussainDr. Shaik Abdul HussainDr. Shaik Abdul Hussain


Course CoordinatorCourse CoordinatorCourse CoordinatorCourse Coordinator Mr. Gunvantsinh Mr. Gunvantsinh Mr. Gunvantsinh Mr. Gunvantsinh Rathod Rathod Rathod Rathod


ICAR-NATIONAL DAIRY RESEARCH INSTITUTE (DEEMED UNIVERSITY)

KARNAL – 132 001, (HARYANA)

Editing and Compilation

Mr. Gunvantsinh Rathod

Dr. Shaik Abdul Hussain

Mr. Sathish Kumar, M.H.

Dr. P.N. Raju

Cover Page Designed By


Dr. Shaik Abdul Hussain

All Rights Reserved © No part of this lecture compendium may be reproduced or transmitted in any form or by any

means, electronic or mechanical, including photography, recording or any information

storage and retrieval system without the written permission from the Director, ICAR-National

Dairy Research Institute, Karnal, India.

FOREWORD

Training of faculty is essential to update them with the latest knowledge and innovations in the relevant field.

The present training programme is aimed at providing necessary insights into current issues and also to

provide a platform for professionals to interact and enhance their knowledge for making research and

education more effective.

India has achieved a remarkable position in the area of food production in the world, being one among the

highest food producers in the world. Now India is self-sufficient in food production, with the current focus

being on nutritional security of people. There is a need for designing and engineering food products having

potential to fill the gap concerning nutritional security. Much workforce is engaged in fulfilling this novel

deed. Today’s population is facing problems with several lifestyle related disorders. There is a need to

develop speciality foods to counter these life style related ailments. Vast quantum of money is flowing in to

fund research that focuses on these issues. In this context, new age dairy foods developed to prevent various

health problems are gaining popularity throughout the world.

Designing criteria for dairy based functional foods and nutraceuticals involve clear understanding of the intricacies

of food formulation, adoption of appropriate technologies, evaluation and validation of developed products. It is

essential that faculty and researchers who are involved in developing Human Resource for the Food Industry, are

also tuned to these changing trends and demands of consumers so that they can impart quality education to the

prospective commanders of the food industry. Current training program covers many topics on food, nutrition and

their application. It focuses on specialized dairy products, multiple micronutrient fortification and application of

bioactive peptides and probiotics. In addition, it also focuses on novel non-thermal processes like high hydrostatic

pressure, nanotechnology in food packaging, use of bio preservatives etc.

I am sure that this 31st CAFT Training in Dairy Processing will help the faculty and scientists of SAUs and

ICAR institutions to keep abreast with the advances taking place in new age dairy foods at National and

International levels. I sincerely wish National Training Programme under the aegis of CAFT in Dairy

Processing a great success. Further, the information compiled by the organizers in the form of compendium

will greatly benefit the participants and serve as a guide to sensitise them on the researchable issues in the

fields of food and nutrition.

November 21, 2015 (A.K. Srivastava)

CENTRE OF ADVANCED FACULTY TRAINING IN DAIRY PROCESSING

ICAR-NATIONAL DAIRY RESEARCH INSTITUTE (Deemed University)

(Indian Council of Agricultural Research) Karnal-132001 (Haryana) India

Dr. Latha Sabikhi

Director, CAFT (DP)

ACKNOWLEDGEMENT

The Indian Council of Agricultural Research accorded Dairy Technology Division of NDRI as a

centre of Excellence in Dairy Technology for its Centre of Advanced Studies programme in the year

1996. The Centre has recently been renamed as ‘Centre of Advanced Faculty Training (CAFT) in

Dairy Processing’ by the ICAR. Thirty training programmes in different areas of dairy processing

have been successfully organised under CAFT Programme so far. This is the 31st course on

‘Designing New Age Dairy Foods’ being conducted during November 28 to December 18, 2015.

This course will be highly useful for the participating researchers and teachers of Agricultural

Universities, national and other academic institutions in further updating their knowledge in the area

of New Age Dairy Foods.

We express our gratitude to the Indian Council of Agricultural Research for awarding CAFT in

Dairy Processing to NDRI, Karnal. We take this opportunity to thank Dr M.B. Chetti, ADG (HRD)

for approving this short course and timely release of funds.

We express our sincere thanks to Dr. A.K. Srivastava, Director, NDRI, Karnal for his constant

encouragement and guidance and also for providing all necessary facilities for organizing this

course. The continuing interest of Dr. R.K. Malik, Joint Director (Research), NDRI, Karnal, in this

CAFT programme is gratefully acknowledged.

Mr. Sathish Kumar, Scientist (DT) and Course Director deserves a special mention for his diligent

efforts that made the initiation of this programme a success. He has been very ably supported by Dr.

S.A. Hussain and Mr. Gunvantsinh Rathod (Scientists (DT) & Course coordinators) in this

endeavour.

Compilation of various lectures into a compendium, its editing and formatting is a stupendous job.

We must convey our special thanks to the faculty of Dairy Technology, Dairy Chemistry, Dairy

Microbiology, Dairy Engineering, Dairy Economics, Statistics & Management and English for

submission of lectures and for actively participating in conducting the theory and practical classes.

We are highly indebted to the guest speakers who contributed the lecture material well in time and

traveled to Karnal to share their valuable expertise with the participants. We also thank M/s Mother

Dairy, New Delhi for permitting the participants to visit their processing facility. Further, the

contribution of Chairmen and members of different committees for smooth organization of this

training programme is highly appreciable. We are grateful to the technical and supporting staff of

Dairy Technology Division for their contribution to day-to-day activities of this CAFT course.

Date: November 19, 2015 (Latha Sabikhi)

CENTRE OF ADVANCED FACULTY TRAINING IN DAIRY PROCESSING

COMMITTEES FOR ORGANIZATION OF THE

National Training

ON


From 28th

November to 18th

December, 2015

ORGANISING COMMITTEE

Dr. Latha Sabikhi (Director, CAFT)

Mr. Sathish Kumar, M.H. (Course Director)

Dr. Shaik Abdul Hussain (Course Coordinator)

Mr. Gunvantsinh Rathod (Course Coordinator)

RECEPTION COMMITTEE

Dr. Kaushik Khamrui (Chairman)

Mr. Writdhama Prasad

TECHNICAL COMMITTEE

Dr. A.K. Singh (Chairman)

Dr. P.N. Raju

Mr. Yogesh Khetra

Mr. Sathish Kumar M.H

HOSPITALITY COMMITTEE

Dr. Shaik Abdul Hussain (Chairman)

Mr. G.S. Meena

TOUR COMMITTEE

Dr. P.N. Raju (Chairman)

Mr. G.S. Meena

PURCHASE COMMIITTEE

Dr. Latha Sabikhi, (Chairperson)

Dr. Shaik Abdul Hussain (Indenting Officer)


AAO (Audit)

AAO (Purchase)

AAO (Central Store)

List of Participants

1. Dr. A. Poorani

Assistant Professor

Department of Livestock Products

Technology

Veterinary College and Research

Institute

Ramayanpatti, Tirunelveli

Tamil Nadu, PIN: 627 358 Email:

[email protected]

2. Mr. Ashish Maruti Shendurse

Asst. Professor, Dept. of Dairy & Food

Chemistry, Shri. GN Patel Dairy

Science & Food Technology College,

Sardarkrushinagar,Dantiwada

Agricultural University (SDAU)

Sardarkrushinagar, Banaskantha (Dist),

Gujarat, PIN: 385506

Email: [email protected]

3. Dr. Dhiraj Hiraman Kankhare

Assistant Professor

Dept. of Animal Husbandry and Dairy

Science, College of Agriculture, Dhule

Maharashtra, PIN: 424001


4. Dr. Dinkar Keshav Kamble

Associate Professor

Division of Animal Husbandry,

Dairy Science College of Agriculture,

Vidyanagar, Kolhapur



5. Dr. Dnyaneshwar Devrao Patange

Assistant Professor

Dept. of AHDS, College of Agriculture

Kolhapur, Maharashtra, PIN: 416004


6. Dr. Kadam Bapurao Raghunath

Assistant Professor

Maharashtra Animal & Fishery

Sciences University, Futala Lake Road,

Telangkhedi, Nagpur, Maharashtra,

PIN: 440001


7. Dr. Narendra Kumar Nayak

Assistant Professor

Department of LPT

Veterinary College, Mhow, Indore

Madhya Pradesh, PIN: 453446 Email:

[email protected]

8. Dr. Neelam Upadhyay

Scientist

Dairy Technology Division,

NDRI, Karnal, Haryana, PIN: 132001


9. Dr. Om Prakash Malav

Assistant Professor

Department of LPT

College of Veterinary Science

GADVASU, Ludhiana

Punjab, PIN: 141001


10. Dr. P. K. Singh

Assistant Professor

Dairy Technology Division,

College of Dairy Science &

Technology, GADVASU,

Ludhiana, Punjab, PIN: 141004


11. Dr. Praneeta Singh

Assistant Professor

Department of LPT

College of Veterinary & Animal

Sciences (GBPUAT)

Pantnagar, Udham Singh Nagar

Uttarakhand, PIN: 263145


12. Mr. Ranjan Bhagorao Yedatkar

Assistant Professor

Dept. of Dairy Science

Shivaji Mahavidyalaya, Udgir

Latur (Dist), Maharashtra, PIN: 413517


13. Dr. Reeta Mishra

Subject Matter Specialist

RVSKVV-KVK, A.B. Road,

Near Commissioner Office, Morena

(M.P.) PIN: 476001 Email:

[email protected]

14. Dr. Rita Narayanan

Associate Professor

Dept. of LPT, Madras Veterinary

College

Chennai-7, Tamil Nadu, PIN: 600007


15. Dr. Ruma Devi

Assistant Professor

Department of Livestock Products

Technology

C.V.Sc. & A.H., NDUAT, Kumarganj

Faizabad, U.P., PIN: 224229 Email:

[email protected]

16. Dr. Sanjaykumar Vitthal Londhe

Assistant Professor

Dept. of LPT

College of Veterinary and Animal

Sciences, MAFSU

Parbhani, Maharashtra, PIN: 431 402

Email:

[email protected]

17. Mr. Sanket Girdharbhai Borad

Scientist

142, Food Technology Lab, DT

Division

NDRI, Karnal, Haryana, PIN: 132 001


18. Dr. Sharanagouda B. Patil

Associate Prof. & Head

Dept. of Dairy Technology

Dairy Science College, Mahagaon

Cross, Kalaburagi



19. Dr. Somesh Kumar Meshram

Assistant Professor

College of Veterinary Science &

Animal Husbandry

Kuthulia, Rewa, M.P., PIN: 486001


20. Mr. Somnath Hanumant Mane

Assistant Professor

Department of Animal Husbandry and

Dairy Science

College of Agriculture, Pune-3



21. Dr. Vadivoo V. S.

Assistant Professor

Department of Animal Biotechnology

Madras Veterinary College, Chennai-7

Tamil Nadu, PIN: 600007


CENTRE OF ADVANCED FACULTY TRAINING

IN

DAIRY PROCESSING

National Training

ON


From 28th

November to 18th

December, 2015

28.11.2015 (Saturday) DAY 1

9:30 AM - 9:55 AM Registration Dr. S. A. Hussain

10:00 AM -12:00 Noon Inauguration Mini Auditorium

12:00 Noon -1:00 PM Metabolites of dairy microbes and their food

application Dr. R. K. Malik

1:00 PM - 2:00 PM Lunch break

2:15 PM -2:40 PM Visit to ATIC Centre Dr. P. S. Oberoi

2:45 PM - 3:30 PM Visit to Experimental Dairy Mr. H.R. Gupta

3:30 PM - 5:00 PM Visit to Model Dairy Plant Mr. Gian Mutreja

29.11.2015 (Sunday) DAY 2 - HOLIDAY

30.11.2015 (Monday) DAY 3

9:45 AM -10:45 AM Novel starters for value added fermented dairy

products Dr. S. K. Tomar

10:45 AM - 11:00 AM Tea break

11:00 AM -1:00 PM Encapsulation of herbal bioactives through double

emulsion technology (Theory and Practical) Mr. Sathish Kumar


2:15 PM - 3:15 PM Newer probiotic organisms for different

physiological conditions Ms. Rashmi H.M.

3:15 PM - 3:30 PM Tea break

3:30 PM - 5:00 PM Molecular characterization of probiotic organisms-

(Practical) Ms. Rashmi H.M.

01.12.2015 (Tuesday) DAY 4

9:45 AM - 10:45 AM Technological advances in fresh cheeses Dr. S.K. Kanawjia


11:00 AM -1:00 PM Preparation of functional fresh cheese (Practical) Mr. Yogesh Khetra


2:15 PM - 3:15 PM Sodium reduction in cheeses Mr. Yogesh Khetra


3:30 PM - 5:00 PM Designing aspects of newer dairy beverages Mr. Sathish Kumar

02.12.2015 (Wednesday) DAY 5

Programme

9:45 AM - 10:45 AM Technological challenges and design aspects of

vitamin and mineral fortified milks Dr. Sumit Arora

10:45 AM -11:30 AM Communicating science Dr. Meena Malik

11:35 AM -11:45 AM Tea break

11:50 AM -1:00 PM

Technochemical and biological properties of

glycomacropeptide (GMP) and its application in

functional Foods

Dr. Richa Singh


2:15 PM - 5:30 PM Preparation and evaluation of DVS starter cultures

(Theory & Practical)

Dr. Surajit Mandal

03.12.2015 (Thursday) DAY 6

9:45 AM - 10:35 AM Application of casein and caseinates in formulation

of specialized foods

Dr. V.K. Gupta

10:40 AM -11:30 AM Nanotechnological applications in development of

novel dairy foods Dr. S. A. Hussain


11:50 AM - 1:00 PM Probiotics in cheeses Dr. Latha Sabikhi


2:15 PM - 3:15 PM

Advances in infant food formulations

Dr. D.K.Thompkinson

(Guest Faculty)


3:30 PM - 4:30 PM Osteoanabolic effect of milk derived bioactive

peptides Dr. Suman Kapila

4:30 PM - 5:30 PM Mentor Interaction (Dr. Latha Sabikhi, Dr. A. K. Singh, Mr. Sathish Kumar,

Dr. S. A. Hussain & Mr. Gunvantsinh Rathod

04.12.2015 (Friday) DAY 7

9:45 AM - 10:35 AM Oligosaccharides in formulation of functional foods Ms. Indumathi, K.P.

10:40 AM - 11:30 AM Advances in drying of functional dairy foods Er. P.S. Minz


11:50 AM - 1:00 PM

Designing of herb based traditional dairy products Mr. Writdhama Prasad


2:15 PM - 5:00 PM Calcium and Iron fortified milk (Practical) Dr. Sumit Arora

05.12.2015 (Saturday) DAY 8

9:45 AM - 1:00 PM Isolation of potential new probiotic bacteria (Theory

& Practical) Mr. Diwas Pradhan


2:15 PM - 5:00 PM

Technological advances in manufacture of low-

calorie dairy foods (Theory & Practical)

Dr. P. N. Raju

06.12.2015 (Sunday) DAY 9 - Holiday

07.12.2015 (Monday) DAY 10

9:45 AM - 10:35 AM Technologies for the manufacture of colostrum

powder and its applications Mr. Sanket Borad

10:40 AM - 11:30 AM Designing dairy foods to combat metabolic disorders Dr. Kaushik Khamrui


11:45 AM - 1:00 PM

Preservation potential of plant essential oils in dairy

foods Dr. Chand Ram Grover


2:15 PM - 3:05 PM ISO & Food safety systems for food processing

plants

Mr. Appaji Rao

(Guest faculty- Food

Safety Consultant)


3:15 PM - 4:05 PM Newer detection tools to detect milk fat adulteration Dr. Vivek Sharma

4:05 PM - 5:00 PM Technology and application of whey protein

hydrolysate

Ms. Priyanka Singh

Rao

08.12.2015 (Tuesday) DAY 11

9:45 AM - 11:30 AM Cell culture technique for evaluation of therapeutic

potential of dairy foods

Dr. Ritu Trivedi

(Guest faculty)


11:45 AM - 1:00 PM Is it difficult to file Patent in India? Dr. Y. S. Rajput


2:15 PM - 3:15PM

Spores / Enzymes based sensors–An innovative

approach for monitoring food safety Dr. Naresh Goyal

3:15 PM - 5:00 PM Detection of pathogens in selected dairy foods using

advanced techniques (Practical) Mr. Raghu, H. V.


9:45 AM - 10:30 AM Soft computing in dairy and food processing Dr. A. K. Sharma


10:45 AM - 1:00 PM Integration of milk with other food groups for

improved nutritional profile Dr. A. K. Singh


2:15 PM - 3:00 PM Cholesterol reduction technologies for milk and milk

products Dr. Vivek Sharma


3:10 PM - 5:00 PM

Detection of foreign fats in ghee by triglyceride

profiling using low- resolution gas -liquid-

chromatography – (Practical class)

Dr. Vivek Sharma

10.12.2015 (Thursday) DAY 13

9:45 AM - 10:35 AM Newer packaging technologies (nanomaterial-based

and edible packaging) for dairy and food products Dr. P.N. Raju

10:40 AM -11:30 AM Protein based fat replacers: Techno chemical aspects

and their applications in dairy foods

Mr. Gunvantsinh

Rathod


11:40 AM - 1:00 PM Designing milk for human health Dr. Latha Sabikhi


2:15 PM - 5:00 PM Preparation of aloe-vera supplemented probiotic lassi

(Practical) Dr. S. A. Hussain

11.12.2015 (Friday) DAY 14

9:45 AM - 10:30 AM Advances in ice cream and frozen desserts Mr. Yogesh Khetra

10:30 AM - 1:00 PM Formulation of low calorie ice cream (Practical) Dr. S. A. Hussain


2:15 PM - 3:15 PM Application of cryogenics in processing for

dairy and food products Dr. P. Barnwal

3:15 PM - 4:15 PM

Feeding strategies and rumen microbial interventions

for enhancing the nutritional & therapeutic properties

of milk

Dr. A.K. Tyagi


4:30 PM - 5:30 PM Strategies for commercialization of milk based health

foods Dr. A. K. Singh

12.12.2015 (Saturday) DAY 15 – Visit to Mother Dairy, Delhi

13.12.2015 (Sunday) DAY 16 - Holiday

14.12.2015 (Monday) DAY 17

9:45 AM - 10:35 AM Bio-functional applications of lactic acid bacteria Dr. Shilpa Vij

10:40 AM -11:30 AM High pressure processing of milk and milk products Dr. A. K. Singh


11:50 AM - 1:00 PM Application of membrane processing for production

of innovative dairy ingredients Mr. G.S. Meena


2:15 PM - 5:30 PM Membrane processing (Practical) Mr. G.S. Meena

15.12.2015 (Tuesday) DAY 18

9:45 AM - 10:35 AM Selected alternative processes in dairy and food

processing Mr. G. S. Meena

10:40 AM -11:30 AM Production technologies of bioactive peptides from

milk proteins

Dr. Rajesh Kumar Bajaj


11:50 AM - 1:00 PM Milk genomics: An approach for enhancing the

nutritional and therapeutic values of milk Dr. Dheer Singh


2:15 PM - 5:00 PM Sensory evaluation of milk and milk products

(Theory & Practical) Dr. Kaushik Khamrui


9:45 AM - 1:00 PM Presentations on ‘New ideas on functional food

developments’

Participants of this

training program


2:15 PM - 5:00 PM An overview of SAS Enterprise Guide in in dairy and

food sciences (Theory and Practical) Dr. Ravinder Malhotra

17.12.2015 14 (Thursday) DAY 20

9:45 AM - 10:35 AM Cost estimation of value added dairy products Dr. A. K. Chuahan

10:40 AM -11:30 AM Marketing strategy of new food products Dr. Smita Sirohi


11:50 AM - 1:00 PM Challenges in developments of high protein milk and

milk products Dr. V. K. Gupta


2.15PM - 5.00 PM

Rapid diagnostic tests for detection of milk

adulterants – Current Status (Theory and Practical) Dr. Rajan Sharma

18.12.2015 (Friday) DAY 21

9:45 AM - 1:00 PM Course evaluation, online feedback submission and

interaction with Faculty Dr. Latha Sabikhi

1.00 PM - 2:00 PM Lunch break

2.00 PM - 5:00 PM Valedictory function Mini Auditorium

CONTENTS

1 Designing milk for human health 01-05

Latha Sabikhi

2 Technological advances in fresh cheeses 06-09

S K Kanawjia

3 Probiotics in cheeses 10-13

Latha Sabikhi

4 Challenges in developments of high protein milk and milk products 14-19

Vijay Kumar

5 Designing dairy foods to combat metabolic disorders 20-24

Kaushik Khamrui

6 Sodium reduction in cheeses 25-30

Yogesh Khetra, S K Kanawjia, Alok Chatterjee and others

7 Nanotechnological applications in development of novel dairy foods 31-34

S A Hussain, Payal Meena and Akanksha Wadehra

8 Application of membrane processing for production of innovative

dairy ingredients

35-41

G S Meena, A K Singh, S Borad and others

9 Application of casein and caseinates in formulation of specialized

foods

42-46

Vijay Kumar

10 Encapsulation of herbal bioactives through double emulsion

technology

47-49

M H Sathish Kumar, Heena Lamba & Latha Sabikhi

11 Designing of herb based traditional dairy products 50-53

Writdhama Prasad and Kaushik Khamrui

12 Selected alternative processes in dairy and food processing 54-62

G S Meena, A K Singh, S Borad and others

13 Milk genomics: An approach for enhancing the nutritional and

therapeutic values of milk

63-67

Dheer Singh and Suneel Kumar Onteru

14 Protein based fat replacers: Techno chemical aspects and their

applications in dairy foods

68-70

Gunvantsinh Rathod, Latha Sabikhi and Sathishkumar M. H.

15 Technologies for the manufacture of colostrum powder and its

applications

71-76

Sanket Borad and Ashish Kumar Singh

16 Advances in ice cream and frozen desserts 77-79

Yogesh Khetra, S K Kanawjia and Gadsingh Shankar Prakash

17 Designing aspects of newer dairy beverages 80-85

M H Sathish Kumar, Latha Sabikhi and Gunvantsinh Rathod

18 Technological challenges and design aspects of vitamin and mineral

fortified milks

86-89

Sumit Arora, Chitra Gupta and Apurva Sharma

19 Advances in drying of functional dairy foods 90-93

P S Minz and P Barnwal

20 Application of cryogenics in processing for dairy and food products 94-99

P Barnwal

21 Oligosaccharides in formulation of functional foods 100-102

K P Indumathi and Rita

22 Osteoanabolic effect of milk derived bioactive peptides 103-106

Suman Kapila, Srinu Reddi and Rajeev Kapila

23 Newer detection tools to detect milk fat adulteration 107-110

Vivek Sharma and Tanmay Hazara

24 Production technologies of bioactive peptides from milk proteins 111-114

Rajesh Kumar Bajaj and Priyanka Singh Rao

25 Technology and application of whey protein hydrolysate 115-121

Priyanka Singh Rao, S Athira and Richa Singh

26 Technochemical and biological properties of glycomacropeptide

(GMP) and its application in functional Foods

122-126

Richa Singh

27 Cholesterol reduction technologies for milk and milk products 127-130

Vivek Sharma

28 Rapid diagnostic tests for detection of milk adulterants – Current

Status

131-135

Rajan Sharma, Bimlesh Mann, K Satya and others

29 Spores / Enzymes based sensors–An innovative approach for

monitoring food safety

136-143

Naresh Kumar, H V Raghu, Pradip Kumar Sharma and others

30 Bio-functional applications of lactic acid bacteria 144-148

Shilpa Vij and Jagrani Minj

31 Preparation and evaluation of DVS starter cultures 149-153

Surajit Mandal

32 Novel starters for value added fermented dairy products 154-165

Sudhir Kumar Tomar, C S Rajani and Hitesh Kumar

33 Newer probiotic organisms for different physiological conditions 166-168

Rashmi H. M

34 Isolation of potential new probiotic bacteria 169-171

Diwas Pradhan

35 Preservation potential of plant essential oils in dairy foods 172-178

Chand Ram Grover and Rohit Panwar

36 Feeding strategies and rumen microbial interventions for enhancing

the nutritional & therapeutic properties of milk

179-183

Amrish Kumar Tyagi and Sachin Kumar

37 Marketing strategy of new food products 184-189

Smita Sirohi and Divya Pandey

38 Cost estimation of value added dairy products 190-195

A K Chuahan

39 Soft computing in dairy and food processing 196-201

A K Sharma

40 An overview of SAS Enterprise Guide in in dairy and food sciences 202-220

Ravinder Malhotra

41 Is it difficult to file Patent in India? 221-224

Y.S. Rajput, Rajan Sharma and Dheraj Nanda

42 Sensory evaluation of milk and milk products 225-230

Kaushik Khamrui and Writdhama Prasad

43 High pressure processing of milk and milk products 231-236

Ashish Kumar Singh, P N Raju, Sanket Borad

44 Advances in infant food formulations 237-248

D K Thompkinson

45 Newer packaging technologies (nanomaterial-based and edible

packaging) for dairy and food products

249-258

Narender Raju Panjagari, Mukesh Kumar Bishnoi, Pavan Kanade

46 Technological advances in manufacture of low-calorie dairy foods 259-262

Narender Raju Panjagari and Ashish Kumar Singh

47 Metabolites of dairy microbes and their food application 263-271

R K Malik, Chhaya Goyal and Taufeeq

48 Detection of foreign fats in ghee by triglyceride profiling using low-

resolution gas -liquid- chromatography – (Practical class)

272-273

Vivek Sharma and Tanmay Hazra

49 Communicating science 274-279

Meena Malik

11

Designing Milk for Human Health

Latha Sabikhi

Dairy Technology Division

1. Introduction

Functional foods, with their extra-nutritional health benefits have started to occupy prime positions in the

manufacturing industries. Milk, with its intrinsic beneficial attributes, is a natural functional food. While

the earlier emphasis was to breed animals to produce more milk, the attention is now tuned to adding

more value to milk and studying its health implications. By a comprehensive understanding of the

biochemistry, genetic makeup and changes in the animals diet that affect milk synthesis and composition,

ways and means to manipulate milk composition to suit specific needs can be found. Using the two

approaches of nutritional and genetic interventions, researchers are now hoping to develop 'designer milk'

tailored to consumer preferences or rich in specific milk components that have implications in health as

well as processing. Some potential means to alter milk composition or 'design' milk by nutritional and

genetic approaches so as to achieve specific health and/or processing opportunities are discussed

hereunder.

2. Applications of ‘designing’ milk

Designer milk may be applied for human health measures and also for better processing abilities. Among

applications of designer milk in diet and human health is that it generates a healthier proportion of

unsaturated fatty acids (USFA) in milk fat, reduced lactose content for the benefit of lactose intolerant

individuals and removal of β-lactoglobulin that is absent in human milk from bovine milk. Better

processing and technological prospects include alteration of primary structure of casein to improve

technological properties of milk, production of high-protein milk, accelerated curd clotting time for

cheese manufacturing, increased yield and/or more protein recovery, milk containing nutraceuticals and

replacement for infant formula etc.

3. Alterations in milk fat

The quantity of milk fat is a determinant of its value and hence a major indicator of the revenue accrued

from milk. However, it has a negative connotation in terms of human health, owing to the negative

publicity that milk fat is receiving. Alternative feeding practices and genetic interventions has now made

manipulation of composition of milk fat to suit the human needs possible. Some of the possibilities are

decreasing the level of saturation in milk fat, increasing conjugated linoleic acid (CLA) and omega 3 fat

levels in milk fat, and reducing the fat content in milk.

The degree of unsaturation of milk fat rises with the increase in the unsaturated fat content in dairy feeds.

Over and above the health benefits, this also provides a boost to the processing industry. For example,

the spreadability of butter made from this milk being higher, owing to the lower melting point of milk fat

containing USFA. Feeding of a special encapsulated blend of canola and soybean meal doubled the

spreadability of butter in an Australisn study in the late 1990s. When taken out of a fridge at 5ºC, the

22

butter was nearly as spreadable as margarine, without losing its special eating qualities. Clinical trials

revealed that consumption of dairy products made from this milk led to decrease in low density

lipoprotein levels in the blood of the consumers. The type of fatty acids present in milk fat also has a

profound impact on the flavour and physical properties of dairy products. Batches of butter produced

from milk of high oleic sunflower seeds- and regular sunflower seeds-fed cows were equal or superior in

flavours to the control butter. Cheddar cheese comparable to control, but with higher concentrations of

USFA could be produced from milk of cows fed on extruded soybean and sunflower diets. Also, the

abundance of C12, C14, C18:1 and C18:2 fatty acids enhanced the safety of cheeses against Listeria

monocytogenes and Salmonella typhimurium.

CLAs reportedly suppress carcinogens, inhibiting proliferation of leukaemia and cancers of the colon,

prostate, ovarian, and breast. The other reported beneficial health effects of CLA are antiatherogenic

effect, altered nutrient partitioning and lipid metabolism, antidiabetic action (type II diabetes), immunity

enhancement and improved bone mineralization. Dairy products are rich in CLA, which is synthesized in

the rumen during the biohydrogenation of linoleic acid. A diet rich in linoleic acid may increase the CLA

levels in milk fat two-fold, while simultaneously reducing the saturated fatty acid levels in milk fat. Milk

from a grass-fed cow can have five times as much CLA as milk from a grain-fed animal.

The polyunsaturated fatty acid content in human diet should not be much greater than 4% of the caloric

total, in approximate proportions of 2% -3 linolenic acid and 2% -6 linoleic acid. Milk from grass-fed

cows contains an ideal ratio of essential fatty acids (EFAs). Replacing grass in the diet with grains or

other supplements increases the proportion of -6 to -3 fatty acids. While deficiency in -3 is

associated with asthma, heart disease and learning deficiencies, high -6 content in the diet can lead to

cancer, cell proliferation, clotting of blood, depressed immune function, high blood pressure,

inflammation, irritation of the digestive tract, sterility and weight gain. Report suggest that nearly equal

amounts of omega 3 and omega 6 fatty acids result in lower risk of allergies, autoimmune disorders,

cancer, cardiovascular disease, dementia, diabetes, some mental disorders and obesity.

Genetic makeup of cows could be altered to enable them to generate milk with only two per cent fat,

which would, in turn, reduce the cost of feed per kg milk by 22%. The methods used to change the fat

composition, enzymes that influence the synthesis of fat are targeted. For example, reducing the activity

of acetyl CoA carboxylase that regulates the rate of fat synthesis within the mammary gland would lead

to reduction in the fat content of milk and also the energy requirement of the animal to produce milk.

4. Alterations in lactose

For many human beings, the level of the hydrolysing enzyme lactase or -galctosidase gradually declines

early in life to become almost absent when they become adults. When such individuals ingest milk or

milk products, the lactose remains undigested and mal-absorbed in the gut. This causes retention of water

by its osmotic action, which, when coupled with the bacterial production of large volumes of carbon

dioxide leads to intestinal upsets and dehydration. Lactose intolerance limits the use of dairy products to

many people, depriving them of this valuable source of nutrients. Moreover, since milk is a major source

of calcium, lactose intolerance is many times associated with osteopaenia in old age. The known

remedies for intolerance are dietary changes such as avoidance of dairy products or consumtion of

33

hydrolysed low-lactose products (post-harvest) or through the use of -galactosidase replacement (pre-

harvest).

The preharvest methodologies of reducing lactose involve either the removal of -lactalbumin (which

helps in lactose synthesis) and gene 'knock-out' methodologies or by introducing the lactase enzyme into

milk via mammary gland specific expression. Although these methods are successful, they reduce the

overall sugar content of the milk, resulting in highly viscous milk. Studies on mice indicated that the milk

of such mice was highly viscous with very high protein (88%) and fat (60%), no - lactalbumin and no

lactose. An alternative to produce low-lactose milk is over-expression of -galactosidase in milk. But, the

monosachharides produced within the milk increases the osmotic pressure within the alveolar lumen,

thereby drawing more water and resulting in further dilution of other milk components. Transgenic

methods that generated mice that selectively produced a biologically active -galactosidase in their milk

led to halving the lactose content of the milk, while the enzyme expression levels were low. These

experiments conducted led to reduction in the lactose content while retaining most of the monosaccharide

content of the milk.

5. Alteration in milk protein

Protein supplementation, in terms of increased content or improved amino acid profile by the addition of

L-taurine, L-leucine and L-phenylalanine offers additional nutritional benefits to milk. -casein, which is

the most abundant milk protein, is involved in binding calcium phosphate and thus controlling milk

calcium levels. Higher -casein content in milk is linked to smaller micelles, better heat stability, and

improved cheese-making properties. Transgenic cows secreting elevated levels of - (8-20%) and -

caseins (two-fold) have been produced by genetic engineering. In one experiment involving transgenic

animals, the total milk protein increased by 13–20% and total milk casein by 17–35% compared to

control cows. This has immense economic benefits for the cheese, casein and milk protein concentrate

industries. Milk augmented with specific inhibitor of either plasmin or plaminogen activator would be a

boon for the process industry, as both these native enzymes hydrolyse proteins, leading to diminished

protein yields.

The consumption of A1 -casein is associated with health risks such as autism or Asperger’s syndrome,

child diabetes, schizophrenia and coronary heart disease. A2 MilkTM from commercial dairy herds is

being marketed in New Zealand and Australia at a small premium over regular or A1 milk. It is claimed

that A2 MilkTM has only negligible amounts of the A1 -casein in it. The animals producing A2

MilkTM are not genetically modified, but achieved by selectively milking only those cows that naturally

produce milk without A1.

6. Bovine milk that resembles human milk

The composition of bovine milk used to produce infant formulae could be greatly improved to suit the

needs of the infant by incorporation ingredients that resemble those of human milk. Lactoferrin, the iron-

binding protein and also lysozyme, have antimicrobial properties and may also mediate some effects of

inflammation and have a role in regulating various components of the immune system. The lactoferrin

level in human milk is about one g/l (in human colostrum about seven g/l), about ten times as much as

there in cow milk. Human milk contains 0.4 g/l of lysozyme. Active human lysozyme has been produced

44

in the milk of transgenic mice at the concentrations of 0.78 g/l. On the processing front, the expression of

lysozyme in milk results in the reduction of rennet clotting time and greater gel strength in the clot. A

double transgenic cow that co-expresses both human lactoferrin and human lysozyme in milk may reduce

the incidence of intra-mammary infection or mastitis. Production of human lipase in bovine milk could be

used as a constituent of formulas to increase the digestibility of lipids especially in premature infants.

Cow milk contains a natural protein, β-lactoglobulin, not present in human milk. Cow milk allerginicity

in children is often caused by the presence of this protein and has been minimised by feeding hydrolysed

whey. However, this is not a perfect process, and some β-lactoglobulin fragments can still induce

allergies in susceptible infants. Removing β–lactoglobulin from cow milk to make it more like human

milk is the obvious alternative. New Zealand researchers have now ‘designed’ a cow that produces little,

if any, β-lactoglobulin in its milk.

7. Human therapeutic proteins in milk

Several human proteins that have high value in terms of health implications have been generated in milk

of cows and other small animals. GTC Biotherapeutics (Framingham, MA) produces more than 60

therapeutic proteins (plasma proteins, monoclonal antibodies, vaccines) from milk of cows and goats.

The company is working on a project to develop a malaria vaccine from goat milk. It is claimed that a

litre of goat milk can contain up to nine grams of the transgenic protein and that eight goats can produce

enough vaccine to inoculate 20 million people. A recombinant human anti-thrombin III (produced in goat

milk), an anti-coagulant protein found in blood is being tested for its efficacy. PPL Therapeutics

(Edinburgh, UK, and Blacksburg, VA) works with rabbits and sheep to produce -1-antitrypsin,

fibrinogen, and a lipase meant for those who cannot digest dietary lipids. Products such as insulin,

growth hormones and blood clotting factors have also been obtained from the milk of transgenic cows,

sheep, or goats. The major advantage of transgenic technology is that proteins can be produced at a very

low cost. The production costs per gram of human tissue plasminogen activator through bacterial

fermentation, mammalian cell culture and cow transgenic technology were estimated at 20000, 10000

and 10 US dollars respectively.

8. Conclusion

If transgenic technology that produced 33% more total solids (40-50% TS) and 17% less lactose in milk

of experimental mice than that of normal ones could be translated into large animals, the idea of cow

milk that contains 6.5% protein, 7% fat, 2.5% lactose and 50% less water is not unimaginable. The

advantages, besides the direct economic benefit in terms of 50% reduction in the cost of shipping milk,

would be less stress on the cow and on her udder (since she would be producing one half her normal

volume of milk) and milk with higher solids content. Despite these promising prospects, the future of

transgenics in dairy animals is far from being accepted, as several ethical, legal and social aspects of

biotechnological research would need to be addressed. Nevertheless, milk designing allows food and

beverage manufacturers to retain all of the best features of milk (texture, taste, protein content and

calcium) intact, while removing or reducing milk lactose and fat inorder to yield value-added ‘designer’

milk.

9. Selected reading

Karatzas C N (2003) Designer milk from transgenic clones. Nature Biotechnology. 21, 138–139.

55

Sabikhi L (2007). Designer Milk. Advances in Food and Nutrition Research. Chapter 5. Vol. 53. Elsevier

Publications, Academic Press/Elsevier, San Diego, CA, USA. pp:161-198.

www.a2corporation.com

www.afns.ualberta.ca

www.nationaldairycouncil.org

http://www.nationaldairycouncil.org/

6

Technological Advances in Fresh Cheeses

S. K. Kanawjia


1. Introduction

Cheese has been classified on the basis of its composition, coagulating agent, extent/manner of

ripening etc. On the basis of ripening, cheese is broadly classified as ripened cheese and unripened or

fresh cheese.Fresh cheese include the products which are ready for consumption shortly after

manufacture. These may be acid coagulated or rennet coagulated. On the basis of manner of

coagulation, fresh cheese again may be classified as rennet curd cheese or fresh acid-curd cheese. For

most of the varieties, starter culture and rennet are used in combination for development of desired

quality characteristics. Some of the popular fresh cheese varieties consumed round the globe are

discussed in this chapter.

2. Quarg Cheese

Quarg – the proper German name is Speisequark – is a natural, unripened, fresh cheese produced on a

large scale in Germany and is very popular there. It is essentially a milk protein paste, manufactured

by acid coagulation of milk by proper bacterial cultures (e.g. Streptococcus cremoris and Leuconostoc

citrovorum) with a small rennet addition for better separation of the protein coagulum from the whey

and thus better yields. It is produced in a variety of fat contents, ranging from an essentially fat-free

type to a variant with as much as 40 per cent fat in the dry matter. This cheese is popular in central

Europe (e.g. Germany, Poland and Austria). Other names for this type of product in different countries

include kvarg, tvarog, tworog, twarog, Sauermilchquark and Speisequark. Chakka and Shrikhand are

the products related to quarg popular in India.

Quarg is milky white in color, may be even faintly yellowish. Body and texture are homogeneously

soft, smooth and mildly supple or elastic. Spreadability must be good. Due to high moisture content (~

82%, w/w), the shelf-life is limited to 2-4 weeks at <80C. There should be no appearance of water or

whey, dryness or graininess, bacteriological deterioration, over-acidification or bitter flavour during

storage (Kroger, 1980; Siggelkow, 1984; Guinee et al, 1993). Odor and taste, i.e. the flavour, must be

clean and may be mildly acidic. Quarg is essentially coagulated, flocculated casein with high water

content. It is manufactured from milk by acid coagulation and/or rennet coagulation and subsequent

separation of whey. The composition is variable and depends largely on the composition of vat milk.

In comparison to most ripened cheeses, Quarg is low in dry matter (DM) and, hence, low in fat and

protein and high in lactose/lactate. As most of the calcium is solubilised during the acid coagulation

and removed with the whey, it is much lower in calcium than rennet-curd cheeses. (Schulz, D. et al.

1999).

2.1 Technology Developed at NDRI for manufacture of quarg cheese

Quarg cheese was manufactured by using method described by Spasenija D. Milanovic et al. (2004)

with some modifications. Good quality standardized buffalo milk ( Fat- 3.0±0.30%, SNF-

9.50±0.25%) was taken in a vat and pasteurized at 850C for 15 minutes. The cheese milk is then

cooled and inoculated with NCDC culture @ 1.0 percent and incubated at temperature 370C. Two and

half hrs (2.5 hrs) after the addition of starter culture (when the pH reaches up to 6.3), Meito rennet @

200-300mg/100kg milk was added and mixed thoroughly. Thereafter the vat content was left

undisturbed for curd setting, which took around 14-16 hrs starting from culturing. The coagulum was

then cut using 1/3 inch cheese knives and it was again left undisturbed for about 10-15 minutes. The

7

temperature of the contents was then slowly and gradually increased to 55-600C @ 10C per minute

and the curd kept hold for 10 minutes at 600C as per the requirement for thermoquarg manufacture.

Cooked curd was then cooled to room temperature and filled in muslin cloth for 3.5 to 4 hrs (TS

around 28-30%) for dewheying. Thereafter fibers, plant sterol esters and salt were added in curd and

homogenization of total mass was carried out in Hobart mixer. The quarg is then filled in PS cups and

stored at 6±10C.

Figure 1: Flow Diagram for manufacture of Fiber and phytosterol enriched quarg cheese.

Buffalo Milk

Standardization

Pasteurization

Cooling (30-370C)

Inoculation

Renneting

Incubation 14-16 hrs

Cutting & Stirring

Heating 600C/10 minutes

Cooling [ Room temp.]

Whey off

Mixing

Quarg Cheese

Salt, Dietary Fibers and

Plant Sterol Esters

8

2.2 Enhancement of functional attributes in Quarg Cheese

The current trend is functional foods development is to enhance the health attributes of widely

consumed foods by fortifying with functional ingredient. It has been established that plant sterols and

stanols lower blood cholesterol levels by partly blocking absorption of cholesterol in the gut. Now

they are widely available in a range of food products for those who want to lower their cholesterol

level. Dietary fiber is another food ingredient gaining lot of attention from the health point of view.

2.2.1 Enrichment with Dietary Fibers

Dietary fiber, especially soluble fibers are associated with carbohydrate and lipid metabolism has

shown to have hypercholesterolemic properties. Keeping in view the reported beneficial effect of

dietary fiber on cardiac disease, inulin (Raftiline), oat (Vitacel) fiber and soy fiber were assessed for

their suitability. Inulin was used @ 8-12%, w/w, of curd and oat fiber and soy fiber @1-3%, w/w, of

the curd in order to provide sufficient concentration of dietary fiber in the product. The study revealed

addition of oat fiber at the level 1.0 % resulted in an increase in all the sensory scores of all attributes

studied and also found very close to control. Further increasing the level of oat fiber from 2.0 to 3.0

percent, there was substantial decrease in the sensory scores. Oat fiber @ 1.0 percent showed the

highest overall acceptability may be because of reduced free whey, whitish to creamy colour, good

body and textural attributes. The lowering of scores for body and texture at higher levels of oat fiber

could have been because of harder body, creamy colour and poor spreadability of the product.

Sensory responses of quarg cheese with different levels of soy fiber inferred that the quarg cheese

containing 1.0 percent soy fiber received highest flavour, body and texture, colour and appearance as

well as overall acceptability score. Further increasing the level to 3.0 percent resulted in significant

(p<0.05) lowering of sensory scores.

2.2.2 Enrichment of Quarg cheese with prebiotic and probiotic attributes

The technology design to develop the probiotic Quarg cheese with enhanced therapeutic dose for

claimed health benefits up to the shelf life of product. Two selected probiotics added in Quarg cheese

in three different forms viz. propagated probiotic culture, concentrated cell biomass and encapsulated

probiotic at two different stages viz. with traditional starter culture (TSC) and at mixing stage, and

results of various parameters compared with control sample. In each process prebiotic inulin was also

added for stimulation of growth of probiotic. Probiotic Quarg cheese manufactured by each process,

evaluated for the sensory, textural, physico-chemical and survivability of probiotic in fresh cheese

sample as well as during storage also. Based on these results method of manufacturing and probiotic

culture were selected for further storage study. The results obtained revealed that probiotic Quarg

cheese with desired quality attributes and therapeutic dose can be made employing M3 and M4

methods. Further, it is inferred that M2 method has adverse effect on quality attributes of Quarg

cheese. It was also observed that Quarg manufactured using probiotic L. casei (NCDC 298) possessed

good overall acceptability and survivability during storage of 30 days.

3. Cream Cheese

Cream cheese is a soft, rich, creamy white, unripened cheese having slightly acidic taste with diacetyl

flavor. It is usually manufactured by the coagulation of cream or mixture of milk and cream by

acidification with starter culture (Guinee et al., 1993). It is very popular in North America, Asia and

Oceania. It is used as spread, salad dressing and often used in place of butter, high fat spread.

According to FSSR (2011), cream cheese (Rahmfrischkase) means soft unripened cheese obtained by

coagulation of pasteurised milk of cow and / or buffalo or mixtures thereof and pasteurised cream

with cultures of harmless lactic acid producing bacteria with or without the addition of suitable

coagulating enzymes. It shall have a soft smooth texture with a white to light cream colour. It may

contain spices, condiments, seasonings and fruit pulp. It shall contain moisture not more than 55.0

9

percent and milk fat on dry basis not less than 70.0 percent. Salts, stabilizers and preservatives may be

added in cream cheese.

3.1 Manufacture of cream cheese

Lundstedt (1954) had described the traditional method for making cream cheese. In this method milk

was first standardized to 12% fat. Then the milk was subjected to single stage homogenization at 1800

psi pressure at a temperature of 50˚C followed by pasteurization at 65˚C for not less than 30 min.

Then the milk was cooled to desired setting temperature of 22˚C. The milk was then inoculated with

lactic acid bacteria and incubated for 16 to 18 hr. until the desired pH of 4.6 was reached. The

acidified gel or curd formed was heated to 54˚C followed by drainage of whey in cloth bags for

overnight. It is observed that the source and amount of fat, the percentage of non-fat Solids, heat

treatments, homogenization pressures and pH have a great impact on final body and texture of cream

cheese.

3.2 Enhancemnent of functionality

Now a days, the demand for functional foods has directed the attention of researchers towards making

the cream cheese functional by addition of inulin, phytosterol etc.

It was observed that addition of inulin in cream cheese as a fat replacer at the rate of 1 g for 25 g of fat

replacement made the product more firm as compared to control cream cheese because increase in

inulin content decreases the moisture content of the product (Fadaei et al., 2012). Sensorialy upto 50%

replacement of fat in cream cheese was reported to be acceptable. In addition to this, inulin increases

the dietary fibre content of cream cheese.

Due to the immense potentiality to reduce LDL cholesterol level in blood, plant sterol esters at the

amount of 2, 3 and 4% were added in cream cheese to enhance its functionality. Though the addition

of phytosterol upto 4% level showed no significant change in sensory quality of inulin enriched cream

cheese, it was reported that phytosterol at a level of 4% contributed to smoother as well as firmer

body and texture as well as enriched flavour to the product.

4. Functional Cheese Spread

Butter, the traditional spread for bread is now avoided due to poor spreadability, high saturated fat and

cholesterol content. The annual growth rate of cheese production in India is 10-15% and 90% cheese

is consumed as processed cheese and processed cheese spread. Processed cheese spread contains a

lower amount of fat and higher amount of protein compared to any low fat table spread. It contains

not only that protein and fat are in pre-digested form, also it contains calcium, phosphorus, riboflavin

and other vitamins in a concentrated form and contains health beneficial bioactive peptides. In this

direction, cheese spread can provide nutritionally superior spread for bread and it can be incorporated

with functional ingredients.

Processed cheese spread has been developed with three functional ingredients viz., inulin,

phytosterols and ω-3 fatty acid. Effect of different levels of inulin, phytosterols and ω-3 fatty acid

additions were investigated. The optimization of the level of the three functional ingredients in

combination was carried out using Central Composite Rotatable Design of Response Surface

Methodology. The consumer acceptance study revealed that the product had been liked very much by

the consumers due to its flavour and spreadability and the product itself was nutritionally and

functionally sound. To validate its hypocholesterolemic effect, developed products were fed to

hypercholesterolemic rats and it was observed that serum total, LDL, VLDL cholesterol and

Atherogenic index decreased and at the same time liver cholesterol and triglyceride decreased

significantly (p<0.05). Cost of the developed product was at par with the products available in the

market.

10

Probiotics in Cheeses

Latha Sabikhi


1. Introduction

The microbial ecology of the human gastrointestinal system has many important functions and establish

interactions that keep them in a state of dynamic equilibrium, the disturbance of which may affect the

human wellbeing. The major aim of consuming probiotics regularly is the restoration of the healthy status

of the gut in particular, and body, in general. The theoretical basis for selection of probiotic micro-

organisms include safety, functional aspects (survival, adherence, colonisation, antimicrobial production,

immune stimulation and prevention of pathogens) and technological details such as growth in milk and

other food base, sensory properties, stability, phage resistance and viability. The nutritional requirements

of the probiotic strains and their tolerance to the manufacturing and storage stress are some of the aspects

to consider while developing new products. Dairy products are frequently chosen as delivery vehicles of

probiotic microorganisms to the intestine. Currently hundreds of probiotic dairy products are

manufactured and consumed around the world, including pasteurized milk, ice-cream, fermented milks,

cheeses and baby foods. Cheese being a 'live' food, is potentially an excellent vehicle for these beneficial

microbial cultures. It can be classified as a nutritional and convenience food, being a potential carrier for

probiotic cultures and a natural source of biological peptides. This lecture, besides listing the

technological challenges faced while attempting to make probiotic cheese, reviews selected studies on the

manufacture of such cheeses.

2. Technological considerations in the manufacture of probiotic cheese

Probiotic bacteria should be technologically suitable for the incorporation into cheese products so as to

retain both viability and functional efficacy during processing on a commercial scale and throughout

consumption. There are significant differences among species and even strains of probiotic organisms

with respect to the acid- and bile-tolerance of some of these organisms. Since most of the probiotic

bacteria are reported to be pH-sensitive, the choice of a strain that would survive the lowered conditions

of pH in the cheese is imperative. Additionally, this strain should also be able to withstand the acidic

conditions of the human stomach and tolerate the high bile salts concentration of the upper small

intestine.

The relation between the normal lactic cultures that are used in cheesemaking and the probiotic bacteria

that would be introduced is another area of technological importance. They should, from the outset,

provide no competition to each other and should be able to survive in harmony, so as to produce no

undesirable flavour or textural trait in the product. The amount of inoculum and the appropriate time of its

addition in milk (or to cheese) are some other parameters to be investigated. Some probiotic organisms,

particularly bifidobacteria being slow growers, some workers suggest that the amount of inoculum must

be large, often to the extent of 3 to 5 per cent, whereas others are of the opinion that such large quantities

of cultures would lead to biochemical fermentations in the cheese that are atypical of the product.

The oxygen sensitivity of probiotic organisms, particularly bifidobacteria, is a major bottleneck in their

successful cultivation in milk. This can be overcome by using super-concentrated cultures. Dairy products

that are direct set with these cultures contain almost 4 million live bacteria per ml. The counts of oxygen-

11

sensitive organisms are reported to increase during the ripening period, probably owing to the anaerobic

conditions produced in the cheese.

From a food processing perspective, it is desirable that the selected strains are suitable for large-scale

industrial cheese production and withstand the mass processing conditions. In addition, a probiotic cheese

should have the same sensory and nutritional qualities as the conventional cheese. This means that the

level of proteolysis and lipolysis must be the same as that in cheese which does not have probiotic

bacteria.

The method of addition of probiotic bacteria into cheese has a crucial effect in the probiotic viability and

functional efficacy during cheese processing and storage. The probiotic bacteria may be added either

before the fermentation, together with the starter culture or after fermentation. In the first option, the

optimal initial inoculum of probiotic to be added and the amount lost in the whey need to be assessed as

per the manufacturing process. In the latter, cheese must be cooled directly after probiotic addition, as

metabolic activities of starters and probiotic bacteria are drastically controlled and reduced at these low

temperatures. Probiotic bacteria have been added semi-hard cheeses by freeze- and spray-drying

techniques. These methods enhanced probiotic viability during cheese processing and storage via the

protecting probiotic bacteria by encapsulating them.

It is recommended that to accrue benefitcial effects, 108

probiotic bacteria must be ingested. Assuming

that approximately 100 g of cheese is consumed daily, about 106-7

CFU/g will assure the ingestion of 108-9

CFU per day. Addition of prebiotic substances has been attempted to maintain and enhance probiotic

viability in cheeses. Oligofructose and/or inulin added to Petit Suisse cheese enhanced the viability of

both Lactobacillus acidophilus and Bifidobacterium animalis subsp. lactis (Cardarelli et al., 2008).

Several techniques have been applied to enhance and maintain the viability of probiotic bacteria under

harsh conditions typical in cheeses, including the selection of probiotic strains tolerant to oxygen, acid

and bile and the addition of amino acids and peptides. Exposure to sub-lethal levels of the stresses

(temperature, pH or bile salts) before use as the starter adjunct has also been successful. Selection of high

oxygen consumers as probiotic strains is also another useful way to increase viability under anerobic

conditions.

Survival of probiotic organisms in salted cheeses is low, owing to the inhibitory effect of salt, especially

when salt level is higher than 4%. Therefore, processing of cheeses with high salt content should be

optimized to minimize the inhibitory effect of salt. Protecting the probiotic bacteria from the hostile

environment by microencapsulation and/or incubating cells under sub-lethal conditions have been

successfully implemented.

The packaging system and environment in which the cheese is stored has an impact on the viability and

stability of probiotic organisms. Probiotic dairy foods, including cheese, are generally packaged in

plastics films which have different levels of oxygen permeability. Films with low oxygen permeability

should be used for storing probiotic cheeses. Vacuum packaging and modified atmospheric packaging or

a combination of the two may also result in higher viability.

The high protein content in cheese provides probiotic bacteria with a good buffering protection against

the high acidic condition in the gastrointestinal tract. The dense matrix and relatively high fat content of

cheese may offer additional protection to probiotic bacteria in the stomach. Also, the relatively high pH

values and lack of antagonistic effects of starter cultures, in rennet set cheese may exert optimal

conditions to maintain probiotic bacteria viability during cheese making and storage.

12

3. Probiotics in cheese

Several soft, semi soft (semi hard), and hard probiotic cheese products have been developed and marketed

in the last few years. Hard cheeses, such as Cheddar, may offer certain advantages over yoghurt-type

products in terms of delivery of viable probiotics, such as the reduced acidity of the cheese compared with

yoghurt environments and the high fat content and texture of Cheddar cheese, which may offer protection

to the microorganisms during passage through the gastrointestinal tract. Studies have demonstrated that

bifidobacteria survived well in Cheddar and Gouda cheeses.

One report (Stanton et al. 1998) documents the incorporation of a number of strains of probiotic

lactobacilli of human origin into Cheddar cheese and assessment of their performance during ripening.

Cheddar-like cheese was produced by using B. infantis (Ross et al., 2002), while Cheddar cheese was

produced by using L. acidophilus, L. casei, L. paracasei and Bifidobacterium spp. (Ong et al., 2006).

Probiotic strains of L. paracasei A13 were used to produce Argentinian Fresco Cheese (Vinderola et al.,

2009). Sabikhi et al. (2014) established that Edam cheese was a good carrier of B. bifidum (ATCC

15696). The probiotic cheese had over 7.5 log cfu/g of viable bifidobacteria after three months of

ripening. Supplementation of cream dressing with freeze-dried concentrates is a suitable method of

incorporating bifidobacteria in cottage cheese. Incorporation of these organisms may also be a way of

making beta-galactosidase available for lactose-intolerant consumers.

Numerous strains of probiotic bacteria have been successfully added into different types of cheeses

including lactobacilli (L. acidophilus, L. casei, L. gasseri, L. paracasei, L. plantarum, L. rhamnosus) and

Bifidobacterium spp. (B. animalis ssp. lactis, B. bifidum, B. infantis, B. longum), and to a lesser extent,

Propionibacterium freudenreichii ssp. shermanii (Karimi et al., 2011).

Some probiotic cultures, (e.g., L. rhamnosus) produce antibacterial substances that act specifically against

undesired micro-organisms such as clostridia. The use of these organisms is a possible replacement for

nitrate addition to suppress the growth of gas-formers in cheeses like Edam and Gouda. Thus they

promote the natural preservation, with minimised use of chemical preservatives.

4. Conclusion

There is abundant scope in the development of diverse range of cheeses that may afford opportunities as

carriers of probiotic organisms in the altered macro-environment of the product. Cheese manufacturers

searching for avenues to diversify their enterprises would gain tremendously, in view of the enormous

value addition that may be envisaged in making and marketing a probiotic cheese. However, certain

technological characteristics of cheese manufacture must be considered when designing a probiotic

cheese in order to maintain cell viability and quality characteristics. Functionality of each new cheese

developed should be assessed in animal models and clinical trials before qualifying the product as a

probiotic cheese.

5. Selected reading

Cardarelli H R, Buriti F C, Castro I A and Saad S M (2008) Inulin and oligofructose improve sensory quality and

increase the probiotic viable count in potentially synbiotic Petit-Suisse cheese. LWT-Food Science and

Technology 41 1037-1046.

Karimi R, Mortazavian A M and Cruz A G (2011) Viability of probiotic microorganisms in cheese during

production and storage: a review. Dairy Science and Technology 91 283–308.

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Ong L, Henriksson A and Shah N P (2006) Development of probiotic Cheddar cheese containing Lactobacillus

acidophilus, Lactobacillus casei, Lactobacillus paracasei and Bifidobacterium spp. and the influence of these

bacteria on proteolytic patterns and production of organic acid. International Dairy Journal 16446–456.

Ross R P, Fitzgerald G, Collins K and Stanton C (2002) Cheese delivering biocultures-Probiotic cheese. The

Australian Journal of Dairy Technology 57 71-78.

Sabikhi L, Sathish M H K and Mathur B N (2014) Bifidobacterium bifidum in probiotic Edam cheese; influence on

cheese ripening. Journal of Food Science and Technology. 51 3902-3909.

Stanton C, Gardiner G, Lynch P B, Collins J K, Fitzgerald G, Ross R P, Klaenhammer T R, Connolly J F and

FitzGerald R J (1998) Probiotic cheese. In Special Issue: Functional foods: designer foods for the future.

International Dairy Journal 8 491-496.

Vinderola G, Prosello W, Molinari F, Ghiberto D and Reinheimer J (2009) Growth of Lactobacillus paracasei A13

in Argentinian probiotic cheese and its impact on the characteristics of the product. International Journal of

Food Microbiology 135 171-174.

14

Challenges in developments of high protein milk and milk products

Vijay Kumar


1. Introduction

Milk proteins are well known for their high nutritional quality and bioavailability and, therefore, milk

and milk products are major source of quality protein in our diet.Due to absence of anti-nutritional

factors and presence of high proportions of essential amino acids, milk proteins are nutritionally

excellent proteins.Milk proteins also exhibit specific physiological functions like transportation of

trace element or vitamins, inhibition of angiotensin I-converting enzyme (ACE), anti-microbial

activity, anti-carcinogenic activity, hypocholesterolemic activity etc. Further, milk proteins are

preferred ingredients for their functional supremacy, their good flavour, colour and nutritional profile.

Major protein of milk, casein has some rather unique properties and cannot be replaced by other

proteins in certain food application. Whey proteins have an exceptional biological value that exceeds

that of egg protein and therefore, whey proteins are choice for body builders and elite athletes

(Buckley et al., 1998; Carey et al., 2006). Whey proteins are considered as rapid digested protein that

gives high concentrations of amino acids in postprandial plasma (Nilsson et al., 2007). In addition,

whey proteins contain a number of other proteins that positively affect immune function such as

antimicrobial activity (Ha and Zemel, 2003). Whey protein also contains a high concentration of

branched chain amino acids (leucine, isoleucine, and valine). These latter amino acids are thought to

play a role as metabolic regulators in protein and glucose homoeostasis, and in lipid metabolism, and

as such may play a role in weight control (Smilowitz et al., 2005; Zemel, 2004). They are also

important for their role in the maintenance of tissue and prevention of catabolic actions during

exercise (MacLean et al., 1994). Further, whey proteins are rich and balanced source of sulphur amino

acids (methionine, cysteine). These amino acids serve a critical role as anti-oxidants, as precursors to

the potent intracellular anti-oxidant glutathione (Shoveller et al., 2005), which has been shown to

have strong antioxidant properties that can assist the body in combating various diseases (Counous,

2000).

Demand of protein enriched food products is increasing with increasing consumer health

awareness.Current trends and changing consumer needs indicate a great opportunity for protein

enriched food product. High protein milk is suitable for consumers, those who require high protein

diet. It also helps in coping problem of malnutrition in children. High protein milk is beneficial for

enhancing the physical fitness, decreasing obesity while maintaining muscle and bone strength of the

consumers. Quantity of protein is an important consideration when we use milk as a sports recovery

beverage. High protein milk provides the dietary requirement of protein for lactating and pregnant

women, sports persons, growing children and muscle development and maintenance in adults. High

protein diet is recommended for pregnant women who are strict vegetarian as it helps in placental

growth and its functioning, proper growth of baby in womb. Protein sources are much more important

for elderly as they need amino acids to repair cells and achieve longevity. High mineral content of

high protein milk avoid osteoporosis and other mineral deficiency condition.

2. Challenges in developments of high protein milk

Enhancement of the protein content of milk can be carried out by addition of conventional milk

protein products such as sodium caseinate and coprecipitates, ultrafiltration of milk and by addition of

UF separated milk protein concentrates, whey protein concentrates or whey protein isolates.

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2.1Addition of conventional milk protein products

Preparation of Sodium caseinate and coprecipitates requires the addition of significant amount of

sodium hydroxide and otherchemicals. Therefore, enhancing milk proteins in milk with the addition

of these protein productscan’t be accepted by FSSA (2006).

2.2Application of UF Technology

McDonough et al. (1976) reported that up to 40% of the total solids of skim milk could be replaced

with a whey protein concentrate (WPC 35%) derived from sweet whey, or 20% with a whey protein

concentrate derived from acid whey without a deleterious effect on sensory quality. The lower

threshold value for whey protein concentrate made from acid whey was attributed to its higher content

of salt and organic acid. Increasing the protein content of 2% fat milk from ~3.4% to at least ~3.8%

by the addition of α-lactalbumin-rich whey protein fraction had no effect on the sensory quality of the

milk (Peter at el., 1987).

High whey protein beverages remain a sensory challenge due often to astringency and an unpleasant

after-taste following consumption. Processing and flavour-masking have both been usefully applied in

addressing this and other challenges in the manufacture of drinks containing a high content of whey

protein (Beecher et al., 2006; Drake, 2006; Johnson et al., 1996).

An attempt was made to develop protein enriched cow and buffalo milk using whey protein

concentrate (WPC) and ultrafiltration (UF) retentate (Bihari, 2012). Effect of different protein

levelsviz. 5, 6 and 7% was investigated on the quality of protein enriched milk. With increased protein

content, there was found a highly significant (p<0.01) increase in total solids, ash content, calcium

content, titratable acidity and viscosity of protein enriched cow and buffalo milks, irrespective of

protein source studied. In case of WPC, enhanced protein content milk exhibited highly significantly

(p<0.01) better sensory scores for flavour, consistency/mouthfeel and colour and appearance at 5%

protein level, irrespective of type of milk. It was observed that, among the two protein sources, UF

retentate enriched milk had a highly significantly (p<0.01) higher sensory scores for flavour,

consistency/mouthfeel and colour and appearance than WPC enriched milk, irrespective of type of

milk. In case of UF retentate enriched cow milk, maximum sensory scores were obtained at 7%

protein level, whereas in buffalo milk, sensory scores were maximum at 6% protein level.

UF retentate enriched milk was stable (27 min) at 140˚C, whereas, WPC enriched milk was thermally

unstable at 140˚C. UF retentate enriched milk contained highly significantly (p<0.01) greater amount

of calcium than WPC enriched milk. Whitening index of UF retentate enriched cow and buffalo milks

was observed highly significantly (p<0.01) higher than WPC enriched milk. Overall, quality of WPC

enriched milk was very much inferior than UF retentate enriched milk.Hence UF retentate was

recommended for the production of enhanced protein content milk.

Addition of UF retentate to milk for the enrichment of proteins was found to be economical both in

cow and buffalo milk. The total estimated production cost per liter of protein enriched cow milk was

₹ 33.50, 40.98 and 48.20 for 5, 6 and 7% protein milk, whereas in protein enriched buffalo milk, at

5, 6 and 7% protein level, total production cost was ₹ 38.78, 45.49 and 52.19, respectively.

3. Challenges in developments of high protein milk products

3.1 Ultrafiltration Process

Ultrafiltration (UF) process is most flexible, convenient and efficient technological method for

enhancement of protein content of milk (Poulsen, 1978). Additionally, UF process has many

advantages like energy saving, improved yield of protein, enhanced nutritive value of the product,

recovery of whey protein and reduction of whey disposal problem. The nutritionally important whey

proteins are recovered in their native and functional state in the product.Ultrafiltration has a wide

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range of applications in the dairy industry. From milk, UF produces a permeate containing water,

lactose, soluble minerals, non-protein nitrogen and water-soluble vitamins and a retentate in which

proteins, fat and colloidal salts content increase in proportion to the amount of permeate removed. The

process has been widely used for the manufacture of WPC, milk protein concentrates, low lactose

powder several fermented dairy products like Yoghurt and Srikhand. UF retentate seems to be a

highly promising base for chhana, long-life paneer.UF technology has also been applied to upgrade

khoa maufacture from cow and buffalo milks.

The milk concentrate obtained by UF has a greatly altered compositional properties compared

to conventionally or RO concentrated milk. Skim milk ultrafiltration retentate is found to have

higher concentration of protein, fat, total solid and minerals content. However, Ultrafiltration does not

alter the natural ratio of whey protein to casein in milk. Patel et.al., (1992) reported that when buffalo

skim milk ultrafiltered to 5.5 fold concentration, the TS content of buffalo skim milk increased

from 10.20% initially to 23.50% after maximum weight reduction of approximately 77%, after which

the flux rate becomes almost zero. Similarly, protein content increased from 3.96% to 16.44%, fat

from 0.10 to 0.4% and ash from 0.92 to 2.10%. Lactose concentration of skim milk was observed to

decrease from 5.22 to 4.56% by UF.Lewis (1986) reported that viscosity is the main factor, which

limits the extent of concentration by UF; the protein fraction makes the main contribution to the

viscosity.

Patel et al. (1999) reported that as the concentration in terms of total solids increased during UF

concentration of buffalo skim milk the heat stability got decreased. The initial HCT at 120°C of

buffalo skim milk was 111 min, at total solids, 10.59% (protein 4.19%) that decreased to 11 min after

UF concentration to total solids, 28.39% (protein, 21.45%). The HCT at 120°C values at the

total solids level of 14.41, 17.79, 25.04 and 28.39% were reported to be 52, 38, 37.5 and 11 min,

respectively.

3.2 Milkprotein concentrates

Typically with a protein content from 50-85% of total solids, MPC can be considered as a functional

ingredient to be used in the manufacture of other foodstuffs. To obtain milk protein concentrates with

85% protein/TS, it is necessary to employ diafilteration treatment. General problems of MPC are its

poor solubility, reconstitutability and stability. The main application of MPC today are in spreads and

dressings .It is also used as a protein base in processed or even recombined cheeses. This high protein

and high calcium ingredient can be used for the preparation of many dietetic foods including foods for

elderly people and sport persons.

3.3 Low lactose powder

Lactose intolerance is a global problem. UF technology is employed for the manufacture of low-

lactose powder. Additional diafiltration treatment is employed to further reduce lactose. During the

ultrafiltration process, some of the soluble salts like calcium, sodium and potassium are bound to go

in the permeate. These salts are important for giving milk its natural taste. To maintain the salt level

and thereby revive the original taste of milk on reconstitution, salts are added to the retentate before

spray drying. The addition of potassium chloride and trisodium citrate at optimized levels of 0.85%

and 0.55% to buffalo milk UF-DF retentate was observed to increase the flavour score of product

from 6.33 (control) to 7.42 on 9 point Hedonic scale. Further, to improve the drying properties of the

retentate and reconstitutability of the powder, 4% malto-dextrin is added to the retentate before spray

drying (Patel et al., 1991).

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3.4 UFChhana

Preparation of good quality chhana using skim milk ultrafiltered-diafiltered retentate and plastic

cream has been reported (Sharma and Reuter, 1991). Skim milk, heated to 95°C for 5 min., is

ultrafiltered (26% TS). The retentate is diafiltered (23% TS) with equal amount of water to reduce

lactose. For preparation of chhana, the retentate is mixed with plastic cream to a protein/fat ratio of

0.722. The mixture is heated to 85-90°C/5 min. and coagulated with dilute lactic acid to develop the

characteristic grain. The granular mass is subsequently pressed to remove free moisture, yielding

chhana. The process is reported to yield about 18-19 percent extra product and also no significant

difference in flavour, body and texture and appearance compared to traditional method.

3.5 UF Paneer

Production of good quality paneer using ultrafiltration (UF) has been reported by Sachdeva et al.

(1993). The process offers advantages like access to mechanisation, uniform quality, improved shelf

life, increased yield and nutritionally better product. The method involves standardisation and heating

of milk followed by UF. The yield increases by about 25 percent due to the retention of good quality

whey proteins and the slightly increased moisture content.

3.6 Processed Cheese Foods

At Federal Dairy Research Centre in Federal Republic of Germany, Gupta and Reuter (1992)

standardised the manufacturing process of processed cheese foods with 20% of their cheese solids

replaced by WPC. An increased amount of WPC and trisodium citrate improved the firmness in a

highly significant manner (p <0.01), but had a highly significant deleterious effect (p <0.01) on the

melting quality of processed cheese foods (Gupta and Reuter, 1993). Diafiltration of WPC had a

negative effect as regards suitability for the product. With the increase of moisture content over a

wide range, the firmness of processed cheese foods decreased in a highly significant manner (p

<0.01), while melting quality increased in a highly significant manner. It was also observed that

increased amount of WPC in processed cheese foods imparted milder flavour in the final product

(Thapa and Gupta, 1996). Trisodium citrate at 2.5% and with a moisture content of 45.2% resulted in

processed cheese foods with the best sensory characteristics (out of a total of 7, the scores were as

follows: flavour, 5.5; consistency, 6; appearance, 5.8; overall acceptability, 5.6. With less than 43.4%

moisture, processed cheese foods were judged to be short, dry, hard, brittle and crumbly in body.

Thapa and Gupta (1992) reported the preparation of processed cheese foods with 15 and 20% cheese

solids replacement by solids of WPC (27.41% TS), obtained through ultrafiltration of cheddar cheese

whey. Instron measurements revealed that increased levels of WPC and emulsifier and decreased

moisture level imparted greater hardness, springiness, adhesiveness, gumminess and chewiness in

processed cheese foods.

3.7 WPC added fermented products

The formulation of yoghurt products with optimum consistency and stability to synersis (whey

separation) is of primary concern to the dairy industry. The viscosity and stability of yoghurt is almost

wholly dependent on the protein content of the milk. Workers at Massey University, New Zealand

concluded that the addition of up to 15% WPC (20%, if the preheating step is modified) would

enhance the desirable property of natural set yoghurt. In general, the addition of WPC resulted in a

firmer yoghurt with less synersis, but yoghurt made with more than 20% WPC exhibited slight

graininess.

Gupta et al. (1994) experimented with preparation of yoghurt samples using 3.5% fat UHT milk with

different quantities (0, 2.5 and 4.0%) of two types of WPC, namely Biolan P-35 (35% protein) and

lactalbumin 80 (80% protein) During incubation, a slightly slower development of acidity was

18

observed in yoghurt with added WPC than in control yoghurt. Addition of about 33% more yoghurt

culture was required in all WPC added yoghurt samples for achieving the similar final pH as in

control after 4 - 4.5 h of incubation. It was observed that the higher the amount of WPC and the

higher the temperature pretreatment of WPC added milk, the firmer was the consistency of the

yoghurt. It was concluded that the deleterious effect of higher protein in yoghurt in terms of too firm

body could be countered by resorting to lower heat pretreatment of WPC added UHT milk. Yoghurt

with 4% Lactalbumin 80 could be prepared of comparable consistency and pH by heat treatment of

UHT milk to 72°C/2 min and with the addition of 10% yoghurt as culture. But the final flavour of

this yoghurt was observed to be musty and was, therefore, unacceptable.

3.8 WPC added Khoa

Patel et al. (1993) investigated the manufacture of khoa from buffalo milk with added 0, 5, 10 and

18% WPC (27.40% TS), prepared by ultrafiltration of sweet cheese whey. Addition of higher than

18% WPC was not possible without the addition of cream for maintaining the desired fat/TS ratio

(0.38) in the product. The addition of WPC to buffalo milk prior to heat processing gave more

uniform product. Slow heating during manufacture of khoa gave better quality product in terms of

white colour and softness of grains. The flavour score of 18% WPC added khoa was marginally

higher (6.5) than 6.25 of the control khoa. The increased amount of WPC gave khoa with increased

grain size, mainly due to which the overall acceptability score of 18% WPC added khoa was lower

(6.1) to 6.9 of control khoa. However, such khoa is desirable for the preparation of kalakand. Hence,

the selection of level of WPC is subject to the requirement of type of khoa intended for further

use.Increased level of WPC also resulted in reduced cohesiveness and increased dryness in the

product. Good quality kalakand could be prepared by adding 0.3% k-carrageenan, which was

necessary to improve the cohesiveness of the product. The total solid (TS) and WPC contents of khoa

had significant effect on its rheological characteristics. The addition of WPC increased hardness and

adhesiveness, but decreased cohesiveness, springiness, and chewiness in khoa. Further, The WPC

added khoa bound more moisture, as a result of which, it was difficult to concentrate it to the same TS

level as the controlled khoa. In other words, the desired body of the WPC added khoa could be

obtained at a lower TS level than the controlled khoa, which resulted in substantial increase in the

yield of the former product.

4. Selected Readings

Beecher J W, Drake M A and Foegeding E A (2006) Factors determining flavour and stability of acidic whey

protein beverages. Proc. of the fourth intl. whey conference, Chicago, USA, 279–291.

Bihari H (2012) Production of Milk with Enhanced Protein Content. A M.Tech. Thesis submitted to NDRI

Deemed University.

Buckley J, Abbott M, Martin S, Brinkworth G and Whyte P (1998) Effects of an oral bovine colostrum

supplement (intacts) on running performance. Australian Conference of Sci. and Medicine in Sport.

Adelaide, South Australia.

Carey K, Larsen A, Rowney M and Cameron-Smith D (2006) Application of whey proteins to enhance the

molecular adaptations and strength gains following resistance exercise training. In Proc. of the fourth intl.

whey conference, Chicago, USA, 36-46.

Counous G (2000) Whey protein concentrate and glutathione modulation in cancer treatment. Anticancer

Research20 4785-4792

Drake M A (2006) Flavor and flavor carry-through of whey proteins in beverages. In Proc. of the fourth intl.

whey conference, Chicago, USA, 292–300.

FSSAI, (2006). Food Safety and Standards Regulation, Gazetted notifications.

Gupta, V.K. and Reuter, H. (1992) Processed cheese foods with added whey protein concentrates. Lait, 72, 201.

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Gupta V K and Reuter H (1993) Firmness and melting quality of processed cheese foods with added whey

protein concentrates. Lait, 73 381.

Gupta VK, Renner E and Renz-SchauenA (1994) Set cream yoghurt with added whey protein concentrates.

Brief Communication, 24th International Dairy Congress, Melbourne, Australia, 404.

Johnson M A, Jelen P, Mitchell I R, Regester G O andSmithers G W (1996) High protein whey drinks. Food

Australia. 48 360–361.

Lewis MJ (1986) Physical properties of dairy products. In: Advances in Milk Products Vol. 2. R. K. Robinson,

Elsevier Applied Science Publishers, London.

MacLean DA, Graham TE and Saltin B (1994) Branched-chain amino acids augment ammonia metabolism

while attenuating protein breakdown during exercise. American Journal of Physiology, 267E 1010-1022.

McDonough, F.E., Alford, J.A. and Womack, M. (1976) Whey Protein Concentrate as a Milk Extender. Journal

of Dairy Science59 34-38.

Nilsson M, Holst JJ, Bjorck IM (2007) Metabolic effects of amino acid mixtures and whey protein in healthy

subjects: studies using glucose-equivalent drinks. American Journalof Clinical Nutrition, 85 996-1004.

Patel RS,Gupta V K, Singh S and Reuter H (1992) Ultrafiltration behaviour of buffalo and cow milk. Indian

Journal of Dairy Science45322-325.

Patel RS, Gupta V K, Singh S and Reuter H (1993) Effect of addition of whey protein concentrates on the

sensory and instron texture profile of khoa made from cow milk. Journal of Food Science and Technology.

30, 64.

Patel R S, Prokopek D, Reuter H and Sachdeva S (1991) Manufacture of low lactose powder using ultrafiltration

technology. Food Science and Technology (Switzerland), 24: 338.

Patel AA, Patel R S, Singh RRB, Rao KK, Gupta, SK, Sindhu JS, Dodeja AK, Kessler H G and Hinrichs J

(1999) Studies on heat stability of membrane concentrated milk meant for ultra high temperature (UHT)

sterilization. Final Report: Volkswagen Foundation Funded Research Project.

Peter S, Rattray W and Jelen P (1987) Heat stability and sensory quality of protein-standardized 2% fat milk.

Milchwisenschaft

Poulsen, P.R. (1978) Feasibility of ultrafiltration for standardizing protein in milk. Journal of Dairy

Science61807-814.

Sachdeva S, Patel RS, Kanawijia SK, Singh S and Gupta VK (1993)Paneer manufacture employing

ultrafiltration, 3rd

Int. Food Conv., IFCON-93, Mysore.

Sharma DK and Reuter H (1991) A method of chhana making by ultrafiltration technique. Indian Journal of

Dairy Science44 (1) 89.

Shoveller AK, Stoll B, Ball RO and Burrin DG (2005) Nutritional and functional importance of intestinal

sulphur amino acid metabolism. Journal of Nutrition135 1609-1612.

Smilowitz JT, Dillard CJ and German JB (2005) Milk beyond essential nutrients: The metabolic food.

Australian Journal of Dairy Technology,60 77–83.

Thapa TB and Gupta VK (1992) Rheology of processed cheese foods with added whey protein concentrates.

Indian Journal of Dairy Science, 45 86.

Thapa TB and Gupta V K (1996) Chemical and sensory qualities of processed cheese foods with added whey

protein concentrates. Indian Journal of Dairy Science, 49 129.

Zemel M B (2004) Role of calcium and dairy products in energy partitioning and weight management.

American Journal of Clinical Nutrition, 79 70–91.

20

Designing Dairy Foods to Combat Metabolic Disorders

Kaushik Khamrui


1. Introduction

Metabolism is the sum of the chemical processes and interconversions that take place in the cells and

fluids of the body that converts food into energy. This includes the breakdown and buildup of large

molecules, their conversion into small molecules, transportation and absorption of nutrients and minerals

and the ultimate production of energy from these absorbed molecules. A myriad of metabolic functions

are constantly occurring in the body of any living organism, as the cells work together to keep their parent

organism healthy. Virtually every chemical step of metabolism is catalyzed by an enzyme. Hence, a major

part of a healthy metabolism depends on the generation of enzymes which break food down into energy

and handle the transport of that energy.

If a genetic abnormality affects the function of an enzyme or causes it to be deficient or missing

altogether, various disorders can occur that are known as metabolic disorders. The disorders usually result

from an inability to break down some substance that should be broken down, allowing some intermediate

substance that is often toxic to build up, or from an inability to produce some essential substance. Some

compounds may build up to toxic levels in the body, because they are not being properly metabolized. In

other cases, the host organism may fail to get proper nutrition, even if it is eating a healthy, balanced diet.

A metabolic disorder can cause a wide range of symptoms including muscle weakness, neurological

problems, intestinal irregularities, and cardiovascular problems, among many others. Typically, a

metabolic disorder is inherited. In some instances, diseases, exposure to toxins, diet, and drug use may

cause metabolic disorders. Since the symptoms can be vague, diagnosis is complicated, especially in

regions where people do not have access to proper health care.

2. Metabolic Syndrome

“Syndrome” means a combination or group of different symptoms that characterize a specific disease or

illness. The metabolic syndrome is connected with a group of symptoms caused by the malfunctioning

metabolic processes and linked to the existence to specific diseases. The World Health Organization

(WHO) criteria of metabolic syndrome are:

1. Fasting plasma glucose: ≥ 100 mg/dl

2. Impaired plasma glucose tolerance: ≥ 140 mg/dl, two hours after 75g glucose challenge. Plus, any

two of the following

1. Dyslipidaemia: Plasma triglycerides ≥150 mg/dL

2. Dyslipidaemia: High density lipoprotein (HDL) cholesterol <35 mg/dL (men) or <39 mg/dL (women)

3. Hypertension: (≥140 mm Hg systolic or ≥ 90 mm Hg diastolic) or taking blood pressure medication

4. Adiposity: Body Mass Index (BMI) greater than 30 and/or waist:hip ratio >0.9 in men >0.85 in

women

http://www.answers.com/topic/buildup

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http://www.wisegeek.com/what-is-a-balanced-diet.htm

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5. Microalbuminuria: Urinary albumin excretion rate ≥20 µg/min or albumin:creatinine ratio≥30 mg/g

3. Dairy foods for Lowering Blood Glucose Level

The effect of foods on blood sugar level is measured by Glycemic Index (GI). Foods that contain types of

carbohydrates that break down quickly during digestion and release glucose rapidly into the bloodstream

have a high GI, whereas carbohydrates that break down more slowly, releasing glucose more gradually

into the bloodstream possesses a low GI. A lower glycemic index suggests slower rates of digestion and

absorption of the food carbohydrates and indicates greater extraction of the products of carbohydrate

digestion from the liver. A lower glycemic response usually relates to a lower insulin demand and helps

long-term control of blood glucose as well as blood lipids. The glycemic index of foods depends on a

number of factors such as the type of starch (amylose versus amylopectin), physical entrapment of the

starch molecules within the food, fat and protein content of the food and organic acids or their salts in the

food. High, medium and low glycemic index foods have GI values > 70, between 56-69 and < 55,

respectively. Table 1 shows the glycemic index of some common foods. Most of the dairy products are

naturally low in GI, however fortifying them with soluble fibers e.g., psyllium, pectin, guar gum, fructo-

oligosaccharides etc. lower GI even further by speeding up the gastric emptying rate.

4. Dairy Foods for Lowering Dyslipidemia

Due to the association with high dietary cholesterol and saturated fat dairy foods have been implicated to

contribute to development of dyslipidemia, one of the most potential risk factors for cardiovascular

diseases. It has been reported that only one percent increase in energy as saturated fatty acids would

elevate blood cholesterol by 2 mg/dl. However, contrary to the traditional belief, recent findings suggest

that dietary patterns with high dairy product intake are associated with reduced risk of the components of

metabolic syndrome. Some studies have also demonstrated direct negative association of dairy food

consumption and components of metabolic syndrome, e.g., high blood pressure and adiposity, which are

also risk factors for type 2 diabetes. There are basically two approaches for development of dairy products

to fight dyslipidemia:

4.1 Low-fat dairy products

A DASH (Dietary Approaches to Stop Hypertension) study conducted in USA evaluated the effects of a

healthy diet that included low-fat dairy products (milk, yoghurt, and cheese), fruits, and vegetables on

blood pressure in 450 subjects for eight weeks. Results showed that, prepared by combining low-fat milk,

cheese or yoghurt with fruits and vegetables “the DASH diet,” resulted in the greatest reductions in blood

pressure compared to a fruit and vegetable diet that excluded the low-fat dairy products was about half as

effective as the DASH diet. Another study examined the effects of the DASH diet in subjects with

metabolic syndrome. Compared with the control diet, the DASH diet led to increased HDL, lower

triglycerides, lower blood pressure, weight loss, and reduced fasting blood glucose in both men and

women.

4.2 Fortification with specified nutraceuticals

Dietary fibers have long been acknowledged as food components that are beneficial to health. Dietary

fibers can be either soluble or insoluble in water. Soluble dietary fibers have been associated with

reducing the blood cholesterol levels. The body uses cholesterol in the production of bile acids some of

which are excreted daily. The consumption of water-soluble fiber binds to bile acids, which result in an

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http://en.wikipedia.org/wiki/Amylose

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increased excretion of cholesterol. A number of soluble fibers, e.g., pectin, β-glucans, polydextrose,

hydrolyzed guar gum etc. could be used in dairy-based beverages to make in hypolipidaemic.

Insoluble fibers increase bulk and lowers the speed of passage of foods through the GI tract, reduces the

risk of colon cancer and diverticulitis (formation of small pouches in the colon). By selecting the

appropriate one, insoluble fibers (like lignin, cellulose, hemi-cullulose, resistant starches) could also be

used effectively in dairy beverage products. Particle suspension using fluid gel

technology/micofluidization could also be used for incorporating insoluble fibers in milk beverages.

Omega-3 fatty acids are polyunsaturated fatty acids essential to human health but cannot be manufactured

by the body, hence must be obtained from food. Omega-3 fatty acids can be found in fish and certain

plant oils. There are three major types of omega 3 fatty acids that are ingested in foods and used by the

body: alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA).

Extensive research indicates that omega-3 fatty acids help to prevent certain chronic diseases such as

heart disease, arthritis, foster brain and visual development and improves immune reaction against

allergies. A number of dairy beverages fortified with omega-3 fatty acids are already available in the

supermarket shelves in US, Canada, Europe and Australia. Fermented milk products and cheeses made

with probiotic cultures could also be fortified with plant sterols and stanol esters. The later ingredients

have been authorized by the US Food & Drug Administration (FDA) for the use of labeling health claims

for their ability to reduce the risk of coronary heart disease (CHD) by lowering blood cholesterol levels.

5. Dairy Foods for Lowering Hypertension

Several bioactive ingredients derived from fractionation of milk protein could play an important role in

reducing hypertension. Hydrolysates of whole milk proteins as well as caseins and whey proteins are

good source of casokinin and lactokinin. Casokinin and lactokinin are Angiotensin-I Converting Enzyme

Inhibitory Peptides (ACE-IP). Inhibition of ACE results in lowering blood pressure and hence, fermented

dairy products or cheese prepared with specific cultures that produces particular enzymes that breaks milk

proteins to generate above-mentioned peptides will help to control blood pressure.

6. Dairy Foods for Lowering Microalbuminuria

Microalbuminuria (MA) is defined as a persistent elevation of albumin content in the urine of ≥20 µg/min

(≥30 mg/d). It is an established risk marker for the presence of cardiovascular disease and predicts

progression of nephropathy (kidney disorder) when urine albumin content increases >300 mg/d. The

presence of MA is basically the kidney's warning to an increased cardiovascular risk. There is a positive

correlation between the protein content in the diet and MA. It has been reported that a 0.1 g/kg body

weight per day reduction in intake of animal protein was related to an 11.1% improvement in MA.

Although beneficial effects from the protein restriction were reported, one study raised concern that too

low a protein intake may cause malnutrition. Patients in the low-protein diet group reported lower energy

intakes and a significant decrease in body weight compared to the control group. Therefore, although the

majority of the studies report that a reduction of protein to 0.8 g/kg body weight per day may improve

MA, this must be done in the context of overall adequate energy and nutrient intake. Increasing the

moisture content in products like cheese and paneer and using ultra and micro-filtration for selective

removal of casein as well as whey proteins could be used for developing lowering protein in dairy

products.

23

7. Lactose Intolerance

Lactose intolerance yet another metabolic disorder (but not life threatening) is the inability to metabolize

lactose, because of a lack of the required enzyme -galactosidase (lactase) in the digestive system.

Lactose, the major component of milk, is a disaccharide with component monoglycerides, viz., glucose

and galactose joined together in , 1-4 linkage. Lactose as such is rarely absorbed by humans; it relies on

the prior conversion to its component monoglycerides carried out by the enzyme -galactosidase of the

mucosal epithelial cells. Because of intestinal -galactosidase inefficiency, some individuals and even the

whole population of some countries, show lactose intolerance and they have difficulty in consuming milk

and dairy products. The prevalence of lactose-malabsorbance is high in East and South India as 60-100%

of among the them are lactose-malabsorbers.

7.1 Dairy Foods for the Lactose Intolerant

Lactose free or low lactose (~30% of normal) dairy products have been developed for the lactose

intolerants by partial or complete hydrolysis of the lactose present in milk. The two main methods of

lactose hydrolysis are: acid hydrolysis and enzymatic hydrolysis using the enzyme -galactosidase. The

first method is characterized by very severe pH and temperature conditions (pH=1-2, t=100 to 150° C),

thus rendering the end product unsuitable for use as a food ingredient. Enzymatic hydrolysis of lactose

using -galactosidase seems to be an attractive method. The enzyme -galactosidase is widely distributed

in nature, e.g., in plants, animal organs, bacteria, yeast and fungi etc. Due to increase expense in using

soluble -galactosidase, the concept of using immobilized enzyme serves as a better alternative to reuse

the enzyme, resulting in lowered enzyme cost, besides making the process continuous.

Several procedures have been developed for immobilization of enzymes. Adsorption of the enzyme onto

an insoluble matrix is the most simple and inexpensive, but suffers from enzyme desorption due to weak

binding. Only very slight improvement could be obtained by cross-linking the enzyme by a reagent. Gel

entrapment, yet another method, where reactor is under diffusion control and thus limited to only small

molecular weight substrates. Covalent bonding of the enzyme to a solid matrix is the most important

method for immobilization, where enzyme is very strongly attached to the support thus processing a very

stable enzyme system.

A range of reduced lactose and lactose free milks are available in American and European Market. The

three most popular lactose-reduced or lactose-free products on the US market are Lactacid, Dairy Esse

and Mootopia. Lactose free brands available in Europe include Hyla, Emmi, Lacto free, etc. The reduced

lactose or lactose free milk manufactured by treatment with -galactosidase possesses a lower freezing

point and sweeter than normal milk. Lactose hydrolyzed milks are more susceptible to Maillard browning

during UHT treatment because the mono-saccharides formed from lactose react faster than lactose with

amino acids, resulting in extensive browning. The -galactosidase treatment of fluid milk increases the

cryoscopic value of milk from 0.054 to 0.650°C making it difficult to assess the adulteration of milk with

water by cryoscopic method. The -galactosidase treatment increases the cost of fluid milk by ~ $0.06-

0.08/L. The dairy company Valio, Finland patented chromatographic separation method to remove lactose

from milk. The milk is low in lactose but tastes like normal fresh milk.

8. Conclusion

On account of increased consumption of energy rich foods having high amount of sugar and fat content

coupled with sedentary lifestyle, has opened gateway for non communicable disorders like obesity,

http://en.wikipedia.org/wiki/Metabolism

http://en.wikipedia.org/wiki/Lactose

http://en.wikipedia.org/wiki/Lactase

24

diabetics and cardiovascular diseases to creep in to the human life which are also known as metabolic

disorders. Developing low fat dairy products, fortifying them with functional ingredients, and using

modern technological interventions it is possible to develop dairy products that can help to combat and

cure metabolic disorders.

9. Selected Reading

Glycemic Index (GI) Food Chart. http://www.carbs-information.com/glycemic-index-food-chart.htm. [Internet

document: Accessed on 12.06.2013]

Khamrui K and Solanki D C (2007) Flavouring with Functionalities. Times Foods Processing Journal 5 23-25.

Metabolic Diseases (2011). In: Sale Genetic Encyclopedia. http://www.answers.com/topic/metabolic-disease.

[Internet document: Accessed on 15.02.2011]

Rehman S U (2009) Reduced lactose and lactose free dairy products. In: Advanced Dairy Chemistry. Vol – 3.

(McSweeney, E. L. H. & Fox, P. F., Editors). Pp. 98-103. Springer Science + Business Media, New York.

Spence L A (2008) The Emerging Role of Dairy Foods in Reducing the Risk ofMetabolic Syndrome and Type 2

Diabetes. Nutrition Outlook.

http://www.innovatewithdairy.com/SiteCollectionDocuments/The%20emerging%20rol%20of%20Dairy%20fo

ost.pdf. [Internet document: Accessed on 14.02.2011]

Table 1. Glycemic Index of common foods

Dairy Products

Milk (whole) 22

Milk (skimmed) 32

Milk (chocolate flavored) 34

Ice Cream (full-fat) 61

Ice cream (low-fat) 50

Yogurt (low-fat) 33

Cheese 0

Paneer 0

Grains

Basmati Rice 58

Brown Rice 55

Fruits

Apple 38

Banana 55

Grapes 46

Mango 55

Orange 44

Papaya 58

Pear 38

Pineapple 66

Watermelon 103

Breads

White Bread 70

Whole Wheat Bread 69

Snacks

Cashews 22

Peanuts 14

Popcorn 55

Potato Chips 55

Noodles (instant) 46

Walnuts 15

Sugars

Fructose 23

Glucose 100

Honey 58

Lactose 46

Maltose 105

Sucrose 65

Vegetables

Beets 69

Cabbage 10

Carrots 49

Corn 55

Green Peas 48

Mushrooms 10

Potato (baked) 93

Sweet Potato 54

Pumpkin 75

Onion 10

http://www.carbs-information.com/glycemic-index-food-chart.htm

http://www.answers.com/topic/metabolic-disease

http://www.innovatewithdairy.com/SiteCollectionDocuments/The%20emerging%20rol%20of%20Dairy%20foost.pdf

http://www.innovatewithdairy.com/SiteCollectionDocuments/The%20emerging%20rol%20of%20Dairy%20foost.pdf

25

Sodium Reduction in Cheeses

Yogesh Khetra, S.K. Kanawjia, Alok Chatterjee and Gadsingh Shankar Prakash


1. Introduction

Sodium is an essential nutrient, the cation that regulates extracellular fluid and plasma volume. It also

indulges in transport of some molecules across cell membrane, essentially by affecting their membrane

potential. Approximately 98% of dietary sodium is absorbed in the intestine and the excess is excreted

mainly through kidneys. Urinary sodium excretion roughly equals dietary intake in healthy humans.

Sodium excretion in sweat and urine is altered by several hormones and sympathetic nervous system, in

response to change in dietary sodium and thus, plasma levels of sodium is maintained in an optimal range.

However, faulty functions of kidney, due to age or some chronic diseases, weaken the efficient excretion

of excess sodium, resulting in increased plasma volume and hypertension. Hypertension, in turn, is

correlated to enhanced risk towards coronary heart disease (CHD), stroke and end-stage renal disease.

2. Physiological role of sodium

Sodium regulates extracellular volume, maintains acid-base balance, neural transmission, renal function,

cardiac output and myocytic contraction [1]. Sodium is consumed mainly in the form of sodium chloride

(NaCl) which comprises a sodium atom and a chlorine atom representing respectively 39.33 % and 60.67

% of its mass.

A proper balance of sodium levels in our body is of vital importance as the osmotic pressure of the

extracellular fluids is mainly dictated by the presence of sodium-ions. The level of sodium in our blood

plasma and interstitial fluids is vital for basic physiological functions of almost all cells in our body.

Sodium regulates many transport processes and slight deviations in the levels affect the electrical activity

of muscle and nerve cells, renal function, capillary exchange, cardiac output, and consequently influence

blood pressure.

3. Salt and public health

Sodium is essential for normal human functioning. However, current intakes of sodium in many parts of

the world is well in excess of the levels recommended. Excessive sodium intake is associated with an

increase in blood pressure, which is a major cause of cardiovascular diseases. It has been estimated that

62% of stroke and 49% of coronary heart disease is caused by high blood pressure [2]. Excess dietary

sodium has also been reported to be associated with other health ailments such as gastric cancer,

decreased bone mineral density and obesity [3]. Asaria [4] calculated that a modest 15% reduction in

population sodium intake could prevent 8.5 million cardiovascular-related deaths worldwide over 10

years. Further, it has been reported that cardiovascular diseases are the most expensive health issues and

accounts for approximately 11% of the total health expenditure around the world. A significant amount of

cost has been saved by implementing salt reduction strategies in various countries [3].

Despite the negative health consequences and associated health care costs of high sodium consumption,

humans consume well above the recommended levels in most developed nations, making sodium

reduction a priority for public health. The daily consumption recommended for adults is approximately

2.4g of Na or 6g of NaCl, which can be found naturally in food [5]. It was reported in the Global Burden

26

Na activates ENaCs on taste receptors

Afferent signal to gustatory region of brain

Weak signal – does not produce noticeable difference Signal strength increases --- Na can be

discriminated from water

Further increase in concentration

Na concentration in the range of perceived saltiness

of Disease Study that excess dietary salt intake is the eleventh leading cause of mortality globally

accounting for 4 million deaths, while in the South East Asian Region it is the seventh leading cause of

mortality [6].

4. Mechanism of Salt perception in human body

Sodium-specific transduction mechanism comprising epithelial sodium channels (ENaCs) on the taste

receptor cells can form the defense for the exclusivity of sodium as a stimulus for salty taste. Specific

ENaC is activated at low concentrations of sodium and is held responsible for the appetitive nature of salt

taste. The other is permeable to multiple cations and is activated at higher concentrations of sodium. This

is accountable for the aversive nature of cations. An afferent signal sent to gustatory processing regions of

the brain upon activation of ENaCs by sodium buds the perception of salt taste. This signal might be too

feeble to be able to assist noticeable difference from a similar solution without sodium, given that the

sodium concentration is low. As the number of sodium ions rises per unit intermolecular space of the

solvent, the signal strengthens enough to drive an individual discriminate it from water but for taste vigor.

This is known as the detection threshold and is often used as a measure of individual sensitivity to

sodium. Concrete perception of saltiness strikes when the concentration of sodium is high enough not

only to activate the taste receptors, but produce electrical impulses too, that can be carried via sensory

neurons to the brain where they are decoded and after which the taste quality can be identified. This is

known as the recognition threshold. The sodium concentrations above the recognition threshold are in the

range of perceived saltiness, which is termed suprathreshold. The concentration of sodium required to

elicit saltiness varies considerably between food matrices [7].

5. Role of sodium in cheese

Salt addition is the major source of sodium in natural cheese. In dry salted cheeses like cheddar, the salt is

added directly to the curds just before hooping and pressing. In other types of cheese, the salt is added by

submersing the cheese in brine for an appropriate period of time. Some cheeses have salt rubbed on the

outer side of the cheese.

Fig 1: Mechanism of salt perception

Incr

easi

ng

Na

Co

nce

ntr

atio

n

27

No matter when or how the salt is added to the cheese, it is used for following purposes in the

manufacture and aging of cheese [8]:

Encourage syneresis and control final moisture of the cheese

Control the metabolism and survival of the starter bacteria

Influence the types of secondary organisms that may grow and create flavors during the ripening

period

Control enzyme activity in the final cheese

Control texture of the final cheese

Be a component of the expected taste of the cheese

Salt, in addition to pH, aw, and lactic acid content, is one of the hurdles inherent in maintaining the food

safety of these traditional cheeses. Low-fat cheeses have a higher moisture content resulting in a lower

salt-in-moisture (S/M) ratio at the same absolute salt content. The amount of salt in the moisture phase of

the cheese controls the growth of microorganisms, not the total amount of salt in the food. Lowering the

S/M hurdle is a safety and shelf-life concern especially in the distribution and serving of cheeses.

6. Strategies for salt reduction in cheese

Most of the sodium in diet comes from processed foods and through exogenously added salt. World over,

efforts are being made to reduce sodium in foods but this involves challenges as salt, generally added in

the form of sodium chloride, is associated with flavour, body and texture, controlled microbial growth,

food safety and shelf life. Thus, stand-alone reduction in salt levels jeopardizes the overall quality of

food and this mandates the use of salt replacers/substitutes in foods to impart salty taste with maintenance

of water activity to control microbial growth. Most of the salt replacers, currently in use, communicate

bitter taste perception and therefore restricts complete replacement of sodium chloride. Potassium

chloride (KCl) is being used as the most common salt replacer in variety of foods but it too has been

reported to endow bitter taste. Thus, many novel ingredients and technological interventions are being

attempted to increase saltiness in foods with lesser levels of sodium chloride.

There are many new technological innovations, such as odor induced saltiness enhancement, changing the

food matrix for enhanced saltiness, using double emulsions to enhance saltiness at reduced levels of salt

and using various novel salt replacers, springing in the area of salt reduction in foods. Conversely, sodium

in cheese is generally controlled by 2 approaches. One is to limit the amount of sodium chloride addition

and the other to use salt substitutes throwing salty taste with low or no sodium.

Several attempts were made to prepare low salt cheese and it has been reported that cheese with lesser salt

content (1.25%) had limitations of bitterness and excessive firmness [9, 10]. Sensory acceptable low salt

(1.3, 1.71 and 2.04%) cheeses were prepared but the product at these levels of salt did not meet FDA

standard for low-salt cheese. Irish Cheddar cheeses prepared with 0.5 and 1.25% salt had slower rate of

flavor formation and higher bitterness. Thus, it can be concluded that cheese prepared with restricted

sodium chloride suffers quality loss and dwindles consumer acceptability as well.

Potassium chloride is the most preferred choice for sodium chloride replacement as it is the most similar

compound to NaCl. Fitzgerald [11] prepared cheddar cheese with different salt substitutes at equivalent

ionic strength of the control sodium chloride level (1.5%). Use of magnesium chloride, potassium

chloride, or calcium chloride at this level resulted in bitter taste, metallic flavor and crumbly body defects.

Proteolysis and lipolysis were also reported to be at higher degree. Partial substitution of NaCl with KCl

has been attempted in several varieties of cheese to reduce sodium level up to varying extent. Consumer

28

Inner aqueous phase

Continuous aqueous phase

Oil phase

acceptable low-sodium Minas fresh cheese was prepared by partial substitution of NaCl by KCl at 25%

(w/w) in the salting step [12]. In an attempt to prepare low sodium feta cheese, it was found that up to a

50% reduction of sodium content is feasible, with partial replacement of NaCl by KCl, without

jeopardizing its quality. The results also indicated that the cheeses made with mixtures of NaCl/KCl

returned no significant differences in compositional (moisture, fat, protein, salt), physicochemical, (pH,

aw), sensory (appearance, body and texture, flavour, overall quality) and textural (force and compression

to fracture, hardness) properties in comparison with the control cheese.

7. Futuristic technologies

7.1 Modification of structure of NaCl

Salty taste has been reported to be influenced by varying size of salt crystals. Rate of salty taste

perception can be increased by using smaller size particles which increases the rate of dissolution of salt

particles. Development of sodium chloride with its physical structure re-engineered into hollow

microscopic particle sizes has also been investigated for reducing sodium in foods without affecting

saltiness perception [13].

7.2 Changing the food matrix

Food matrix can be effectively altered to increase saltiness perception. It has been reported that

continuous delivery of salt from food leads to a gradual decrease in taste sensitivity. However, pulsed

delivery of salt in foods increase the perception of salt and thereby less salt can be used with same

saltiness perception [14]. Stiegar [15] patented a work on three different breads – 1 control with

homogeneous salt (1.5%) distribution and two with inhomogeneous distribution having 2.75% and 0.25%

salt. Salt in later samples was applied in layers of 0.5 or 1 cm thickness such that each salted layer is

followed by an unsalted layer. It was found that breads with heterogeneous salt distribution were saltier

than the homogenous salt distribution bread.

7.3 Encapsulation of salt in emulsion

Water/oil/water emulsions can be successfully used either for salt reduction or salt replacement. Salt

release can be controlled by distributing salt in either inner aqueous phase or continuous outer phase.

Figure 2: Water/oil/water emulsion

29

These phases can be stabilized by an oil phase. Saltiness perception can be increased when same quantity

of salt is present in continuous water phase. For replacement of salt, KCl is most commonly used but at

higher levels of this compound, bitterness occurs. KCl can be encapsulated in inner aqueous phase

surrounded by oil phase. The bitterness due to KCl can thus be masked allowing more sodium chloride to

be replaced in food products.

7.4 Odor induced saltiness enhancement (OISE)

The overall perception of flavor is considered as an integration of taste and odor. It has been demonstrated

that taste can increase odor intensity and conversely, the perception of taste can be enhanced by odor.

Lawrence [16] studied the effect of taste odor interaction on saltiness of solutions. It was found that

expected flavors could enhance saltiness in solutions containing low levels of NaCl through odor –taste

interaction and change in overall perception of flavor. OISE can be effectively used for salt reduction by

using aromas that consumers associate with saltiness. Cheese, sardine, soy and bacon have this ability to

enhance saltiness perception of food products [17].

8. Conclusion

Cheese is a sodium rich food and world over efforts have been made to reduce sodium in cheese.

Potassium chloride has been the most preferred salt replacer for cheese industry till date. However, it is

associated with bitterness and increased firmness thereby, limiting the required replacement. Current

research to decrease sodium based salts in foods include gradually decreasing the salt content in food

products, altering the food matrix, using salt substitutes of plant origin, inclusion of water-in-oil-in-water

emulsions (wow emulsions) as well as the inclusion of aromas giving the increased saltiness perception.

9. Selected Reading

1. Dotsch M, Busch J, Batenburg M, Liem G, Tareilus E, Mueller R and Meijer G (2009) Strategies to reduce

sodium consumption: a food industry perspective, Critical Reviews in Food Science and Nutrition, 49841-851,

2009.

2. He F, G and MacGregor (2010)Reducing population salt intake worldwide: From evidence to implementation”,

Progress in Cardiovascular Diseases, 52 363–382.

3. Liem D G, Miremadi F and KeastR S J(2011)Reducing Sodium in foods: The effect on flavour”, Nutrients, 3 694-

711.

4. Asaria P, Chisholm D, Mathers C, Ezzati M and Beaglehole R (2007)Chronic disease prevention: health effects

and financial costs of strategies to reduce salt intake and control tobacco use, Lancet, 3702044–2053.

5. Kaplan N M (2000)The dietary guideline for sodium: should we shake it up? American Journal of Clinical

Nutrition, 711020-1026.

6. Lim S S, Vos S T, Flaxman A D, Danaei G, Shibuya K, Adair-Rohani H (2010)A comparative risk assessment of

burden of disease and injury attributable to 67 risk factors and risk factor clusters in 21 regions, 1990–2010: a

systematic analysis for the Global Burden of Disease Study, Lancet, 380 2224–2260.

7. Keast R S J and BreslinP A S (2003) An overview of binary taste-taste interactions, Food Quality and

Preferences, 14 111–124.

8. Guinne T P(2004) Salting and the role of the salt in cheese, International Journal of Dairy Technology, 57 99-

109.

9. Banks J M,Hunter E A and Muir D D (1993) Sensory properties of low-fat cheddar cheese: effect of salt content

and adjunct culture, Journal of the Society of Dairy Technology, 46 119–23.

30

10. Mistry V V and Kasperson K M(1998) Influence of salt on the quality of reduced-fat cheddar cheese, Journal of

Dairy Science, 811214–21.

11. Fitzgerald E and Buckley J (1985) Effect of total and partial substitution of sodium chloride on the quality of

Cheddar cheese, Journal of Dairy Science, 683127-3134.

12. Gomes A P, Cruz A G, Cadena R S, Celeghini R M S, Faria J A F, Bolini H M A and Granato D (2011)

Manufacture of low-sodium Minas fresh cheese: Effect of the partial replacement of sodium chloride with

potassium chloride, Journal of Dairy Science, 94 2701-2706.

13. Busch J L H C, Yong F Y S and Goh S M (2013) Sodium reduction: Optimizing product composition and

structure towards increasing saltiness perception, Trends in Food Science and Technology, 29 21–34.

14. Morris C, Koliandris A L, Wolf B, Hort J and Taylor A J (2009) Effect of pulsed or continuous delivery of salt

on sensory perception over short time intervals, Chemosensory Perception, 21-8.

15. Stieger M A, Bult J H F, Hamer R J and Noort M W J (2011) Food product with inhomogeneous tastant bulk

distribution and method for making such food product. US Patent Application US2011/0045157 (A1).

16. Lawrence G, Salles C, Septier C,Busch J and Thomas-Danguin T (2009) Odour–taste interactions: A way to

enhance saltiness in low-salt content solutions, Food Quality and Preference, 20 241-248.

17. Wallis K and Chapman S (2012) Current innovations in reducing salt in food products, Food Health and

Innovation Service. Campden, BRI.

31

Nanotechnological Applications in Development of Novel Dairy Foods

S.A. Hussain, Payal Meena and Akanksha Wadehra


1. Introduction

Nanotechnology is defined as the design, production and application of structures, devices and

systems through control of the size and shape of the material at the nanometer (109 of a meter) scale

(Ravichandran, 2006). Nanotechnology has revolutionized health care, textile, information

technology, and energy sectors etc. but in food sector it is still at infancy.Nanotechnology has the

potential to revolutionize the food and dairy processing sector in upcoming days. The potential

applications of nanotechnology in dairy and food sector comprises of two types: 1. Nano inside -

includesmodifying natural (organic) protein, carbohydrate and fat molecules to achieve added or

altered functionality in foods and preparation of nanosized functional ingredients including food

additives for targeted delivery, 2. Nao outside - includesuse of nanoparticulate materials in food

packaging to improve strength and barrier properties andnano sensors for ensuring the food safety.

Some of the nnao technological research initiatives took place in agri-food sector around the globe

includes sensory improvement of food products (flavour/colour enhancement, texture modification),

increased absorption and targeted delivery of nutrients and bioactive compounds, stabilisation of

active ingredients such as nutraceuticlas in food matrices, packaging and product innovation to extend

shelf-life, sensors to improve food safety and antimicrobials to kill pathogenic bacteria in food.

2. Nanotechnology in Dairy and Food Sector

Food industry is the largest manufacturing sector in the world. There is a great scope for application

of nanotechnology in the food sector. In the present manuscript, application of nanotechnology in the

field of food and dairy industry has covered in two major heads viz. food additives (nano-inside) and

packaging (nano-outside).

2.1 Food Additives (Nano-inside)

Functional ingredients (for example, drugs, vitamins, antimicrobials, antioxidants, flavorings,

colorants, and preservatives etc.) comes in different molecular and physical forms such as polarities

(polar, nonpolar, amphiphilic), molecular weights (low to high), and physical states (solid, liquid,

gas). These ingredients are rarely utilized directly in their pure form; instead, they are often

incorporated into some form of delivery system,which must perform a number of different roles

(Qureshi et al., 2012). Nanoemulsions, nanofibers, nanotubes, nanoencapsulates, nanoceuticals,

nanostrcutres and nanoparticles are the delivery systems generally applied in nanotechnology to

deliver food additives or functional ingredients into food products.

Emulsions with droplet diameter below 100 nm are referred to as nanoemulsions. Due to the reduced

size of droplets, nanoemulsions are less likely to destabilize. Nanoemulsions possesses characteristic

rheological and textural properties, which offer them application in food sector. In the recent past,

supplementation of functional ingredients in food products through nanoemulsions has gained

considerable interest among the researchers. Nanoemulsions offer several advantages like targeted

delivery of the bioactives and also help in their controlled release at specific site. Food-grade

ingredients (such asproteins, polysaccharides, and phospholipids) are also used for the formation of

nanoemulsions usingsuitable processingoperations involving high energy inputs, membrane

technology and interfacial engineering. Nanoemulsions are also used to develop low-fat products

32

without compromising the creaminess. Low-fat nanostructuredmayonnaise, spreads and ice creams

are developed using this technology (Chaudhry et al. 2008).

In recent past, encapsulation of biologically active substances into nanostructures to enhance the

flavour and other sensory characteristics of food and dairy products has been the major research

interest. Nanoencapsulation of functional ingredients is done to protect them from environmental and

processing factors as well as from harsh conditions occurring in GI tract. Nanoencapsulation also

helps in targeted delivery of the bioactives, greater residence time and greater absorption at specific

site (Chen et al., 2006). The protection of bioactive compounds, such as vitamins, antioxidants,

proteins, and lipids as well as carbohydrates may be achieved by using this technique for the

production of functional foods with enhanced functionality and stability (Radha et al., 2008).

Nanoencapsulation can make significant savings for formulators, as it can reduce the amount of active

ingredients needed (Huang et al., 2011). α-lactalbumin, a globular milk protein is beneficially used in

the production of nanotubes. At specified conditions α-lactalbumin forms nano tubes with a diameter

of only 20 nm by self-assembling of the partially hydrolysed molecule (Otte et al., 2005). These

nanotubes can be used to encapsulate several vitamins, enzymes and other bioactive molecules

(Srinivas et al., 2010) for their incorporation into food products. α-lactalbumin nanotubes also mask

the undesirable flavour/aroma and colour imparted by nutraceutical compounds when added to food

(Bikker and Kruif, 2006).

Food grade biopolymers such as proteins and polysaccharides form nanoparticles through self-

association or aggregation or by phase separation in mixed biopolymer systems. These biodegradable

nanoparticles can be used to encapsulate deliver drugs, micronutrients and herbal bioactives etc. like

iron, vitamin, protein etc. (Riley et al.,1999).Nanoceuticals, which deals with nano-sized bioactive

molecules,isalso gaining popularity. Bioactives at their nano-scale were reported to perform better

stability during processing operations and storage. Some examples of the commercial food/dairy

supplements containingnano particles include Omega-3 fatty acids, vitamins, probiotics etc.

(Neethirajan and Jayas, 2011). Recently, a functional butter was developed by supplementing nano

sized herbal supplements into it (Ivanovand Rashevskaya, 2011).

2.2 Food Packaging (Nano-outside)

Food packaging makes up the largest share of current markets of the use of nanotechnology in the

food sector. Incorporation of nanoparticles into packaging materials improves their durability,

temperature resistance, flame resistance, barrier properties, optical properties and recycling properties.

Plastic materials have been dominating the food packaging industry due to their low-cost, low density,

resistance to corrosion, desirable physical (e.g. barrier and optical) and mechanical properties and

ease of processing.However, they are absolutely non-degradable. A variety of renewable biopolymers

such as polysaccharides, proteins, lipids, and their composites, derived from plant and animal

resources have been investigated for the development of edible/biodegradable packaging materials to

substitute for their non-biodegradable petro-chemical counter parts. When biopolymers are combined

with nanoparticles, the resulting bio-nanocomposites exhibit significant improvements in the

mechanical properties, dimensional stability and solvent or gas resistance with respect to the pristine

polymer. The application of nanocomposites promises to expand the use of edible and biodegradable

films produced from agro-processing products and by-products based on polysaccharides such as

starch, cellulose, and chitin and on proteins such as milk proteins including whey proteins, soybean

protein and gelatine. The use of these materials serve a number of important functions such as

extending the food shelf-life, enhancing food quality because they can serve not only as barriers to

moisture, water vapour, gases and solutes but also serve as carriers to some active substances such as

antioxidants and antimicrobials. Milk-protein based bio-nanocomposites in combination with other

33

agro-processing products with or without antimicrobial or antioxidative effects has yet to be

investigated in large-scale (Sorrentino et al., 2007).

A nano-titanium dioxide plastic additive (DuPont light stabilizer210) which can reduce UV damage to

foods in transparent packages has been developed by Du Pont. Incorporation of nanoparticles in food

packaging to create barrier properties against selective gases is a current research programme at

Norwegian Institute of Technology (SINTEF, 2004). Nanoscale edible coatings invisible to naked eye

have also been developed for use on fruits, vegetables and other food products (Rhim,

2004).Nanolaminates coated with functional agents such as antimicrobials, antioxidants, enzymes and

etc. to extend the shelf-life of foods were also developed by several workers. Further, these

nanolaminated coatings could be created entirely from food-grade ingredients (proteins,

polysaccharides, lipids) by using simple processing operations thus creating interest among the

industries (Ravichandran, 2009).Nanotechnology is also used to design nano-sensors for tracking the

internal or external environments of packaging to find out the condition of food products and package

throughout the supply chain (Nachay, 2007). Nano-sensors can detect the gases released when the

food spoils and the package itself or the nano-sensor changes their colour to alert the customer.

Researchers are developing intelligent packaging, which will release a preservative if the food inside

starts to spoil. This “release on command” a bioswitch developed through nanotechnology will

operate the preservative packaging.

3. Conclusion

Potential applications of nanotechnology in dairy and food sector are under research. In the near

future, the entire scenario of dairy and food industry will be transformed by nanotechnology.

However, there are some problems associated with this technology, with respect to the accidental or

deliberate use of nanoparticles in food, or food-contact materials, it can cause some health issues.

Nanotechnology has to face several critical challenges such as to decide optimal intake levels,

development of adequate food delivering matrix, finding out of beneficial compounds, novel product

formulation and safety of product as well as safety of consumer.

4. Selected Reading

Chaudhry Q and Castle L (2011) Food applications of nanotechnologies: an overview of opportunities and

challenges for developing countries. Trends in Food Science and Technology, 22595-603.

Chen H, Weiss JandShahidi F (2006) Nanotechnology in nutraceuticals and functional foods. Food

technology.

Graveland-Bikker J F and De Kruif C G (2006). Unique milk protein based nanotubes: food and

nanotechnology meet. Trends in Food Science and Technology, 17 196-203.

Huang CNotten A andRasters N (2011). Nanoscience and technology publications and patents: a review of

social science studies and search strategies. The Journal of Technology Transfer, 36 145-172.

Ivanov S V andRashevskaya T A (2011). The nanostructure's management is the basis for a functional fatty

foods’ production. Procedia Food Science, 1 24-31.

Mishra U K (2012). Application of nanotechnology in food and dairy processing: an overview. Pakistan

Journal of Food Sciences, 22 23-31.

Nachay K (2007). Analyzing nanotechnology. Food technology, 61 34-36.

Neethirajan SandJayas D S (2011). Nanotechnology for the food and bioprocessing industries. Food and

bioprocess technology, 4, 39-47.

Otte JIpsen R Bauer R Bjerrum M J andWaninge R (2005). Formation of amyloid-like fibrils upon limited

proteolysis of bovine α-lactalbumin. International dairy journal, 15 219-229.

34

Ravichandran R (2009). Nanoparticles in drug delivery: potential green nanobiomedicine applications.

International Journal of Green Nanotechnology: Biomedicine, 1(2), B108-B130.

Ravichandran Rand SASIKALA P (2006). Nanoscience and nanotechnology: perspectives and overview. First

Indian at the South Pole 3 PS Sehra Madame Marie Curie–Radioactivity and 10 Atomic Energy, 44.

Rhim J W (2004). Increase in water vapor barrier property of biopolymer-based edible films and coatings by

compositing with lipid materials. Food Science and Biotechnology.

Riley T Govender T Stolnik S Xiong C D Garnett M C Illum L and Davis S S (1999). Colloidal stability

and drug incorporation aspects of micellar-like PLA–PEG nanoparticles. Colloids and surfaces B:

Biointerfaces, 16 147-159.

SINTEF. (2004). Space station technology applied to food packaging.

http://www.foodproductiondaily.com/news/ng.aspn=5 4940-space-station technology.

SorrentinoA Gorrasi G andVittoria V (2007). Potential perspectives of bio-nanocomposites for food

packaging applications. Trends in Food Science and Technology, 18 84-95.

Srinivas G Zhu Y Piner R Skipper N Ellerby M andRuoffR (2010). Synthesis of graphene-like nanosheets

and their hydrogen adsorption capacity. Carbon, 48 630-635.

Thangapazham R L Puri A Tele S Blumenthal R andMaheshwari R K (2008). Evaluation of a

nanotechnology-based carrier for delivery of curcumin in prostate cancer cells. International journal of

oncology, 32 1119-1123.

35

Application of Membrane Processing for Production of Innovative Dairy Ingredients

G S Meena, A K Singh, S Borad, N Kumarand and P T Parmar

Dairy Technology Division and NIFTEM

1. Introduction

Very less substances are present in nature which are fit for direct human consumption. Hence, the raw

material has to subject to single or a set of different unit operations to convert it into safe and eatable

form. Moreover, if the target componentsareprone to lose its typical desired functional, nutritional and

sensorial characteristics during processing,than selection ofappropriate separation method and processing

conditions become crucial. Milk, which is considered as natural treasurefor goodness for all age groups, is

a good example. Very harsh processing conditions (thermal or chemical) have a detrimental effect on its

nutritionally rich, heath promoting constituents. Although, processes are available to harvest these desired

milk constituents, but their commercial viability depends on factors like separation capability

withoutlosing key ingredients activity and lower energy, capital and labor costs (Akin et al.,

2012).Membrane processing has got the potential to efficiently deal with separation requirements of milk

and the same has made dairy industry as one of the early adopter and potent consumer of membrane

technology. This process require a particular set of equipments and process conditions for the production

of a specific innovative dairy ingredient. With the development of more robust membrane systems like

mineral and hybrid membrane systems, membrane applications in dairy industry is continuously

growing.Several applications of this novel technology in the production of novel dairy ingredients have

been discussed in following sections.

2. Concentration of Milk Fat Globule Membrane (MFGM) and milk fat fractionation

From industrial point of view, lower value products like sweet cream buttermilk (SCBM), butter serum

and whey forms raw materialof MFGM.Several researchers have been tried the isolation of MFGM

fragments from buttermilk using cross flow microfiltration(MF) and ultrafiltration (UF) membrane

processes seems suitable for MFGM purification capable owing to their capability to particles separation

from dissolved solids. Passage of buttermilk constituents is affected by separation parameters like pH,

temperature and type of feed followed by pore size and membrane material (Morin et al., 2004, 2006;

Rombautet al., 2007). For the purification of polar lipids, all other buttermilk constituents needs their

selective separation that can’t be achieved by MF alone.Proteins, particularly similar size casein micelles

in buttermilk causes problem during membrane based purification, thus these separation failed to remove

casein micelles completely. Phospholipids enriched fractions of reconstituted buttermilk was produced by

Sachdeva and Buchheim (1997) through the coagulation of buttermilk with rennet, citric acid and lactic

cultures. Either rennet coagulation with added 0.1% CaCl2or acid coagulation(citric acid, pH-5.2) of

heated (0.1% CaCl2,70°C) reconstituted butter milk transferred about 80 % of the total phospholipids

present in buttermilk into serum phase as compared to lactic cultures induced coagulation that recovered

only 53% phospholipids in to the serum phase. Two MF systems (polysulfone membrane, 0.1µm pore

size and ZrO2membrane, 0.2µm pore size) were used to isolate the phospholipids enriched fractions. With

diafiltration, 67% recovery of phospholipids was obtained in ZrO2 membrane.Further better results (77%

recovery)were obtained with the combined use of 0.1µm ZrO2membrane andUF of the buttermilk

serum.They emphasized on the use of optimal conditions during MF (like high speed of passage of the

36

retentate, low transmembrane pressure (TMP)) to ensure almost constant membrane filtration behavior.

About 97% of phospholipids present in buttermilk serum was recovered by them. As a different approach,

diameter of casein micelle was reduced by Corredig et al. (2003) and Roesch et al. (2004) through

itsdissociation into sub micelles by the addition of sodium citrate and its subsequent MF to obtain MFGM

rich retentate.About 80% MFGM containing material with markedly reduced caseins was obtained

(Corredig et al., 2003). Rombaut et al. (2007) used whey obtained from the acid coagulation of SCBM

(mostly free from caseins) as raw material to concentrate MFGM fraction by MF. MFGM retention was

significantly affected by the filtration conditions, membrane material and its pore size. They observed

best results with negatively charged, <0.15 μm membrane due reduced loss of MFGM. Of the total polar

lipids present in acidified whey, 98% was recovered during optimal conditions. A summary of the

methods (membrane based or other) employed for the isolation of MFGM has been shown in Figure 1.

MF will be used for the fractionation of milk fat globules according to their size in future as till now, it is

not used on commercial scale while classical centrifugal separation is unable to perform the same. Milk

fat fractionation without damaging their native fat globule membrane using ceramic MF membranes has

been proposed (Goudedranche et al., 2000; Michalski et al., 2006). Lopez et al., (2011) prepared native

fractions of varying size fat globules using MF operation with or without diafiltration. They concluded

that unstable (fat globules > 6.6 μm size) and highly stable(1.6 μm) fat globules could be produced.

Moreover, these different fragment will deliver altered techno functional and better health properties that

still needs detailed investigation.

Figure 1. Summary of isolation methods of MFGM (Dewettincket al., 2008;Sichienet al., 2009)

37

3. Concentration and fractionation of milk proteins

Two main class ofproteins present in milk are caseins and whey proteins which contribute to 80% and

20% of the total milk proteins. Further, these two classes are made of different fractions of major and

minor proteins which are unique in their nutritional, biological and functional potential. These protein

fractions are represented with some of their physical properties in Table 1. For the fractionation of caseins

and whey proteins in their native state, UF and MF has been used but MF has been widely used for this

particular application. The fractionation is based on two approaches i.e. protein size and their

molecularweight. Although caseins have lower molecular weight but the same exists as micelles with

average diameter of 0.05 to 0.5 μm (majority lies between 0.13-0.16 μm) as reported by Fox and

McSweeney (2003), greater than whey proteins, hence can be separated on the basis of differed particle

size (Table 1).During MF, different casein fractions, fat, and enzymes are concentrated in retentate while

native whey proteins or serum proteins ( free from chemical and physical changes and have pH similar to

that of milk) that are free from casein peptides, enzymes, fat, and denaturized whey proteins are pass

through the membrane into permeate. This permeate act as an excellent raw material for the further

fractionation of other fragments of whey protein. The native serum proteins have been reported to possess

better functional properties (Maubois, 2002) than classical whey protein concentrate (WPC) and whey

protein Isolates (WPI). The separation caseins and whey proteins from skim milk has been shown if

Figure 2.

Figure 2: Separation of Casein and whey protein from skim milk using MF (Adapted from Hu et

al., 2015)

In this process, generally skim milk is subjected to MF that separates it into caseins and lactose enriched

retentate as well as whey proteins and lactose containing permeate. Upon subsequent stages and

diafiltration, purity of caseins are increased by reduction in lactose content. All types of available MF

processes either equipped with ceramic membranes (0.1-0.22μm pore size) i.e. conventional (0.22μm

ceramic membrane), uniform Transmembrane pressure-TMP (UTP) process (developed by Sandblom,

1978), GP process (developed by Garcera and Toujas, 2002),Isoflux process (developed by Grangeon et

al., 2002) or with polymeric based MF processes (PVDF, 0.3-0.5 μm) has been used for the fractionation

of these proteins. However, all these processes has their own merits and demerits in terms of energy

consumption, throughput and other several operational parameters. This process is also used to produce

native calcium phosphocaseinate that can be used in the cheese manufacture. The solution enriched in

38

serum proteins are further concentrated in UF membrane to increase the concentration of different whey

proteins fractions. From the UF retentate, the different fractions like lactoferrin, β-lactoglobulin (β-lg) and

α-lactalbumin (α-la) are produced using Ion exchange Chromatography as shown in Figure 3.

Figure 3: Potential applications of membrane processing in milk processing (Adapted from

Lipnizki, 2010)

4. Milk protein concentrate (MPC), Milk Protein Isolate (MPI), Whey Protein concentrate and

Whey Protein Isolate (WPI)

MPC and MPI are the two total milk protein enriched powders which have the same ratio of casein and

whey proteins as it was present in the milk from which they were produced. The major difference

between these two powders are in their protein content. MPC contains protein on dry matter basis in the

range of 42-89% while MPI have minimum protein content ≥89%. Both of them are produced from

pasteurized skim milk (raw material) that subjected to removal of fat either by centrifugation or by MF

process followed by subsequent UF of defatted skim milk for the concentration of total milk proteins by

removing lactose, and other water soluble vitamins and salts into permeate. The MWCO of UF membrane

is selected in such a way that it allows either zero or minimum loss of proteins into permeate. The degree

of concentration in UF is governed by the desired protein content in final MPC. The UF retentate is

usually subjected to diafiltration for the improvement in protein purity to produce higher protein

containing MPC and MPI. This retentate is optionally subjected to conventional evaporation for increase

in TS. Finally either UF retentate or evaporator concentrate is subjected to spray drying to convert them in

respective MPC and MPI.Similar to MPC and MPI, WPC and WPI are also produced using UF process

but the raw material for these whey protein rich powders are preferably defatted whey rather than

skimmed milk. Moreover, defatted WPC act as a very good raw material for the isolation of Lactoferrin

(Lf) and Lacto peroxidase(Lp) through Ion exchange chromatography.

39

Figure 4: Potential applications of membrane processing in whey processing (Adapted from

Lipnizki, 2010)

5. Fractionation of other Innovative dairy Ingredients

Another important application of membrane processing is in the development of reduced lactose based

dairy products. Apart from hydrolysis of lactose with Lactase (β-D-galactosidase, β-D-

galactosidegalactohydrolase, EC 3.2.1.23) enzyme and its Chromatographic separation, membrane

processes are have the potential to reduce/modify the lactose content of milk. It is widely accepted fact

that about 70% population from the different parts of the world are suffering from the problem of lactose

intolerance. Membrane process particularly ultrafiltration (0.001- 0.2 μm pore size; 1–1 000 kDa MWCO,

2-10 bar operational pressure) passes lactose into permeate and thus reduces lactose content in retentate.

This reduced lactose retentate can be added into number of food formulations developed for the lactose

intolerant group. Several patented processes has been already available for the production of low lactose

containing dairy products. Glycomacropeptide (GMP) is separated by ultrafiltration having its MWCO

between 20-50kDa.Combined with lactase, UF performs better for the manufacturing of an array of

reduced lactose food products.

Membrane processes are also used to produce demineralized whey based products for the persons having

problems with salts (Figure 4). Moreover, natural milk salts can also be concentrated by combined use of

membrane processes. Membrane processes are one of the integral part during the hydrolysis of milk

proteins for the production of desired peptides where these membrane based processes are used to

concentrate the peptides prior to their further purification and isolation. Moreover, membrane based

40

chromatography is gaining acceptance in biopharmaceutical and protein food production and its wider use

in other sector is yet to be seen.

6. Suggested Readings

Akin O, Temelli F,Köseoğlu S (2012) Membrane Applications in Functional Foods and Nutraceuticals, Critical

Reviews in Food Science and Nutrition52 347-371.

Corredig M, Roesch RR, Dalgleish DG (2003) Production Of A Novel Ingredient From Buttermilk. Journal of

Dairy Science86 2744–2750.

Dewettinck K, Rombaut R, Thienpont N, Le TT, Messens K, Van Camp J (2008) Nutritional And Technological

Aspects Of Milk Fat Globule Membrane Material. International Dairy Journal, 18 436–457.

DOI:10.1016/J.Idairyj.2007.10.014.

Etzel MR (2004)Manufacture and Use of Dairy Protein Fractions. Published in a supplement to The Journal of

Nutrition. Presented as part of the 94th American Oil Chemists’ Annual Meeting & Expo held in Kansas City,

MO, May 4–7, 2003. (http://jn.nutrition.org/content/134/4/996S)

Fox, P.F. and McSweeney, P.L.H. (eds.) (2003) Advanced Dairy Chemistry, Vol. 1, Proteins, third edition, Kluwer

Academic/Plenum Publishers, New York.

Garcera D, Toujas E (2002) Graded permeability macroporous support for crossflow filtration. US patent,

US6375014.

Goud´edranche H, Fauquant J, Maubois JL (2000) Fractionation of globular milk fat by membrane microfiltration.

Lait, 80, 93–98.

Grangeon A, Lescoche P, Fleischmann T, Ruschel B (2002) Cross-flow filter membrane and method of

manufacturing it. US patent US6499606.

Hu K, Dickson JM, Kentish SE (2015)Microfiltration for casein and serum protein separation InMembrane

Processing for Dairy Ingredient Separation, First Edition. Edited by Kang Hu and James M. Dickson. Published

by John Wiley & Sons, Ltd. 1-34.

Lipinzki F (2010)Cross-Flow Membrane Applications in the Food Industry In Membrane Technology Volume 3:

Membranes for Food Applications, Edited by Peinemann,K,V., Nunes,S.P.,andGiorno, L. WILEY-VCH Verlag

GmbH & Co. KGaA, (Weinheim), 1-27.

Lopez C, Briard-Bion V, M´enard O, Beaucher E, Rousseau F, Fauquant J, Leconte N, Robert B (2011) Fat globules

selected from whole milk according to their size: different ompositions and structure of the biomembrane,

revealing sphingomyelin-rich domains. Food Chemistry, 125 355–368.

Maubois JL (2002) Membrane microfiltration: a tool for a new approach in dairy technology. Australian Journal of

Dairy Technology, 57 92–96.

Morin P, Jiménez-Fl Ores R, Pouliot Y (2004) ‘Effect of Temperature And Pore Size On The Fractionation Of Fresh

And Reconstituted Buttermilk By MicrofiLtration’, Journal of Dairy Science87 267–273.

Roesch RR, Rincon A, Corredig M (2004) ‘Emulsifying Properties Of Fractions Prepared From Commercial

Buttermilk By MicrofiLtration’, Journal of Dairy Science87 4080–4087.

Rombaut R, Dejonckheere V, Dewettinck K (2007) Filtration of Milk Fat Globule Membrane Fragments from Acid

Buttermilk Cheese Whey. Journal of Dairy Science90 1662– 1673. DOI:10.3168/Jds.2006-587.

Sachdeva S, Buchheim W (1997) Recovery of Phospholipids from Buttermilk Using Membrane Processing.

KielerMilchwForsch49 47–68.

Sichien M, Thienpont N, Fredrick E, Trung Le, T, Camp, Dewettinck K (2009) In Dairy-derived ingredients Food

and nutraceutical uses, Edited by Milena Corredig, book Chapter: Processing means for milk fat fractionation

and production of functional compounds, 68-95.

41

Table 1.Physical properties of dairy proteins ( Source: Linvey*, 2010; Etzel

#, 2004 and Fox and McSweeney

$, 2003)

Protein

Content

in milk*

(g/l)

Molecular

weight*

(k Da)

Molecular

mass#

(Kg/mol)

Concentration#

(g/L)

Isoelectric#

point

(PI)

Conc.$

(g/L)

Caseins

(Spherical, dia.-0.05 to 0.5 μm)

24–28

αs1 (αs1-CN) 12–15 22.1–23.7 24 13 4.9/5.3

10

αs2 (αs2-CN) 3–4 25.2–25.4 2.6

β (β-CN) 9–11 23.9–24.1 24 9.3 5.2 9.3

κ (κ-CN) 3–4 19.0 19 3.3 5.8 3.3

γ( γ-CN) 0.8

Whey proteins

5–7

β-

lactoglobulin(β

-lg)

2–4 18.3 18 3.2 5.4 3.2

α-lactalbumin

(α-la) 1–1.5 14.2 14 1.2 4.4 1.2

Bovine serum

albumin (BSA) 0.1–0.4 66 66 0.5 5.1 0.4

Immunoglobuli

ns (Ig) 0.6–1.0 146–1,030 150 0.7 5-8 0.7

Lactoferrin (Lf) ∼0.1 80 77 0.1 7.9

Casenomacrop

eptide - - 7 - - -

Lactoperoxidas

e - - 78 0.03 9.6

Glycomacrope

ptide (GMP) - - 8.6 1.5 <3.8

MFGM proteins ∼0.4 13–200 - - -

Total milk proteins 30–35 - - - -

42

Application of Casein and Caseinates in Formulation of Specialized Foods

Vijay KumarDairy Technology Division

1. IntroductionWith a content of 0.7 - 0.9% phosphorus, covalently bound to the casein by a serine ester linkage,casein as a phospho-protein is a member of a relatively rare class of proteins. Moreover, due to highproportions of essential amino acids, casein is nutritionally excellent protein. Its protein efficiencyratio reported is 2.5, which is mostly unaffected by the processing conditions usually employed duringthe dairy operations. Casein has some rather unique properties and cannot be replaced by otherproteins in certain food application. Edible casein and caseinates are long established dairy by-products finding use in many dairy and food products. World production of casein is around 2.5 lakhtonnes. The biggest importer of casein is United States of America, where about 1 lakh tonnes ofcasein is imported, most of which is utilised for the manufacture of imitation cheeses.

Acid casein is insoluble in water. Its soluble form caseinates may be prepared from freshlyprecipitated acid casein curd or from dry acid casein by reaction with dilute solution of alkali (such assodium, potassium, calcium or ammonium hydroxide). Sodium caseinate is the most commonly usedwater-soluble form of casein and is used in the food industry. The two main reasons for using sodiumcaseinate as an ingredient in foods are its functional properties and nutritive value. Sodium caseinateis valued for its ability to emulsify fat in the production of modified dairy products such as coffeewhiteners, whipped cream and ice cream. It also possesses very good water binding and whippingproperties. Industries of meat processing, baking and modified dairy products are the largest consumerof sodium caseinate. The various food products in which sodium caseinate is used consist of variouskinds of sausages, meat-based and milk-based instant breakfasts, modified milk, whipped cream,coffee whiteners, non-dairy creams, desserts, sauces, soups, bread, doughs, crackers (biscuits), dieteticproducts and various protein-enriched products. . Other casein products, used in a descending order inthe food industry are calcium caseinate, potassium caseinate, other caseinates, and, finally, purecasein.

Other soluble forms of casein are produced using phosphates, carbonates, and other salts as thesolubilizers. Magnesium caseinate is prepared from casein and a magnesium base or basic salt suchas magnesium oxide, magnesium hydroxide, carbonate or phosphate or by ion exchange. Compoundsof casein with aluminium may be prepared for medicinal use or for use as an emulsifier in meatproducts. Heavy metal derivatives of casein, which have been used principally for therapeuticpurposes, include those containing silver, mercury, iron and bismuth. Iron and copper caseinates havealso been prepared by ion exchange for use in infant and dietetic products

One another form of casein that is commonly used in the food industry is the hydrolysate. Casein canbe hydrolysed with acid, alkali or proteolytic enzymes. Today, casein hydrolysates have assumed anew dimension in food industry. They find use in wide ranges of soups, gravies, sauces, drinks,vegetable and fruit juices, flavourings and nutritional, dietetic and formulated foods. They are alsoused as ingredients in crackers, snack foods, and other food products. Casein hydrolysates obtained byacid hydrolysis have a meat-like flavor. These are, therefore, used to accentuate the meat flavor inheat-treated canned and dried meat products such as soups.

43

2. Bakery productsCasein and casein derivatives are mainly used in bakery products to enhance flavour and othersensory properties and also for nutritional fortification of the wheat flour. The limiting amino acid(lysine) in most cereal proteins can be very well complemented with dairy proteins. Casein/caseinatescan be added to breakfast cereals, milk biscuits and protein-enriched bread. The PER of wheat flour isonly 1.1, compared with 2.5 for casein. By supplementing the wheat flour with casein, it is possible toincrease considerably the PER of the mixture. For instance, for a 50:50 mixture of casein and wheatprotein, the PER can be raised to 2.2 - 2.3. One of the most important functional characteristics ofcasein products in bakery products is its water binding capacity.

Acid and rennet casein, sodium caseinate and calcium caseinate can be used in bread making and areadded at a level of 15-20% of the wheat flour. A satisfactory loaf volume can be obtained by the useof casein products. The physical structure of bread reflects the unique properties of the major proteinsof wheat flour. Upon hydration, gluten forms a stretchable viscoelastic network that can entrap gasproduced by yeasts. The structure stabilizes during baking. Bread, with milk proteins added in oneform or another, shows a good crumb structure, bread yield, flavour and keeping quality. In themanufacturing of high protein biscuits, milk proteins play an important role as they increase thenutritive value and also the texture.

Milk proteins are often incorporated into the base flour for pasta manufacture for the purpose ofenhancing nutritional quality and to improve texture. Products fortified by addition of sodium orcalcium caseinate prior to extrusion include macaroni and pasta.

3. Modified dairy productsThe use of sodium caseinate in the dairy industry and in the manufacture of modified milk productshas increased all around the world. The addition of 1% sodium caseinate into UHT low-fat milkfortifies this dietetic drink with protein. The addition of sodium caseinate to cultured milk productssimultaneously increases the nutritive value and improves the technological quality of products.

Milk Protein products are widely used to supplement the protein content and, therefore, enhancesensory characteristics of conventionally processed dairy products and are also used in the productionof a range of imitation dairy products. Imitation cheeses are made from vegetable fat, caseins, saltsand water and are used in pizza, lasgne and sauces and on burgers, grilled sandwiches, macaroni etc.,at a significant cost-saving compared to the use of natural cheese. The functional properties of rennetcasein that favour its use in imitation cheese include fat and water binding, texture enhancing, meltingproperties, stringiness and shredding ability.

Sodium caseinate is used in powdered coffee creamers, which also contain vegetable fat, acarbohydrate source and added emulsifier and stabilizers. These creamers are cheaper, have a longershelf life and are more convenient to use (e.g. they require no refrigeration) than fresh coffee creams.In these products, sodium caseinate acts as an emulsifier/fat encapsulator and whitener, imparts bodyand flavour and promotes resistance to feathering.

Sodium caseinate is used to increase gel firmness and reduce synersis in yoghurts, and is added tomilk shakes for its emulsifying and foaming properties. Sodium caseinate is also used as anemulsifying and fat encapsulating agent in the manufacture of high-fat powders for use as shorteningsin baking or cooking. Dry whipping fats or whipping creams contain casein products.

A procedure for the manufacture of a soluble casein concentrate suitable as an ingredient in babyfoods and dietetic foods was developed in the former USSR. Precipitated casein is dissolved byadding 2.8% sodium citrate, 3.2% calcium citrate, and 5% sodium bicarbonate to it and the product isdried and ground.

44

4. BeveragesCasein products are used as stabilizers or for their whipping and foaming properties in drinkingchocolate, fizzy drinks and fruit beverages. There is also alarge market for sodium caseinate as anemulsifier in cream liqueurs and to a lesser extent in wine aperitifs. Cream liqueurs typically contain16% (w/w) milk fat, 3.3% sodium caseinate, 19 % added sugar and 14 % ethanol. Trisodium citrate isalso added to prevent calcium-induced age gelation. Casein products have also been used in the wineand beer industries as fining agent, to decrease colour and astringency and to aid in clarification.

5. ConfectioneryCaseins are used in toffee, caramel, fudge and other confections as they form a firm, resilient, chewymatrix on heating and they contribute water binding and aid emulsification. Casein hydrolysates areused as foaming agents in place of egg albumen in marshmallow and nougat as they confer stability tohigh cooking temperatures and good flavour and browning properties.

6. Meat productsThe possibility of the production of simulated meat by using artificial protein fibers based on caseinhas been investigated for several years. This field is protected by a variety of patents. The basicprinciple described in one of them consists of the extrusion of a sodium caseinate solution through adie with small openings into an acidic medium. Care must be taken that the continuity of casein fibersis maintained at all times. The patent covers the production facility scheme and 26 products based onsodium caseinate. Using characteristic flavors, coloring agents, and other ingredients, it is possible toproduce simulated beef, chicken meat, pork, bacon, ham, and even fish, all of which have the tasteand texture of the products being simulated.

The use of caseinates in meat products is traditional and has been used for 30 years as a potentialfunctional protein ingredient in meat industry and it has gone far since then. Today the best-documented non-meat ingredient in meat products is caseinates round the globe. Besides nutritionalvalue, they are mainly used due to their excellent water binding and emulsifying properties and,therefore, the major application field is that of the rather comminuted meat products. Amongst manyother functional properties, the bland flavour and neutral colour of caseinates deserve a specialmention. The caseinates get their functionality from their unique molecular characteristics. Theyhave a random coil structure with a low percentage of helix. They show no heat gelation ordenaturation and have a high viscosity in solution. The ability of caseinates to bind moisture throughH-bonding and entrapment thereby enhancing the yield of end products, has been used beneficially invarious meat and sausage preparations. In addition, their salt tolerance and high protein contentattract many meat traders.

The functional behaviour of milk proteins in comminuted meat products have been studied by Hungand Zayas (1992). Milk proteins have been utilized as fillers, binders and extenders in cookedcomminuted meat products to reduce cook shrink and formulation cost, as well as to improveemulsifying capacity, emulsion stability, water binding, potential nutritive value and slicingcharacteristics. The dairy proteins can also improve or alter the consumer acceptance (flavour,mouthfeel, colour, appearance etc.) of the finished product. These proteins significantly increase thegel strength of meat proteins and it has been shown that there is a synergistic effect between milkproteins and salt soluble meat proteins, through covalent cross-linkages. The functionally designeddairy ingredients, especially milk protein products, have held their position in this competitive sectorbecause they exhibit good functionality features and affecting the final product in a desirable way.Choice of dairy by-products in meat industry is often more guided by economic criteria. Surpluscaseins have been used for substituting more expensive meat and egg proteins in Western countries.Even in this area, competition from vegetable proteins is increasing. Milk proteins, however, are

45

preferred ingredients for their functional supremacy, their good flavour, colour and nutritional profile,because of which they are able to keep their foothold with vegetable protein products.

In comminuted meat products, a considerable amount of free fat is released during the manufacture ofthese products, which must be stabilized. Caseinates, when used in the desired way, not only stabilizethe fat, but also impart water binding and consistency. They do retain a part of the soluble fraction ofmeat proteins in their native form, which otherwise prone to denaturation at the interface, enabling itavailable for gel formation upon heating (Schut and Brouwer, 1971) possibly through protein-proteininteractions of meat and milk analogues. Consequently caseinates, a milk protein derivative, is awidely accepted functional protein giving the meat product a better stability. This kind of protein alsohas many hydrophobic groups. This could be one of the several reasons why they are perfectemulsifiers, and are active at the fat-water interface to prevent fat separation. Besides they areperfectly water soluble, a truly desirable characteristic in several restructured meat products. It furthersupplements and complements the native meat proteins and does not doubt the naturality of productswhen utilised in functional proportions, i.e. less than 3 percent (Visser, 1984).

Though caseinates may be employed in dry or pre-solubilised form at the beginning of thecomminuting process, optimum stability results are obtained when they are processed in the form ofpreviously prepared caseinate/fat/water emulsion (Schut and Brouwer, 1975; Visser, 1984). Sodiumcaseinate is the most versatile of all milk proteins and disperses well in water or melted fats. Uponaddition of warm water during processing, sodium caseinate dispersion hydrate and the resultingcolloidal solution forms a base for subsequent emulsions. The technologically more difficult andcheaper type of fat,such as of beef and sheep may be perfectly used by pre-emulsifying withcaseinates. The rationale of using sodium caseinate in emulsion technology is based on its manifoldchemical reactivity. The reactivity of caseinates lies in the unique distribution of the electricalcharges on the polymeric molecule, its hydrogen bonding ability and its richness of hydrophilic aswell as lipophilic bonding sites. The more complex colloidal chemical performance of caseinates canbe accentuated by its reactivity with lecithin and carrageenan.

Addition of caseinate stabilizes the meat emulsion as required in the sausage mix. It thickens thegravy during frying and prevents it running out, but excess incorporation of caseinate may result indrying up of the sausages. Further addition of water-absorbent materials becomes essential whensodium caseinate concentration in sausages exceeds 5% (Salavatulina et al., 1983). The greater waterholding capacity, lower viscosity and lower cooking losses of sausage batters containing 2% sodiumcaseinate in comparison to all meat control were observed by Hung and Zayas (1992).

The nutritional value of sausage products, in which part of the meat protein replaced with otherproteins was studied by Safronova (1983). The nutritional value of proteins in sausages dependeddirectly on the levels of replacement; for sodium caseinate 50% of the meat protein could be replacedwithout any adverse effect on it. Usage of a very high viscosity sodium caseinate as an effective meatbinder has been found to be desirable in some meat products, because of its greater water binding andgelling properties.

The effect of sodium and calcium caseinates on the functional quality of nuggets made from spent henmeat has been studied by Rao et al. (1994a,b). They observed that with 2% incorporation of sodiumcaseinate or 1% calcium caseinate, the yields were improved, protein content was higher andbrightness scores were higher for nuggets containing caseinates as compared to control nuggets. Thefrying losses were also lesser for caseinate added nuggets. The texture profile analysis revealedhigher firmness and springiness for sodium caseinate containing chicken nuggets and highercohesiveness and gumminess for nuggets having calcium caseinate (Rao et al., 1996).

46

7. Dietary, pharmaceutical and medical applicationSince milk protein products are of high nutritional quality, they are used extensively in dietarypreparations for people who are ill or convalescing, for malnourished children in developing countrieson a therapeutic diet and for people on weight-reducing diets. Caseins are used in special preparationsto enhance athletic performance and have been incorporated into formula diets for space feeding.

While casein products are not generally used in infant formulae, they are used extensively inspecialized preparations for infants with specific nutritional problems. Caseinates are used in low-lactose formulae for lactose-intolerant infants while various types of caseinates have been used ininfant foods with a specific mineral balance, e.g. low-sodium infant formulae for children withspecific renal problems. Casein hydrolysates are used in specialized foods for premature infants, informulae for infant suffering from diarrhea, gastroenteritis, galactosaemia and malabsorption. Aspecial casein hydrolysate, low in phenylalanine, has been prepared for use in formulae for feedinginfants with phenylketonuria. Casein products are also added to various foods for children and infantsand to drinks as a nutritional supplement.

Diets that are suitable for geriatrics, high-energy supplements, weight-control diets, hypoallergenicinfant formulas and therapeutic or enteric diets are some of the areas in which casein hydrolysates aremost useful. casein hydrolysates are boon to people who are suffering from protein allergy orstomach disorders and to those who require easily digestible foods. The production of hydrolysedprotein provides an opportunity for the dietary management of patients with various digestivedisorders as a result of pancreatic malfunction, pre- and post-operative abdominal surgical patients,patients on geriatric and convalescent feeding and others who for various reasons are not able toingest a normal diet. Casein hydrolysates also have pharmaceutical applications in intensive carefoods, anemia treatment, prevention of blood cholesterol, treatment of dental diseases and inadministration of amino acid mixture intravenously.

Specific drugs have been produced from casein; -casein is useed as raw material for production of -casomorphins, tetra- to hepta-peptides which can regulate sleep, hunger or insulin secretion.Sulphonated glycopeptides prepared from casein have been used for the treatment of gastric ulcers. Itis claimed that the use of casein in toothpaste prevents dental caries, in cosmetics it conceals facialwrinkles and in special therapeutic creams it heals wounds.

8. Selected ReadingHung, S.C. and Zayas, J.F. (1992) Functionality of milk proteins and corngerm protein flour in comminuted

meat products. J. Food Qual., 15: 139-152.Mann, E.J. (1989) Dairy ingredients in meat products. Dairy Indus. Inter., 54(2): 9-10.Rao, K.V.S.S., Anjaneyulu, A.S.R., Singh, R.R.B., Rao, K.H. and Yadav, P.L. (1994a) Effect of sodium

caseinate on the fuctional quality of nuggets from spent hen meat. Ind. J. Poultry Sci., 29(1): 115-116.Rao, K.H., Singh, R.R.B., Anjaneyulu, A.S.R., Rao, K.V.S.S. and Yadav, P.L. (1996) Evaluation of caseinates

and refined wheat flour as emulsion stabilizers in chicken nuggets. Proc. XX World's poultry Cong., Vol-IV,PP 435.

Schmidt, D.G. (1986). "Association of Caseins and Casein Micelle Structure." In Developments in DairyChemistry. 1. Proteins, P.F. Fox, ed. Elsevier, London, pp. 61-86.

Mulvihill, D.M. (1992) "Production, functional properties and utilization of milk protein products." In AdvancedDairy Chemistry. Vol. 1. Proteins, P.F. Fox, ed. Elsevier Applied Science, London, pp. 369-404.

Caric, M. (1990). Technology of Concentrated and Dried Dairy Products, 3rd ed. Naucna Knjiga, Beograd,Yugosla via, 293 pp. (in Serbian).

Mann, E.J. (1991). Dairy Ind. Int. 56, 13-14.Fichtali, J., Voort van de, F.R., and Khuri, A.I. (1990). J. Food Process Eng. 12, 247-258.

47

Encapsulation of Herbal Bioactives through Double Emulsion Technology

Sathish Kumar, M.H., Heena Lamba and Latha Sabikhi


1. Introduction

Plant bioactive refers to those plant components that incur some health benefits apart from maintaining

the normal metabolism of the body. Source of plant bioactive substances include plant derived foods like

fruits, vegetables, food grains, tree nuts, pulses and drinks like tea, coffee, cocoa, wine, beer, edible oils,

spice and herbal extracts. Plant bioactives fall under following major categories i.e. fatty acids (ω-3 fatty

acids), carotenoids (α-carotene, β-carotene, β-cryptoxanthin, lycopene, lutein and zeaxanthin),

antioxidants (tocopherols, flavonoids and polyphenols), flavonoids (flavonols, flavan-3-ols, flavones,

flavanones and anthocyanidins), glucosinolates, phytoestrogens (isoflavones and lignans), sulphur

containing bioactive compounds, terpenoids, dietary fibres, fat soluble vitamins (A, D, E and K) and

phytosterols (stigmasterol, β-sitosterol and campesterol). Bioactive compounds belong to ‘non-nutritive’

group and have been shown to provide protection against oxidative degenerative diseases like coronary

heart disease, cardiovascular disease, ischemic stroke, cancer, Alzheimer’s disease and lung disorders

such as obstructive pulmonary disease. There is a growing need of incorporation of such bioactive

compounds in functional foods, but this poses several challenges for the researchers because of the

physical and chemical instability when extracted from its source. Due to its instability when incorporated

in a food matrix, it is difficult to maintain its bioactivity or effectiveness through processing, storage,

transport and consumption. Bioactivity of the components could be maintained using appropriate delivery

systems, to which properties such as solubility, polarity and charge of the bioactive component are

compatible. Delivery system functionality refers to the sensory and physical impact it will have when

added in final food matrix.

Double emulsion, in this respect, is gaining popularity since the last two decades as a delivery vehicle for

target delivery, because of the following reasons:

1. It allows the substance to be released at a controlled rate in the desired location

2. Variation in composition and particle size affected the availability of bioactive components

Double emulsions, are the emulsions in which the dispersed phase is itself an emulsion present as fine

droplets. They are mainly of two types: oil-in-water-in-oil (O/W/O) and water-in-oil-in-water (W/O/W).

In O/W/O, oil is first dispersed in water and then this emulsion is dispersed in a second oil phase.

Similarly for W/O/W, water is first dispersed in oil which is then dispersed in another water phase.

Further, based on number of internal droplets they are divided into 3 major types i.e. type A, B and C.

Type-A or ‘core-shell’ type multiple emulsion has internal phase as only one large internal droplet,

whereas type-B and type-C multiple emulsions consists of few and several number of small internal

droplets respectively.

2. Formation of Double Emulsion

A number of methods can be employed for double emulsion formation. Different methods have been

adopted for formation of stable double emulsions. These can be broadly divided into two categories: high

shear devices and membrane emulsification. High shear devices used for double emulsion preparation

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includes: ultrasonicator, high pressure homogeniser, ultraturrax (high shear mixer) and microfluidiser.

General principle behind use of such devices is the high shear produced by cavitation, collision and

turbulence, which causes breakdown and uniform dispersion of the dispersed phase. Microfluidics is a

field which sums up the principles of engineering and physics to bring innovative techniques in handling

smaller volumes of fluids in order to achieve certain objectives/ applications. A lot of innovation has been

made in microfluidics to broaden its application in stable double emulsion preparation. Microfluidiser is

by far the most efficient equipment forming designed double emulsion (intended for specific purpose and

having uniform internal structure). It makes use of different flow rates inside interaction chamber to

simulate formation of desired internal structure.

2.1 Oil Phase

Food based double emulsions usually have vegetable oil as oil phase. Canola oil medium chain

triglyceride (MCT) rich oil (MIGLYOL), rice bran oil, soybean oil and rapeseed oil have been tried

successfully as oil phase in double emulsions.

2.2 Aqueous Phase

It usually composed of Milli-Q water, RO water or demineralised water along with 0.01-0.02% of sodium

azide as preservative. Acidity regulators are used to adjust the pH to desired value for encapsulating

bioactives. Buffers can also be used as aqueous phase to maintain the pH of the system.

2.3 Emulsifiers

They are amphiphillic compounds containing both hydrophilic and lipophilic groups that can accumulate

in interfacial area. It thus has the capability of lowering interfacial tension and stabilise the interface

formed between two immiscible liquids in an emulsion. Amount of emulsifiers has prominent effect on

emulsion properties, as lower amounts may result in unstable system, whereas higher amounts may also

lead to destabilization. A combination of hydrophilic and hydrophobic emulsifiers is usually employed to

stabilise double emulsions. Polymeric emulsifiers are preferred over monomeric emulsifiers owing to

their minimal or negligible migration. Double emulsions have three phases and thus require use of both

hydrophilic as well as hydrophobic types of emulsifiers to stabilise the two interfaces. Hydrophilic

emulsifiers are mostly proteins or polysaccharides in nature, like whey or soy, sodium caseinate bile acid,

polysorbitan-monolaurat 80 or Tween 80, polysorbiton-monolaurat 20 or Tween 20, SUPER GUMTM

which is modified gum Arabic, Panodan SDK which are esters of monoglycerides and diglycerides of

diacetyl tartaric acid, and β-lactoglobulin isolated from whey protein isolates (WPI). Hydrophobic

emulsifiers having HLB value of four or less are used to emulsify water droplets in oil phase. They have

higher proportion of non polar groups and thus are soluble in oil or lipid phase. The most commonly used

hydrophobic emulsifier in food based double emulsions is PGPR i.e. polyglycerol ester of polyricinoleic

acid, which is a high molecular weight compound obtained from castor beans.

3. Stability of Double Emulsion

Degree of shear produced by the device dictates the stability of double emulsion. High shear rate device is

required for formation of primary emulsion, while a low shear rate device is used for the dispersion of

primary emulsion to avoid breaking of double emulsion structure. Type of dispersing device also has

great impact on the particle size of the double emulsion which in turn effect encapsulation efficiency and

emulsion stability

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4. Size of the Double Emulsion – Crucial for stability

Particle size is a very crucial parameter in deciding the application of double emulsion. Nano-sized

particles are most suitable for delivery systems. Device like ultraturrax produce particle size in the range

of micrometers, whereas nano-sized double emulsions can be prepared using devices like high pressure

homogeniser, sonicator and microfluidiser. Membranes along with microporous glass or ceramic can also

be used for emulsification. Double emulsion prepared through membrane emulsification showed higher

stability, uniform particle size and no leakage of the contents from inner aqueous to outer aqueous phase.

5. Applications of Double Emulsions

Double emulsions provide protective encapsulation to bioactive substances (hydrophilic and hydrophobic)

in the internal droplets. They are mainly used in applications where controlled (sustained and delayed)

release of bioactive ingredients is desired. They have established industrial applications in various fields

such as pharmaceuticals and cosmetics as a means of microencapsulation of drugs. Applications of double

emulsions in the food industry have been reported in areas like:

Encapsulation and controlled release of flavours, bioactive compounds (both hydrophilic and

hydrophobic), microorganisms or probiotics and nutrients like vitamins and minerals.

Manufacture of low calorie food products e.g. low-fat dressing, low-fat whipping cream and low-fat

cheese.

Manufacture of food products with improved sensory characteristics Encapsulation of substances

having objectionable flavours

Prevention of exposure of sensitive substances from destabilising factors like light, oxygen and heat

Target delivery of drugs and antigens

6. Conclusion

Instability has always remained the key issue regarding commercialisation of this technique. Its

application in food is restricted to the use of all food based ingredients, especially emulsifiers for the

preparation of stable double emulsions. However in the last two decades, a remarkable improvement in

the stability of double emulsions were evident with the use of biopolymer complexes and conjugates as

emulsifiers. Difficulties in scaling up of the process have been tackled by the entry of new processing

equipments like microfluidiser. Plant bioactives are emerging in the field of health and nutrition because

of their vast therapeutic benefits. There is a growing interest in development of functional foods enriched

with plant bioactives. Double emulsions, because of their properties such as controlled release and target

delivery under suitable conditions are considered appropriate delivery vehicles for plant bioactives in

food.

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Designing of Herb Based Traditional Dairy Products

Writdhama Prasad and Kaushik Khamrui


1. Introduction

Herbs and spices are common food adjuncts, which have been used as flavoring, seasoning, and coloring

agents and sometimes as preservative, throughout the world for thousands of years, especially in India,

China, and many other southeastern Asian countries. While bringing color and taste to the food, some

spices have long been considered to possess medicinal value and have been effectively used in the

indigenous systems of medicine. Studies on herbs and spices have viewed their bioactive compounds

from the perspective of antioxidants. The antioxidant property of these molecules was explained on the

basis of the availability of hydroxyl group and the system of conjugated double bonds present in these

molecules. Further investigations into the mechanism of action of these molecules have shown that

polyphenols may not merely exert their effects as free radical scavengers, but may also modulate cellular-

signaling processes during inflammation or may themselves serve as signaling agents.

2. Oxidation and Health

The concept of ‘oxidative stress’ hypothesizes that exposure to adverse physiochemical, environmental

and pathological agents disrupts the body's natural balance, and if excess free radicals are not eliminated

by antioxidants, they may damage crucial extracellular or cellular components. Free radicals are highly

unstable chemical species containing one or more unpaired electrons. In order to attain stability they

extract electrons from other chemicals, which damages the latter compound(s). One of the many free

radicals important for human health is Reactive Oxygen Species (ROS). (ROS) are: (1) generated during

irradiation by UV light, by X-rays and by gamma rays; (2) products of metal-catalyzed reactions; (3)

present as pollutants in the atmosphere; (4) produced by neutrophils and macrophages during

inflammation; (5) continuously produced in our body through various endogenous enzymes; (6) by-

products of mitochondria-catalyzed electron transport reactions and other mechanisms. ROS level in our

body is maintained by various regulatory mechanisms but, higher levels of ROS could be induced by

exposure to external oxidant substances or a failure in the defense mechanisms.

About 5% or more of the inhaled oxygen is converted to reactive oxygen species (ROS) such as O2-, H2O2

and OH- by univalent reduction of inhailed oxygen. ROS are potentially damaging transient chemical

species due to the fact that upto certain levels, they are essential to us for energy supply, detoxification,

chemical signaling and immune function, while on other hand, at higher levels, they damages cell

structures, DNA, lipids and proteins, they attack cellular components involving polyunsaturated fatty acid

residues of phospholipids, side chains of all amino acid residues of proteins, in particular cysteine and

methionine residues and all the components of DNA molecule, damaging both the purine and pyrimidine

bases and also the deoxyribose backbone and thus increases risk of more than 30 different diseases. The

most notorious among them being neurodegenerative conditions like Alzheimer’s disease (AD), mild

cognitive impairment (MCI) and Parkinson’s disease (PD). Other diseases include highly disabling

vascular pathologies like cardiovascular disease (CVD) and cardiac failure.

The harmful effects of ROS are balanced by the antioxidant. Thus, there appears a regular need of anti-

oxidants in our body, to prevent such oxidation associated diseases. Epidemiological evidence indicates

existence of correlation between increased dietary intake of antioxidants and a lower incidence of

51

morbidity and mortality. An anti-oxidant is a substance that significantly delays or prevents oxidation of

cell contents including proteins, lipids, carbohydrates and DNA (Gupta et al., 2006). In nature, there are

a wide variety of naturally occurring anti-oxidants which are different in their composition, physical and

chemical properties, mechanism and site of action (Naik, 2003). Kinetically antioxidants can be

classifieds into six categories as: (1) Antioxidants that break chains by reacting with peroxyl radicals

having weak O-H or N-H bonds: phenol, napthol, hydroquinone, aromatic amines and aminophenols. (2)

Antioxidants that break chains by reacting with alkyl radicals: quinones, nitrones, iminoquinones. (3)

Hydro peroxide decomposing antioxidants: sulphide, phosphide, thiophosphate. (4) Metal deactivating

antioxidants: diamines, hydroxyl acids and bifunctional compounds. (5) Cyclic chain terminating

antioxidants: aromatic amines, nitroxyl radical, variable valence metal compounds. (6) Synergistic action

of several antioxidants: phenol sulphide in which phenolic group reacts with peroxyl radical and sulphide

group with hydro peroxide.

3. Findings for potential application of herbs or herbal components for food preservation:

With the concept of hurdle technology, a number of potential synergists have can be studied for use with

herbs such as low pH, low water activity, chelators, low oxygen tension, mild heat and raised pressure,

even some of these have been studied in foods, viz., effect of additives and with preservation techniques

of mild heat treatment, high hydrostatic pressure and anaerobic packaging.

Table: Biological activities of some commonly used herbs

Botanical name Chemical constituents Biological activities

Curcuma longa

(Turmeric)

Curcumin, beta-pinene,

camphene, eugenol, beta-

sitosterol

For cleaning blood, in cough and dysponoea

management, antifertility agent, for malarial fever

Cuscuta reflex

(Akashbela)

Flavonoid, dulcitol,

bergenin, coumarins,

glycosides, lactone

Carminative, anthelmintic, purgative, diuretic,

jaundice, bilious disorder, anti-fertility drug

Emblica

officinalis

(Amla)

Vitamin C, polyphenols

(ellagic acid, gallic acid

and tannins)

Useful in burning sensations, vomiting, urinary

discharges, leprosy, constipation, inflammations,

piles, anemia.

Glycyrrhiza

glabra

(Mulethi)

Glycyrrhizin, flavonoids,

liquiritin, isoliquiritin,

rhamnoliquiritin

Diuretic, emmenagogue, vomiting, asthama,

bronchitis, acute conjunctivitis, curing wounds, in

peptic ulcers.

Psoralea

corylifolia

(Babchi)

Essential oil, fixed oil,

resin, bakuchiol

Purgative, stomachic, anthelmintic, stimulant,

asphrodisiac, leucoderma, scabies, biliousness, in

various blood and skin diseases.

Santalum album

(Safed-chandan)

Volatile oil, santalol, beta-

sitosterol

Antipyretic, aphrodisiac, useful in diseases of heart,

bronchitis, small pox, seeds in the skin diseases

Combinations of oregano EO with sodium nitrite have been studied for their effect on growth and toxin

production by C. botulinum. It was found that Oregano oil acted synergistically with nitrite to inhibit

growth in broth, whereas oregano oil applied alone at up to 400 ppm had no significant inhibitive effect

on growth. The proposed mechanism of synergism depends on oregano EO reducing the number of spores

which germinate and sodium nitrite inhibiting the outgrowth of spores.

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Synergistic effect of the essential oil from different herbs were also found to have higher activity than

their individual application in certain cases, viz., simultaneous application of nisin and carvacrol or

thymol caused a larger decline in viable counts for strains of B. cereus than was observed when the

antimicrobials were individually applied. Also, it was found that at pH 7 the synergistic action of nisin

and carvacrol was significantly greater at 30OC than at 8

OC, which would appear to indicate temperature-

induced changes in the permeability of the cytoplasmic membrane.

Thymol and carvacrol was shown to have synergistic effect with high hydrostatic pressure (HHP).

Growth of L. monocytogenes cells were reduced more by combined treatment with 300 MPa HHP and

thymol or carvacrol addition than by the separate treatments. This could be due to the fact that both the

herb as well as HHP causes damage to the cell membrane.

Antibacterial activity of Essential Oil was also influenced by the degree to which oxygen is available

(Paster et al., 1990), this could be due to in the absence or at lower oxygen concentrations, oxidative

changes are reduced and also microbes shift to anaerobic metabolism for meeting energy requirements, at

which they are more sensitive. The authors have reported increased antibacterial activity of oregano and

thyme against S. typhimurium and Staph. aureus at low oxygen levels.

4. Incorporation of herbs in milk based products

Fat rich products deteriorates by fat auto-oxidation which leads to generation of off flavors accompanied

by the formation of hydroperoxides which are harmful to human health. Phenolic compounds such as

catechin, catechol, resorcinol, quercetin, and kaempferol inhibit oxidative rancidity in margarine, milk

powder, ghee and buttermilk. Sage (Salvia officinalis) and rosemary (Rosmarinus officinallis) extracts are

one of the most widely used source of natural anti-oxidants. Their extracts are reported to have

antioxidant activity many times stronger than synthetic antioxidants like BHA or BHT.

Addition of rosemary (Rosmarinus officinalis L.) at 7.5% (DM basis) in sheep ghee has shown an

antioxidant effect equivalent to that of mixture of BHA and BHT (250 p.p.m. of a 1:1 mixture of BHA

and BHT) during storage at 78°C. Addition of sage ethanolic extract at 0.1-0.2% (w/w) to sweet cream

directly before churning affected lower level of peroxide value during storage of butter and butter fat at

20 and 60 °C, respectively. Herbal ghee incorporating functional attributes of arjuna has also been

developed for providing beneficial effects against cardiovascular diseases and the product was more

stable to oxidative deterioration as compared to conventional ghee (Rajnikant, 2005; Parmar, 2012).

Freeze-dried hydrodistilled extract of clove (Syzygium aromaticum L.), caraway (Carum carvil L.) and

coriander (Coriandum sativum L.) exhibited antioxidant effect in terms of acid value, peroxide value and

thiobarbituric acid test in the order of clove > coriander > caraway when added at 400 ppm level in butter

oil. Similarly, methanolic extracts of sage, rosemary and oregano were also found to be effective in

retardation of oxidation (TBA test, peroxide value) and lypolisis (FFA) processes in butter. Addition of

Satureja cilicica Essential Oil in butter exhibited strong antioxidant activity. However, the addition of

dried sage and rosemary did not prevent the lipolysis and formation of peroxides during low temperature

storage of the sour-cream butter.

Herbs viz turmeric (Curcuma longa L.), coriander (Coriandrum sativum L.), curry leaf (Murraya koenigii

L.), spinach (Spinacia oleracea) and aonla (Emblica officinalis) incorporation into Sandesh to induce the

antioxidant properties into the product. The authors have reported that the total antioxidative status of

herbal sandesh was lower than samples with TBHQ but similar to those with 200 mg/kg BHA: BHT

(1:1), and even with relatively relatively less anti-oxidative status, all the anti-oxidant added samples

exhibited increased shelf life.

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5. Conclusion and Future Prospects

Herbs are also rich sources of beneficial compounds including antioxidants and components that can be

used in functional foods. In India more than 70% of the population use herbal drugs for their health,

which is based upon experience-based evidences. Much remains to be done to understand the mechanisms

of action for antioxidants, their impact on various types of tumors, the degree to which antioxidants are

absorbed from foods, and what effective concentrations are needed in humans to reduce oxidative stress at

the tissue or cellular level and how the antioxidant capacity of foods and food components relates to

physiologic events in humans.

To facilitate research in this area, there remains a need to collect additional data on the antioxidant

capacity of herbs, spices, and their bioactive components from in vivo, in vitro, and clinical studies and

establish more detailed research databases to serve as a repository for this data from a variety of

disciplines to promote trans-disciplinary research and ultimately aid in fostering new discoveries.

The need of the day is to encourage to encourage incorporation of these herbs to as many food items as

possible and validate their potential by human trials. Due to the rising consumer interest towards non-

synthetic chemical additives, these herbal foods should be supported by government schemes to increase

their marketability, availability and consumption trend.

6. Suggested readings:

Bin Shan, Yi-Zhong Cai, John D. Brooks, and Harold Corke. (2011) Potential Application of Spice and Herb

Extracts as Natural Preservatives in Cheese. Journal of Medicinal Food, 14 284-290.

Flora J S (2009) Structural, chemical and biological aspects of antioxidants for strategies against metal and metalloid

exposure, Oxidative Medicine and Cellular Longevity, 2(4): 191-206.

Halliwell B, & Gutteridge J M C (1999) Free radicals in biology and medicine (3rd ed.). Oxford University Press.

Maxell S RJ (1995) Prospects for the use of anti-oxidants therapies, Drugs, 49 345-361.

Thakur, R. (2013). Study of antioxidant, antibacterial and anti-inflammatory activity of cinnamon

(Cinamomumtamala), ginger (Zingiberofficinale) and turmeric (Curcuma longa). American Journal of Life

Sciences, 1(6): 273-277.

Tuba, A., and Ilhami, G. (2008). Antioxidant and radical scavenging properties of curcumin. Journal of Chemico–

Biological Interactions, 174: 27–37.

Vankar P S, Shanker R, Srivastava J and Tiwari V (2006) Change in Antioxidant Activity of Spices- Tumeric and

Ginger on Heat Treatment. Journal Environment of Agriculture and Food Chemistry, 5 1313-1317.

Yadav D, Kumar S, Krishen R and Mohammad M (2013) Turmeric (Curcuma longa L.): A promising spice for

phytochemical and pharmacological activities. International Journal of Green Pharmacy, 7 85-89.

54

Selected Alternative Processes in Dairy and Food Processing

Meena, G.S., Singh, A.K., Borad, S.G. and Parmar, P.T.


1. Introduction

From decades, foods are thermally being processed to enhance their shelf life and also to ensure food

safety. From different thermal processes (pasteurization, sterilization, boiling, canning, drying and others)

only a single or their combination can be employed on the ground of thermal severity to process a

particular food item in dairy or food industry which ultimately governed by desired food form and its

physico-chemical, textural, sensorial, nutritional attributes and microbial quality. Microbial safety of

these processes have established over the years but the destruction of desired nutritional and sensorial

attributes in the treated food as well as consumption of time and energy; environmental protection (i.e.

minimum waste generation) are the remained limiting factors for such processes. Vast difference in the

food habits of ever-growing population and their awareness towards food quality (nutritional, functional,

sensorial or wholesomeness), safety, environment have forced the global scientists and researchers to

develop new or improved food processing technologies. In last decades, several such technologies which

are thermal (Microwave and radio frequency heating, Ohmic heating, High pressure treatment and others)

as well as non-thermal (Pulsed electric field treatment, Pulsed or white light heating, Ultrasound, UV

treatment, irradiation, Osmotic dehydration, Membrane processing etc.) in nature are being evolved and

evaluated as an alternative to the classical traditional processes (Ahmed and Ramaswamy, 2007). Some

of these thermal and non-thermal processes are being discussed in the following section.

2. Irradiation

Irradiation was developed around hundred years ago and presently, it is considered as a well-established,

scientifically proven safe technique being used for food processing. In general, irradiation is a process

accomplished with the help of different radiations (i.e. γ rays, UV rays, X-rays etc.). In this process, target

food is usually exposed to both ionizing (having enough energy to remove the electron from the orbitals)

and non- ionizing (lack in sufficient energy to remove the electron from the orbital) radiations to destroy

micro-organisms, viruses and other insects that might be present in target food without inducing major

chemical changes. This technology is not only helpful in prevention of food products decay through

sterilization of micro-organisms, but also improves both safety and shelf-stability with the retention of

products wholesomeness. Thus, it falls under minimal processing category, globally being used for food,

pharmaceutical and medicinal applications except Europe where it has got least utilization. Currently,

beside food preservation, irradiation is gaining popularity in other applications also viz., cross-linking of

synthetic and biopolymers, elimination of toxic materials including food allergens, carcinogenic volatile,

N-nitrosamines, embryotoxicity of gossypol and modification of proteins etc. Microwave processing that

has become quite popular up to domestic level has been discussed here. Microwaves (MW) are, non-

ionizing type radiation that stands between Radio and Infrared waves and represents one part of the

electromagnetic wave spectrum. The frequency (ᶂ) of microwaves ranges from 300 MHz-300 GHz and

their wavelength (λ) ranges from 1mm-1m. (Meredith, 1998; Hoogenboom et al., 2009). A particular set

of frequencies is allotted for Microwave heating commonly known as ‘ISM’ (Industrial, Scientific and

Medical) applications, namely 915 (896 in the UK) and 2450 MHz. Domestic microwaves are usually

55

operated on 2450 MHz frequency having penetration depth of 3-8 cm. Penetration depth of microwaves

which depends on several factors including composition of food is 8-22 cm at 915 MHz frequency

(Decareau, 1985). It had become quite popular in food processing owing to capability to attain

volumetric heating at higher heating rates that results in substantially reduced cooking time, more uniform

heating, safe handling, easy operation and lower maintenance than conventional heating (Salazar-

González et al., 2012; Zhang et al., 2006). Lesser changes in flavor profile and nutritional qualities of

foods are encountered during their microwave heating than conventional heating particularly during

cooking or reheating of foods as reported by Vadivambal and Jayas, (2010).

In microwave heating, food materials acts as an electrical conductor and when they are placed in

microwave cavity, they absorb as well as dissipate heat under the electromagnetic field which, increases

their temperature. The energy of the microwaves is transferred to the foods being processed at a

molecular level via molecular interaction between electromagnetic field and foods due to the friction that

generated from the rotation of dipole molecules. The main mechanisms behind the heating of foods

during microwave heating are dipole rotation and ionic polarization. Water is one of the important

component of almost all food materials and plays a vital role during their dielectric heating due to its

dipolar nature (Oliveira and Franca, 2002). Water in the food is the primary dipolar component

responsible for the dielectric heating. When food material containing positive and negative molecules or

polar and nonpolar ions subjected to continuously changing electric field at the rate of microwave

frequency, its molecules tries to align themselves with the ever changing field. Ultimately, rapid as well

as “inside out” generation of heat takes place within the food due to molecules internal friction between

molecules. Polarization of ions i.e. back and forth movement of the ionic molecules trying to align

themselves with the oscillating electric field is the another main mechanism for microwave heating of

foods. The physical state (bound or free) of the food molecules as free ions has better microwave

absorption compared to bound ions (Hippel, 1954; Decareau and Peterson, 1986; Ahmed and

Ramaswamy, 2007).

2.1 Application of Microwave in Dairy and Food Processing

2.1.1 Microwave pasteurization and sterilization

Pasteurization and sterilization are the two most important milk preservation techniques usually used for

the eradication of pathogenic microorganisms and spore formers, inactivation of enzymes and total

destruction of yeasts and molds from milk by exposing it to suitable time-temperature combination in

conventional or alternative processing techniques which also results in providing food safety as well as

enhancement of the milk shelf life. Hamid et al., (1969) studied the pasteurization of milk in a microwave

system and after that among the reported several studies, higher number of studies were conducted to

check shelf-life enhancement of pasteurized milk, ability of a microwaves to inactivate milk pathogens, to

check the impact of microwave heating on milk nutrients, its sensorial attributes as well as non-uniform

temperature distribution at the time of microwave treatment (Jyanes, 1975; Sieber et al., 1996). Recently,

Al-Hilphy and Ali (2013), carried out flash pasteurization of cow milk using microwave (type 956

Kenwood) at 1000 W power level. They also studied the chemical (moisture, fat, lactose, ash and

protein), microbial (TPC & E. coli) and thermo physical (specific heat, viscosity, thermal conductivity

and density) characteristics of the treated sample.

Buffler (1993) reported that Microwave sterilization is superior over conventional retorting as it very

short heat-up time. Moreover, Ohlsson (1987) showed that microwave HTST process (128°C and 3

minutes cooking time) produced better products superior compared to the product that were either canned

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(120°C/ 45 minutes) or retorted in foil pouches (125°C/ 13 minutes). The slow adaptation of

microwave sterilization on commercial scale was due to non-uniform heating as well as shortage of

consistent methods to validate commercial thermal processes for food safety. Microwave have large

number of applications in food processing such as cooking, blanching, microwave assisted air, vacuum

and freeze drying etc. A detailed review on ‘microwave food processing’ had recently published by

Chandrasekaran et al., (2013). More details about microwave application in dairy industry as well as its

merits and demerits has been earlier reported by author elsewhere (Meena et al., 2014)

3. Application of Membrane Processing

Membrane processing has got the potential to efficiently deal with separation requirements of milk and

the same has made dairy industry as one of the early adopter and potent consumer of membrane

technology. This process require a particular set of equipment’s and process conditions for the production

of a specific innovative dairy ingredient. With the development of more robust membrane systems like

mineral and hybrid membrane systems, membrane applications in dairy industry is continuously growing.

Application of ultrafiltration in dairy processing (Meena et al., 2015), membrane application in the

development of innovative dairy ingredients (Meena et al., 2015) as well as advances in membrane

processing (Meena et al., 2014) has been already reported by author that can be seen. Moreover, potential

applications of membrane processing in milk and whey processing has been shown briefly in Figure 1.

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Figure 1: Potential applications of membrane processing in milk and whey processing (Adapted

from Lipnizki, 2010).

4. Ohmic heating

In the current competitive situation of food processing sector due to an rapid increasing number of global

consumer that keeps a constant watch on processed food quality as well as the impact of processing

methods on environmental balance motivates the worldwide researchers/scientists to evolve and evaluate

novel processing tools capable to satisfy their customized needs in terms safe, healthy foods without

disturbing the eco-system. Over the decades, a number of alternative thermal as well as non-thermal

processes have been developed to produce foods retaining “wholesomeness” yet the search of other such

novel process is still ongoing. Ohmic heating, microwave as well as the infrared heating, high pressure

processing etc are considered as novel thermal processes while ultrasonication, irradiation, white/ pulsed

light technology, UV rays are falls under the non-thermal novel processes. Barbosa-Cánovas and

Bermúdez-Aguirre (2010) reported that these processes have the potential to provide pasteurized milk

(most common dairy product) that is at par with classical pasteurized milk in terms of nutritional and

sensorial quality attributes and with enhanced shelf-life.

Ohmic heating is one of the novel thermal process which is also known as Joule heating, Electric heating,

electroheating, and electroconductive heating (Vicente et al. 2006). Moreover, recently Varghese et al.,

(2012) reported that this process is one out of different electromagnetic oriented processes like capacitive

dielectric, radiative dielectric, inductive and radiative magnetic heating. The process which, is exclusively

developed to generate as well as dissipate heat within the material (which are suitable for OH, not all)

itself using its own resistance by subjecting it to an alternating current is known as Ohmic heating. Thus,

it is totally differed from other conventional processes which raise the temperature of target food material

through three classical modes of heat transfer i.e. conduction, convection and radiation because the

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generated heat directly transferred to food omitting the need of any solid-liquid interfaces (Knirsch et al.

2010).

4.1 Principle of Ohmic Heating

In simple words, the basic principle of Ohmic heating consists, flow of electric current (AC) through the

suitable (from which electrical current can pass) food material. As per Ohm’s law (V= I×R) the electrical

energy will convert into thermal energy owing to resistance of food material and also get dissipated

volumetrically there itself. Moreover, the electrical device which uses the resistance of the treated

material to heat it is known as Ohmic or Joule heater (Sakr and liu, 2014). Different components of an

Ohmic heater and its block diagram of this process is shown in Figure 2 while, the relationship between

electrical conductivity and ohmic heating of different foods are given in Table 1.

4.2 Important Terminology Related to Ohmic Heating

4.2.1 Electrical Conductivity (σ)

It is the main parameter that enables the Ohmic heating to occur and also decides the rate of heating in it.

It is measured by the quantity of electricity transferred across a unit area, per unit potential gradient and

per unit time. The electrical conductivity of any substance is typically given by the sum of the electrical

conductivity of individual ions, molar equivalent concentrations of individual ions and molar equivalent

conductivity (Robinson and Stokes, 1959). Ohmic heating is only possible in range 0.01-10 S/m σ while,

it works optimally in the range 0.1-5 S/m of σ. It is the ratio of current density (J) and electrical field

strength (E) and represented as Siemens per meter (S/m) in SI units. It can be calculated by following

formula (Zell et al., 2009).

……....1

Where A is the cross -section area of the material in the heating cell (m2), L- gap between two electrodes

(M), I – electric current (AC) passing through the material (A), V- voltage across the material (V).

4.2.2 Heating Power

The energy (P) given to the ohmic heating system to prescribed temperature are calculated by using the

current (I) and voltage (ΔV) values during heating time (Δt) as reported by Icier and Ilicali, (2005).

4.2.3 Heating Rate

Due to the flow of electrical current through the heating sample, a sensible heat is generated causing the

temperature of the sample rise from Ti (initial) to Tf(final), the amount of heat given to the system can be

calculate from the following equation as reported by Ghnimi et al., (2008):

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59

Figure 2: Batch Ohmic Heater© (Meena et al., 2014)

Table 1 Relationship between electrical conductivity and ohmic heating of different foods.

Products σ (S/m) Ohmic heating

Condiments, eggs, yoghurts, milk desserts, fruit juices,

wine, gelatine, hydrocolloids, etc.

> 0.05 Good

Margarine, marmalade, powders, etc. 0.005 < σ < 0.05 Low

Frozen foods, foam, fat, syrup, liquor, etc. < 0.005 Poor

Source: Goullieux and Pain, 2005

4.2.4 Energy Efficiency

Nguyen et al., (2013) defined energy efficiency in OH as

4.2.5 Merits and limitations of Ohmic heating

Merits and limitation of OH system has been recently reported by Sakr and liu, (2014) and shown in

following Table 2.

60

Merits demerits Suggestions for betterment

Very quickly achieve desired

temperature

Lack of generalized

information

Develop predictive, determinable

and reliable models of ohmic heating

patterns

Rapid uniform heating of liquid

with faster heating rates

Requested adjustment

according to the conductivity

of the dairy liquid

Further research should be

conducted to develop a reliable

Feedback control to adjust the supply

power according to the conductivity

change of the dairy liquid

Reduced problems of surface

fouling

Narrow frequency band Developing real-time temperature

monitoring techniques for locating

cold-spots and overheated regions

during ohmic heating

No residual heat transfer after

shut off of the current

Difficult to monitor and

control

Developing of adequate safety and

quality- assurance protocols in order

to commercialization ohmic heating

technology

Low maintenance costs and high

energy conversion efficiencies

Complex coupling between

temperature and electrical

filed distribution

Instant on -off facility

Reduced maintenance costs

because the lack of moving parts

A quiet environmentally

friendly system

Reducing the risk of fouling on

heat transfer surface

Source: Sakr and liu, (2014).

4.3 Applications of Ohmic heating in dairy industry

Recently, Lien et al., (2014) investigated the suitability of ohmic heating to heat soya milk (10 ˚Brix) up

to 90˚C for tofu manufacture. They observed that as the result of voltage increase, rise in temperature of

soya milk was ranged from 1.46-3.82˚C/min. this research group concluded that OH is an efficient and

convenient technique for tofu making. Sun et al., (2008) investigated the efficacy ohmic heating on

microorganism’s destruction in milk and compared with classical heating under equal temperature

history. They sterilized milk samples by both methods and observed that reduction in microbial counts

and D value of OH treatment were significantly lower than classical heating method. Their results

revealed that OH had thermal and non-thermal effects on MO’s due electrical current. Knirsch et al.,

(2010) also proposed that OH induces electroporation of cell membranes. The application of Ohmic

heating in milk pasteurization and sterilization has been investigated by number of researchers. To

compare the inactivation effects of ohmic heating and conventional heating on total plate count, yeasts

and molds, coliforms, E. coli and salmonella, in buffalo milk under identical temperature conditions,

Kumar et al. (2014) carried out trials on heating of buffalo milk using ohmic heater and subsequent

manufacture of paneer from that milk. Buffalo milk was heated from 20°C to 72°C using ohmic heater as

well as conventionally. Then the paneer manufactured conventionally as well as using ohmically treated

milk was evaluated sensorially as well as microbiologically. Paneer manufactured employing direct

acidification process. The sensory evaluation of the revealed that all the sensory attribute score of paneer

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made from ohmically treated milk were significantly higher than paneer manufactured conventionally.

Hardness value was found lower in case of paneer made from ohmically treated milk than that made

conventionally. The microbial quality of ohmically treated milk was superior than raw milk and milk

heated conventionally. The results of this study suggest that ohmic heating technique is more efficient in

microbial inactivation than conventional heating.

Lien et al., (2014) investigated the suitability of ohmic heating to heat soya milk (10 ˚Brix) up to 90˚C for

tofu manufacture. They observed that as the result of voltage increase, rise in temperature of soya milk

was ranged from 1.46-3.82˚C/min. this research group concluded that OH is an efficient and convenient

technique for tofu making. Milk was pasteurized using OH at the early 20th century (Quarini 1995).

4.4 Selective applications of Ohmic Heating in dairy processing:

Several researchers had reported different exists applications of Ohmic heating is presently being used for

blanching, evaporation, dehydration, fermentation, extraction, sterilization, sterization and heating of

foods to serving temperatures as well as for space foods (Knirsch et al., 2010).

OH had great scope in in dairy processing to replace traditional sterilization/canning/ retorting as it can

aseptically process different liquid products like milk, lassi, butter milk, condensed and evaporated milks

etc because they fall in the prescribed electrical conductivity range. Ohmic heating is well known to

process foods containing particulates and fragile as well as viscous products. It has wide potential in the

manufacture of traditional dairy products like Kheer, Payasams and Basundi. Inactivation kinetics of

different milk enzymes by Ohmic heating still awaited. Investigation to accesses the suitability Ohmic

heating in fermentation process should be taken more vigorously as it is already reported that OH had

potential advantages during fermentation process which is the main process to produce several fermented

Indian dairy products like curd/yoghurt, lassi, Shrikhand etc. It has been already established by Loghavi et

al., (2009) that due to electroporation carried out by OH, is helpful in faster and more efficient nutrient

transport to the interior of the cell and reduced the lag phase of the fermentation process of L. acidophilus.

This particular application must be studied for different dairy starters and probiotic cultures. Finally the

pasteurization as well as sterilization of dairy fluids can be studied by paying attention particularly

towards the destruction of minor milk components in OH in comparison traditional heating methods. Till

now, systematic and scientific evaluations of the series of changes that occurs in pasteurization/

sterilization of buffalo milk needs to be studied.


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Preservation, 2nd

Ed. CRC Press, Taylor & Francis Group, New York.

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Irradiation, Microwave, Radio Frequency, Ohmic Heating, Ultraviolet Light and Bacteriocins. In: Improving the

Safety and Quality of Milk. Volume 1: Milk production and processing. 420-450.

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International, 52 243–26.

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Milk Genomics: An Approach for Enhancing the Nutritional and Therapeutic Values of

Milk

Suneel Kumar Onteru and Dheer Singh

Animal Biochemistry Division

1. Milk genomics

Milk genomics deals with the characterization of genes, genetic variations, epigenetic changes and gene

products, including proteins and others (e.g., miRNA), that are responsible for milk yield and

composition, which are the key factors for defining milk nutritive value as well as its technolgical

processability to various milk products (Figure 1).

Figure 1. A broad view of milk genomics

2. Need for milk genomics

Inflammation, a silent killer, is one the key factors underlying the major non-communicable diseases like

cancers, cardiovascular, respiratory, digestive and metabolic diseases such as diabetes in the current

world. Milk is a gift from the mother nature to combat such silent killer due to its composition comprising

of proteins, peptides, lactose, vitamins, minerals and oligosacchairdes that act as growth factors, toxin

binding factors, antimicrobial factors, immune regulatory factors and prebiotics (German et al., 2006).

With its elegant ingredients in optimal proportions, milk can prevent several diseases. For instance, milk

poly unsaturated fatty acids (PUFA) can combat inflammation, which is an underlying factor all non

communicable diseases as mentioned above. Malnutrition and infections can be prevented by milk

because of its high bioavailable protein, peptides, essential amino acids, Zn and antimicrobial factors.

Similarly, milk vitamins such as B12, A and D can prevent arthritis, night blindness and anemia,

respectively. More importantly, milk CLA, CLA + probiotics + calcium, calcium + vitamin D + trans-

oleic acid, and oleic acid + selenium can prevent cancer, obesity, type II diabetes and cardiovascular

diseases, respectively (Hung et al., 2007; www.milkfacts.info ). In addition to these health promoting

http://www.milkfacts.info/

64

factors, a caution is needed for dairy products usage due to its other components like allergic proteins,

saturated fats and fatty acids.

It has been known that a mutation in the lactase gene in ancient human population drove the Nature’s

milk revolution and dairy industry (Curry, 2013). According to the OECD-FAO agricultural outlook

2013-2022 highlights, the world milk and milk products production and consumption are projected to

drastically be increased 40% more than the current situation with a share of 51% of dairy products

production by developing countries. With this scenario, milk is providing an opportunity to prevent the

growing non-communicable diseases effectively. As the genes of the production animals regulate milk

composition, genomics is one of the ways to make the milk with efficient helath promoting composition

for addressing the world health issues and projected growing dairy industry.

3. Concept of designer milk with nutritional and therapeutic values

Designer milk is a kind of functional milk targeting specific public need with elegant milk composition

and it is produced directly from animals. Functional milk is the milk that provides health benefits beyond

what regular milk provides, when the milk is consumed as part of a varied diet and on a regular basis at

effective levels. The basic difference between functional milk and the regular milk is in the milk

composition. Some of health benefits of few milk ingredients are shown in Table 1. Functional milk can

be obtained by fortification, transgenetic and non-transgenetic approaches. If the functional milk is

obtained by transgenetic and non-transgenetic approaches directly from animals, it can be called as

designer milk. For instance, milk with low lactose can be designed milk for lactose intolerant people.

Similarly, modification of milk protein composition can change the milk processing attributes.

Table 1. Health benefits of few milk ingredients

Milk Ingredient Health benefits

Proteins Essential amino acids, bioactive proteins & peptides, enhanced

bioavailability

Few saturated fatty acids Anti-cancerous (C4:0), anti-inflammatory (C10:0; C12:0),

Anti-bacterial and antiviral (C8:0, C10:0, C12:0)

Mono unsaturated fatty acids Decreases plasma cholesterol, LDL cholesterol and triacylglycerol

(Oleic acid)

Omega -3 fatty acids Anti cancerous, anti inflammatory and anti stress effects (CLA)

Oligosaccharides Prebiotics

Minerals Bone development and weight control (Ca); Prevent Asthma in elders

(Mg); Better immunity ( Zn)

Vitamins Vision & cell differentiation (Vit. A); Antioxidants (Vit. A & E); Anti

anemic (Vit. B12 & Folic acid) & Anti-arbifalvinosi (Riboflavin)

4. Genetic Selection, a non-transgenic tool for designer milk

Genetic selection of dairy animals is a way to enhance the production of functional milk with better milk

composition. The basic requirements for genetic selection of animals for better milk composition are the

information on variability in milk composition, heritability of milk ingredients, chromosomal regions

(QTLs) that explain the variation in phenotypes, breeding strategies and breeding response. The variation

in milk composition could be due to genetic and epigenetic factors. The genetic factors might be species

(e.g., High content of antimicrobial peptides in Kangaroo milk than other species), breed (e.g., high

content of A2 milk in Indian breeds than exotic breeds), and within the breed (e.g., vitamin B12 variation

in Dutch HF cows). The epigenetic factors include environment, nutrition and state of lactation. Several

studies indicated that milk constituents have moderate to high heritabilities, which are further providing

65

clues that genetic selection can improve the milk composition traits. So far, a total of 1907 quantitative

trait loci (QTL) for milk composition traits have been deposited in AnimalQTLdb. Among them, 823,

648, 4 and 10 QTL represent milk protein, fat, processing traits and other milk compositional traits,

respectively.

Genome wide association studies established that the QTL containing the DGAT1 (Diacylglycerol O-acyl

transferase -1) gene could explain at least 18%, 14% and 51% variance in sire daughter yield deviations

of milk yield, protein and fat percentages, respectively (Pimental et al., 2011). This QTL could also

explain nearly 62% of genetic variance in C18:1 mono unsaturated fatty acids. Similarly, a possible

quantitative trait nucleotide (QTN) in lactalbumin-A gene (LALBA_g.242T>C) was identified for milk

protein in Churra ewes (Garcia-Gamaz et al., 2012). The genetic markers like single nucleotide

polymorphisms (SNPs) at a QTL on bovine chromosome 6 could explain 96% variation in milk β-casein

content. Similarly, the SNPs at a QTL on bovine chromosome 11 could explain nearly 100% of genetic

variation casein index in cow milk (Schopen et al., 2011). A total of 68 SNPs on 16 bovine chromosomes

were identified to be associated with vitamin B12 content in cow milk. The SNPs in such QTL are great

tools for marker-assisted selection of cows for better milk composition especially milk protein and fat.

Such genomic studies are very limited to establish variability and heritability in milk minerals and

oligosaccharides.

In India, the number of publications in Pubmed on genes related to milk protein, fat and carbohydrates are

less than 200. This condition indicates that the current research has to be much focused on genes and

genetic selection for improving the milk composition of indigenous cattle and buffaloes to provide

functional milk for large Indian populations. To achieve such goal, our future research should be directed

towards (i) phenotyping of milk composition by advanced technologies in Indian dairy breeds; (ii)

Animal pedigree, feeding and other related data recording; (iii) Genotyping of dairy animals for reported

candidate genes or genome wide genetic markers; (iv) Association analyses by designing the novel

statistical models for Indian livestock production systems; (v) Selection of dairy animals by associated

genetic markers for improvement of specific milk ingredient; (vi) Effective breeding strategies; (vii)

Evaluation of selection response; (viii) Designing new breeding policies for functional milk production.

5. Milk Epigenetics, another non-transgenic approach for designed milk

Milk genomics not only deals with genetic variation at DNA level but also with chemical changes

occurring on top of the DNA and its associated proteins. But studies on these lines are very scanty. It is

well known that age, stage of lactation, feeding regimen, completeness of milking, disease, milking

frequency and disease affect the milk production and composition. For example, two times milking could

cause less methylation of lactation genes than one time milking in mammary gland and promote more

milk yield than one time milking scenario (Singh et al., 2010). It was observed that DNA-re-methylation

around a STAT5- binding enhancer site in the αS1-casein promoter could shutdown of αS1-casein

synthesis abruptly during acute mastitis (Vanselow et al., 2006). Such epigenetic studies exploring the

molecular mechanisms would not only help to understand the animal physiology at different conditions,

but also assist the management of dairy production systems in an effective manner to obtain more milk

yield with better composition.

6. Scope of milk genomics

By exploring the genes, genetic variations, epigenetic changes and gene products, including proteins and

others (e.g., miRNA) responsible for milk yield and composition, milk genomics would be the future area

for the benefits towards dairy industry and consumers as illustrtated in Figure 2.

66

Figure 2. The scope of milk genomics

Figure 3. Flowchart for designer milk production

67

7. Selected Reading

Bouwman et al., (2011) BMC Genetics 12: 43

Curry A (2013). Nature. 500: 20–22

Garcia-Gamaz et al., (2012). PloS One 7: 47782.

German et al., (2006). Trends Food Science and Technology 17: 656-661

Haug et al., (2007). Lipids in Health and Disease 6: 25.

Melzer et al., (2013). PLoS ONE 8: 70256

Pimental et al., (2011). Frontiers in Genetics 2: Article 19.

Rutten et al., (2013). PloS One 8: 62382

Schopen et al., (2011). Journal of Dairy Science 94: 3148–3158.

Singh et al., (2010) Journal of Mammary Gland Biology and Neoplasia 15:101–112.

Vanselow et al., (2006) Journal of Molecular Endocrinology. 37:463-477.

http://jme.endocrinology-journals.org/

68

Protein Based Fat Replacers: Techno Chemical Aspects and their Applications in Dairy

Foods

Gunvantsinh Rathod, Latha Sabikhi and Sathishkumar M. H.


1. Introduction

As a food component, fat contributes key sensory and physiological benefits contributing flavour,

mouthfeel, taste, and aroma/odour (Lucca and Tepper, 1994; Mistry, 2001; Sampaio, 2004). It also

contributes to creaminess, appearance, palatability, texture and glossiness of foods and increases the

feeling of satiety during meals (Romanchik-Cerpovicz, 2002; Sipahioglu, 1999). Fat can also carry

lipophilic flavour compounds, act as a precursor for flavour development (e.g., by lipolysis or frying),

and helps to stabilize flavour (Romeih, 2002; Tamime, 1999).

2. Fat Replacer:

A fat replacer is an ingredient that can be used to provide some or all the functions of fat but

contributing fewer calories. Fat replacers need to be able to replicate all or some of the functional

properties of fat in a fat modified food (Schwenk and Guthrie, 1997).

2.1 Types of fat replacer:

They are either fat substitutes or mimetic. Fat substitutes are lipid like substance intended to replace

fat on mass to mass basis, while fat mimetics are protein or carbohydrate ingredients which function

by imitating the physical, textural, mouth feel and organoleptic properties of true fats (Owusu-

apenten, 2005).

Fig. 1. Classification of fat replacers (Owusu-apenten, 2005)

3. Protein Based Fat mimetics

A contribution of protein in fat replacement is determined by the degree of denaturation, which

influences flavour, as well as the protein solubility, gelling properties, and temperature stability.

Proteins are important whipping agents, emulsion stabilizers, and dough strengtheners. Several fat

mimetics are derived from a variety of protein sources, including egg, milk, whey, gelatine, soy,

wheat gluten and corn zein. Some of these products are microparticulated to form microscopic,

coagulated, round, deformable particles that mimic the texture and mouthfeel of natural fats and oils.

Protein-based fat mimetics are commonly used in margarines, butter, cheese, dairy products, sour

cream, salad dressings, mayonnaise containing products, soups, sauces, baked goods, and frozen

desserts. These substances generally give a better mouthfeel than do carbohydrate-based counterparts.

However, similar to carbohydrate-based substances, protein-based fat mimetics cannot be used for

69

frying. Typical examples of various protein-based fat mimetics used in foods and their applications

are reported in Table.

Type of fat replacer Commercial names Applications

Microparticulated

Protein

Simplesse® Dairy products (ice cream, butter, sour cream,

cheese, yogurt), Baked goods, milk/dairy

products, salad dressings, frozen desserts,

mayonnaise type products, margarine type

products, coffee creamer, soups sauces

Modified Whey

Protein Concentrate

Dairy Lo™ Dairy Products, mayonnaise-type products,

baked goods, frostings, salad dressing

Others K-Blazer®,

ULTRA-BAKETM

,

ULTRA-FREEZETM

,

Lita®, Trailblazer

Frozen desserts, baked goods, spreads, butter

salad, dressing

3.1 Simplesse®:

It is manufactured from whey protein concentrate by a patented micro-particulation process. In this

process they undergo heating and blending, egg protein and milk protein are combined and formed

into minute particles that are 1–1.5 mm in size. These particles are spherical and smooth, which

allows the mouth to perceive them as fat. The product was introduced in 1988 by the NutraSweet

Corporation which is a subsidiary of Monsanto Corporation (St. Louis, Missouri), and is currently

marketed by CP Kelco US, Inc. (Wilmington, Delaware). Simplesse® received FDA GRAS status in

1990 and is approved for use as a thickener or texturizer in ice creams and other frozen dessert

products. Up to one-third of fat can be replaced in frozen foods. This product is also suitable for use in

yogurt, cheese spreads, cream cheese, and sour cream as well as oil-based products such as salad

dressings, mayonnaise, and margarine. The caloric value of Simplesse® is 1–2 kcal/g. It provides fat-

like creaminess. However, similar to other proteins, it tends to mask flavour. As it is made from

proteins, it cannot be used in foods that require high-temperature applications such as frying or

baking. When it is heated, protein gel and the texture effects are lost. Products containing Simplesse®

may not be suitable for people on protein restricted diets. People who are allergic to milk proteins or

egg proteins may have an allergic reaction to this product.

3.2 Dairy Lo™:

Modified whey protein concentrate (Dairy-Lo), a GRAS substance, is manufactured from high-quality

whey protein concentrate. This product contributes only 4 kcal/g. Modified whey protein helps

improve texture, flavour, and stability of low-fat foods. It is typically used in sour cream, frozen dairy

desserts, cheese, baked goods, yogurts, dips, and sauces. Its ability to prevent shrinkage and iciness in

frozen foods makes it especially desirable as a fat replacement ingredient in those products.

3.3 Other protein based fat mimetics:

Other than Simplesse® and Dairy Lo™, there are other recently developed fat replacer: Trailblazer

(Kraft general foods, Glenview, IL) derived from egg white, whey protein and xanthan gum and LITA

from Corn Zein.

4. Application in dairy foods

Romeiha et al., 2002 has prepared low fat cheese using Simplesse® and they found that Simplesse®

had a marked improving effect on cheese appearance, the product was rated as a harder cheese than its

full-fat counterpart. Yazici and Akgun, 2004 has used Simplesse® and Dairy Lo™ in yoghurt and

they found good flavour, appearance and colour score to Dairy Lo™ than Simplesse®. Koca and

Metin 2004 has reported that Simplesse® D-100 and RaftilinesHP can improve the texture and

sensory properties of low-fat fresh kashar cheese. Zoulias et al., 2002 has analysed textural properties

70

of low-fat cookies. They have reported that an increase in polydextrose or Dairytrim content resulted

in harder cookies, while an increase in C-deLight, Simplesse® or Raftiline content has the opposite

effect. So, C deLight, Simplesse® or Raftiline could be used as fat replacers to prepare tenderer low-

fat cookies. They also found that increase in brittleness of the cookies with increase of all fat

mimetics, but a moderate increase was obtained with C-deLight, Simplesse® or Raftiline. Prindiville

et al., has studied effect of whey protein based fat replacer on sensory characteristic of low fat and

non-fat Ice cream. They found that Simplesse® was more similar to milk fat than was Dairy Lo™ in

its effect on brown colour, cocoa flavour, cocoa character, and textural stability but was less similar in

terms of thickness and mouth coating.

5. Selected Reading

Koca N, Metin M (2004)Textural, melting and sensory properties of low-fat fresh kashar cheeses produced by

using fat replacers, International Dairy Journal 14 365–373

Lucca P A and Tepper B J (1994) Fat replacer and the functionality of fat in foods. Trends in Food Science and

Technology 5 12-19.

Mistry V V (2001) Low fat cheese technology. International Dairy Journal 11 413-422.

Owusu-apenten R (2005) Introduction to Food Chemistry, CRC Press, Washington, D.C.

Prindiville E A, Marshall R T and Heymann H (2000) Effect of Milk Fat, Cocoa Butter, and Whey Protein Fat

Replacers on the Sensory Properties of Low fat and Non-fat Chocolate Ice Cream, Journal of Dairy

Science 83 2216–2223.

Romanchik-Cerpovicz J E, Tilmon R W, Baldree K A (2002) Moisture Retention and Consumer

Acceptability of Chocolate Bar Cookies Prepared With Okra Gum as a Fat Ingredient Substitute,

Journal of the American Dietetic Association, 102 1301-1303.

Romeih E A, Michaelidou A, Biliaderis C G, Zerfiridis G K (2002) Low-fat white-brined cheese made from

bovine milk and two commercial fat mimetics: chemical, physical and sensory attributes. International

Dairy Journal 12 525-540.

Romeiha E A, Michaelidoub A, Biliaderisb C G, Zerfiridisb G K (2002) Low-fat white-brined cheese made

from bovine milk and two commercial fat mimetics: chemical, physical and sensory attributes,

International Dairy Journal 12 525–540.

Sampaio G R, Castellucci C M N, Silva M E, Torres E A F S (2004). Effect of fat replacers on the nutritive

value and acceptability of beef frankfurters. Journal of Food Composition and Analysis 18 469-474

Schwenk N E and Guthrie J F (1997) Trends in marketing and usage of fat-modified foods implications for

dietary status and nutrition promotion. Family economics and nutrition review 10 16-32.

Sipahioglu O, Alvarez V B and Solano-Lopez C (1999) Structure, physico-chemical and sensory properties of

feta cheese made with tapioca starch and lecithin as fat mimetics. International Dairy Journal 9 783-789.

Tamime A Y, Muir D D, Shenana M E, Kalab M, Dawood A H (1999) Processed Cheese Analogues

Incorporating Fat-Substitutes 2. Rheology, Sensory Perception of Texture and Microstructure.

Lebensmittel-Wissenschsft und Techologie 32 50-59.

Yazici F, Akgun A (2004) Effect of some protein based fat replacers on physical, chemical, textural, and

sensory properties of strained yoghurt, Journal of Food Engineering 62 245–254.

Zoulias E I, Oreopoulou V, Tzia C (2002)Textural properties of low-fat cookies containing carbohydrate- or

protein-based fat replacers, Journal of Food Engineering 55 337–342

http://journals.ohiolink.edu/ejc/search.cgi?q=authorExact:%22Michaelidou%2C%20Alexandra%22

http://journals.ohiolink.edu/ejc/search.cgi?q=authorExact:%22Biliaderis%2C%20Costas%20G.%22

http://journals.ohiolink.edu/ejc/search.cgi?q=authorExact:%22Zerfiridis%2C%20Gregory%20K.%22

71

Technologies for the Manufacture of Colostrum Powder and its Applications

Sanket Borad and Ashish Kumar Singh


1. Introduction

Colostrum is the first natural secretion by mammal immediately calving. It is the most important food

containing bioactive components like antimicrobial proteins and growth factors present in colostrum.

Colostrum contains immunoglobulins, a principal antimicrobial factor of colostrum, lactoferrin,

lysozyme and lactoperoxidase (Shams, 1994).

Composition of colostrum (Table 1) varies greatly from same milch animal. The best quality of

colostrum, containing the highest proportion of bioactive components, is obtained if milking is carried

out within six hours after parturition. As compared to milk, it is very rich in protein especially

immunoglobulins, growth factors and certain hormones. The basic composition of colostrum is

changed after birth due to maternal reabsorption.

Table 1. Physical properties and chemical composition of Bovine colostrum and milk

Parameter Colostrum (No. of post-partum milkings)

Milk 1 2 3 4

Specific gravity 1.056 1.040 1.035 1.033 1.032

pH 6.17 6.28 6.28 6.38 6.5

Acidity, %LA 0.46 0.28 0.25 0.23 0.14

TS, % 23.9 17.9 14.1 13.9 12.9

Fat, % 6.7 5.4 3.9 4.4 4.0

SNF, % 16.7 12.2 9.8 9.4 8.8

Protein, % 14.0 8.4 5.1 4.2 3.1

Casein, % 4.8 4.3 3.8 3.2 2.5

Total Igs, % 6.0 4.2 2.4 - 0.09

IgG, % 3.2 2.5 1.5 - 0.06

NPN, % of total N 8.0 7.0 8.3 4.1 4.9

Lactose, % 2.7 3.9 4.4 4.6 5.0

Ash, % 1.11 0.95 0.87 0.82 0.74

(Foley and Otterby, 1978 and Tsioulpas et al., 2007)

2. Bioactive components in colostrum 2

Colostrum is reservoir of several bioactive components that can influence immunity, cell growth,

differentiation and other cellular function. Some of the major bioactive protein components of

colostrum are listed in Table 2.

Table 2. Major bioactive protein components of bovine colostrum and milk

Constituent Concentration (g/L)

M.W. (kDa) Colostrum Milk

Casein (αs1-, αs2-, β- and κ-) 26 28 14-22

β-lactoglobulins (β-lg) 8 3.3 18.4

α-lactalbumin (α-la) 3 1.2 14.2

Immunoglobulins (Ig) 20-150 0.5-1.0 150-1000

Glycomacro peptide (GMP) 2.5 1.2 8

Lactoferrin (LF) 1.5 0.1 80

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Lactoperoxidase (LP) 0.02 0.03 78

Lysozyme (LZ) 0.0004 0.0004 14

Serum albumin (BSA) 1.3 0.3 66.3

Growth factor (GF) 50 µg/L-40 mg/L <1 µg/L-2 mg/L 6.4-30

(Park, 2009)

Number of studies have proved that colostrum components stimulate and modulate the immune

system. Bovine colostrum has been found to exert positive effects in the treatment of certain side

effects of Type-2 diabetes, during surgery; in respiratory infections and on bone growth and

development. Safe and legal supplement of colostrum components has been recommended to improve

athletes’ performance with improved immunity against the stresses of intense training and

competition.

2.1 Immune factors

The antimicrobial activity of colostrum is attributed to mostly immunoglobulins; however, minor

immune components such as lactoferrin, lysozyme and lactoperoxidase also contribute to the same

(Donovan and Odle, 1994).

2.1.1 Immunoglobulins (Igs)

The major classes in bovine and human lacteal secretions are IgG, IgM, and IgA. Igs account for 70 –

80% of the total protein content in colostrum, whereas in milk they account for only 1 – 2% of total

protein (Korhonen et al., 2000). The colostral Igs protect against microbial infections and bacterial

toxins, provide systemic immunity (Butler, 1994; McConnell et al., 2001). The concentrations of Igs

in colostrum and milk of cow and buffalo are listed in Table 3.

Table 3. Concentration of Immunoglobulins in colostrum and milks

Igs

Molecular

Weight

(kDa)

Concentration (g/L)

Colostrum Milk

Cow Buffalo Cow Buffalo

IgG1 146-163 15-180 27.72-34.08 0.3-0.6 0.36-1.15

IgG2 146-154 1-3 1.91-2.03 0.06-0.12 0.1-0.19

Total IgG - 20-200 29.75-36.03 0.15-0.8 0.46-1.34

IgA 385-430 1-6 0.18-0.57 0.05-0.1 0.01-0.03

IgM 900 3-9 0.47-0.57 0.04-0.1 0.04

(Elfstrand et al., 2002; Marnila and Korhonen, 2011)

2.1.2 Lactoferrin (LF)

Lactoferrin is an iron-binding glycoprotein (80 kDa) found in colostrum, milk, other body secretions

and cells of most mammalian species. It is present in human colostrum at about 7 g/L level whereas

mature milk contains about only 1 g/L of it.

The antimicrobial activity of LF and its derivatives has been attributed mainly to three mechanisms:

a) Iron - binding from the medium leading to inhibition of bacterial growth;

b) Direct binding of LF to the microbial membrane, especially to lipopolysaccharide in Gram

- negative bacteria, causing fatal structural damages to outer membranes and inhibition of

viral replication and

c) Prevention of microbial attachment to epithelial cells or enterocytes.

2.1.3 Lysozyme (LZ)

Lysozyme, a muramidase enzyme, damages bacterial cell walls by cleaving the β- 1,4-glycosidic bond

in peptidoglycan layer of the bacterial cell wall (Priyadarshini and Kansal, 2002a,b) and thus exerts

73

antimicrobial effects. It is abundantly found in a many secretions such as saliva, tears, human milk,

etc. It is part of the innate immune system.

It is a major component of the whey fraction in human milk (i.e. 0.4 g/L) although its concentration in

bovine milk is approx. 300 times less (0.13 mg/L). Specific activity of buffalo milk LZ is 10 times

that of bovine milk LZ, 5 times that of camel milk and 3 times that of mare’s milk.

2.1.4 Lactoperoxidase (LP)

Lactoperoxidase is secreted from mammary gland into colostrum. It is a glycoprotein that occurs

naturally in colostrum, milk, and many other human and animal secretions. LP is the most abundant

enzyme present in milk. Lactoperoxidase is an effective antimicrobial agent. In in-vitro studies, the

LP system has been shown to be active against a wide range of microorganisms, including bacteria,

viruses, fungi and protozoa (Seifu et al., 2005)

2.2 Growth factors (GF)

EGF (epidermal growth factor), FGF1 and FGF2 (fibroblast growth factor), IGF-I and IGF-II (insulin-

like growth factor), TGF-β1 and TGF-β2 (transforming growth factor) and PDGF (platelet-derived

growth factor) have been identified in bovine mammary secretions. The concentrations of all known

growth factors are highest in colostrum during the first hours after calving and decrease substantially

thereafter. Basically, the growth factors are polypeptides and their molecular masses range between 6

to 30 kDa with amino acid residues varying from 53 (EFG) to about 425 (TGF - β2), respectively. It is

noteworthy that the growth factors present in milk seem to withstand pasteurization and even UHT

heat treatment of milk relatively well (Gauthier et al., 2006).

2.2.1 Epidermal growth factor (EGF)

EGF is a peptide containing 53 amino acids. Human colostrum (200 mg/L) contains 4-6 time higher

EGF than human milk (30–50mg/L). It is also found in many other species, but is not found in

significant amounts in bovine secretions although related molecules have been identified and

characterised. EGF acts as a ‘luminal surveillance peptide’ in the adult gut, readily available to

stimulate the repair process at sites of injury (Playford et al., 1995).

Table 4. Concentration of growth factors for bovine colostrum and milk

Growth factor Concentration (ng/mL)

Colostrum Milk

Epidermal growth factor 4-325 1-150

Insulin-like growth factor – I 100-2000 5-100

Insulin-like growth factor – I 150-600 5-100

Transforming growth factor – β1 10-50 <5

Transforming growth factor – β2 150-1150 10-70

Gauthier et al., 2006

2.2.2 Insulin-like growth factor (IGF)

IGF-I and IGF-II promote cell proliferation and differentiation. They are similar in structure to pro-

insulin and it is possible that they also exert insulin-like effects at high concentrations. Bovine

colostrum contains much higher concentrations of IGF-I than human colostrum (500 mg/L compared

to 18 mg/L) (Vacher and Blum, 1993), with lower concentrations in mature bovine milk (10 mg/L)

(Collier et al., 1991).

74

2.2.3 Transforming growth factor (TGF)

TGF-α is a 50 amino acid molecule that is present in human colostrum and milk at much lower

concentrations (2.2–7.2 mg/L) than EGF (Okada et al., 1991). Systemic administration of TGF-α

stimulates gastrointestinal growth and repair, inhibits acid secretion, stimulates mucosal restitution

after injury and increases gastric mucin concentrations (Barnard et al., 1995). Within the small

intestine and colon, TGF-α expression occurs mainly in the upper (non-proliferative) zones, which

suggests that its physiological role may be to influence differentiation and cell migration rather than

cell proliferation. TGF-α may therefore play a complementary role to that of TGF-β in controlling the

balance between proliferation and differentiation in the intestinal epithelium.

3. Health benefits of colostrum to human being

Colostrum has been known to offer passive protection against enteric pathogens since long. Number

of studies have proven colostrum as an effective in the prevention or treatment of human and animal

diseases, efficacy of which is mainly based on the antimicrobial activity of the specific antibodies and

growth factors present.

Many researchers have found colostrum and its products suitable for treatment and prevention of

certain health related aspects like immunity development, wound healing, T-cell activation, auto-

immune diseases, respiratory diseases, CVDs, etc.

Colostrum also contain low molecular weight proline rich polypeptides (PRPs). Colostrinin, one of

PRPs, was found to exert immunomodulatory properties, maturation and differentiation of murine

thymocytes (Janusz and Lisowski, 1993) and the production of tumour necrosis factor alpha (Inglot et

al., 1996). Leszek (2002) found colostrinin promising natural agent that can prevent the development

of Alzheimer disease, but the mode of action is not clear. Many researchers reported attractive

properties of colostrinin that may prove it worthy pharmacological agent.

4. Processing and preservation of colostrum

Recently processing and preservation of colostrum have been targeted with an aim to utilize its virtue

for human health and nutrition. Refrigeration, freezing, chemicals, microwave vacuum evaporation,

freeze-drying, spray drying and pasteurisation methods etc. have been studied (Chelack et al., 1993;

Johnson et al., 2007; McMartin et al., 2006; Stewart et al., 2005).

Pasteurization (72°C/15 s) resulted in denaturation of 12 to 30% of colostral IgG and increased

viscosity (Meylan et al., 1996; Godden et al., 2003). Elfstrand et al. (2002) observed 28% loss of Igs

in lipid fraction upon centrifugal cream separation, due to flocculation of fat globules by the Igs.

Upon membrane filtration, (MF followed by UF/DF), IgA and IgG2 contents were reduced by 30%

while IgG1 was remained unaffected. 82% of total IgG1 was recovered in the freeze-dried colostrum

whey prepared using UF retentate of colostrum whey. Upon high-speed shearing using Ultra-Turrax

at 20,000 rpm followed by pasteurization at 60°C/30 min did not affect the IgG1, IgG2, IgA and TGF-

β but decreased IGF-1 by 33%.

Fukumoto et al. (1994) supplemented UHT milk with membrane sterilized bovine immunoglobulins

under aseptic condition. It was stable up to 5 months at 4, 25 and 35°C. Pereira et al. (2014) observed

negative linear association between duration UVC treatment and IgG concentration.

4.1 Freezing of colostrum

Freezing of colostrum provides the maximum retention of Ig and nutrients. Lyophilized colostrum

was found to be stable, easy to handle, and suitable for passive immunization (Husu et al., 1993).

Freezing can be successfully used to store colostrum without significant changes in pH, % acidity,

Fat, Total solids, total nitrogen and NPN. Effect of one-time freezing and thawing of colostrum on

IgG is negligible (Arguello et al., 2003).

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4.2 Chemical preservation of colostrum

Chemical preservatives like formalin, sodium benzoate, sodium propionate, sodium acetate, benzoic

acid, sorbitol, gluconic acid lactone (GAL), and combinations thereof were studied during early 1980s

but they resulted in alteration in physico-chemical properties and losses of nutrients.

4.3 Thermal processing

Most studies suggested the Igs are thermo-labile, which are vulnerable to heating above 65°C

therefore commercial pasteurization destroys the activity of Igs (Indyk et al., 2008). Lindstrom et al.

(1994) determined the denaturation temperature of Igs in the range of 62.6 to 67.6°C depending upon

pH. The kinetics of thermal denaturation of Igs are still not clear. D and Z values of denaturation of

immunoglobulins undergoing thermal processing is listed in the table.

Table 5. D and Z values for the thermal denaturation of Igs.

Reference Mainer et al., 1997 Fukumoto et al., 1994

Temp Ig G Ig A Ig M Temp Ig G

62 - - 303.33 62 76418

65 - 2966.67 50 66 8543

69 255 200 10.63 70 702

72 58.17 34.83 3.42 74 81.6

77 10.77 2.93 - 78 12.9

81 3.02 - - 80 6.6

Z value (°C) 6.29 4 5.17 - -

4.4 Freeze-drying

Freeze-drying is used to preserve heat sensitive material. Chelack et al. (1993) reported 10 % losses in

biological activities of Igs upon freeze-drying of colostrum. Das (2013) supplemented ice cream and

dahi with freeze-dried colostrum powder. The disadvantages of freeze-drying are a lengthy process

time, high energy consumption and limited capacity of equipment.

4.5 Spray dried colostrum powder

Chelack et al. (1993) observed 94% retention of the immunolgobulins with 5% loss of bioactivity in

spray dried colostrum while freeze drying and microwave evaporation retained 99% and 93% Igs with

10% and 20% loss of bioactivity respectively. Swaroop (2010) reported 71.24-79.60 and 86.61-92.15

% Igs retention in skim milk colostrum powder and whole colostrum powder, respectively.

Spray dried colostrum powder (SDCP) prepared using optimized processing parameters (feed

temperature of 32°C, feed rate of 160 mL/h, inlet air temperature of 125°C, outlet air temperature of

49°C and drying air flow rate of 0.78 m3/min) contained 3.83 % moisture on dry basis. IgG content of

SDCP decreased progressively upon storage (Yu et al., 2013). Keogh et al., (2003) prepared spray-

dried powder from the conventionally concentrated whole milk UF retentate.

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5. Applications

According to the WHO report 2013, infant mortality rate (per 1000 live births) in India was 43 as

compared to 37 of the global average, whereas under five-mortality rate (per 1000 live births) in India

was 55 as compared to 51 of the global average for the year 2011. The mortality was attributed to

primarily by HIV, diarrhoea, measles, malaria, pneumonia and prematurity, birth asphyxia, neonatal

sepsis conditions, congenital anomalies and other diseases.

Colostrum products can be used as feed supplements or colostrum substitutes to give effective

protection against different enteric diseases in calves and human. The infant mortality rate in India

can be lowered down by feeding colostrum powder fortified milk and other food products.

Immuno-Dynamics, Inc., New life vitamins and APS BioGroup are market leader in the colostrum

based products in USA whereas Lal Nutraceuticals (Delhi), Suboneyo Chemicals Pharmaceuticals (p)

Limited (Jalgaon), The Bangalore Sales Corporation (Bangalore), Avni Food Products (Mahesana)

and many other suppliers are available that can provide range of colostrum powder and other

products.

6. Conclusion

There are increasing concern over nutraceuticals and colostrum is one of the potent reservoir of the

bioactive and immune factors. In order to preserve the immunoprophylactic or therapeutic potential of

immunoglobulins of colostrum, colostrum must be stored in form of dried powder. Colostrum based

products are, thus, having great market demand but they are available in very limited quantity.

77

Advances in Ice Cream and Frozen Desserts

Gadsingh Shankar Prakash, Yogesh Khetra and S.K. Kanawjia


1. Introduction

According to FSSR (2011), Ice-cream means the product obtained by freezing a pasteurized mix prepared

from milk and/ or other products derived from milk with the addition of nutritive sweetening agents e.g.

Sugar, Dextrose, Fructose, Liquid Glucose, Dried liquid glucose, maltodextrin, high maltose corn syrup,

honey, fruit and fruit products, eggs and egg products, coffee, cocoa, ginger and nuts. It may also contain

Chocolate, and bakery products such as Cake, or Cookies as a separate layer and / or coating. It may be

frozen hard or frozen to a soft consistency. It shall be free from artificial sweetener. It shall have pleasant

taste and smell fee from off flavor and rancidity.

Ice-cream is popular in almost all of the age groups but is highly acceptable by children and adolescents.

Moreover, due to recent developments ice-cream industry has become a profitable industry. There are two

main steps in ice cream production. In the first step a mix is prepared, which is aerated and frozen in the

second one. Ice cream and related products are popular throughout the world, because of several

characteristics such as partial freezing, cooling, and refreshing sensation when the product is consumed,

its sweet taste, and the lack of a preconditioning aroma. Today, more than 200 types of ice-cream are

available in the market.

According to FSSR (2011), frozen dessert means the product obtained by freezing a pasteurised mix

prepared with milk fat and / or edible vegetable oils and fat having a melting point of not more than 37.0

degree C in combination and milk protein alone or in combination / or vegetable protein products singly

or in combination with the addition of nutritive sweetening agents e.g. sugar, dextrose, fructose, liquid

glucose, dried liquid glucose, maltodextrin, high maltose corn syrup, honey, fruit and fruit products, eggs

and egg products coffee, cocoa, ginger, and nuts. It may also contain chocolate, cake or cookies as a

separate layer or coating. It may be frozen hard or frozen to a soft consistency. It shall be free from

artificial sweetener. It shall have pleasant taste and flavor free from off flavor and rancidity.

Ice-cream is a member of family of frozen dessert. The only difference between ice-cream and frozen

dessert is ice creams contain at least 10 percent milk fat and 6 percent non- fat milk solids. Anything less

than those are considered to be different frozen desserts.

2. Advances in Ice cream and Frozen Dessert

2.1 Probiotic Ice cream

Probiotics can be incorporated into ice cream either in free or microencapsulated form. In the first case,

probiotics can be supplied by either blending an acidified/fermented milk base (such as probiotic yogurt,

acidified milk, or cream) with the ice cream mix base at the onset of production, or by direct inoculation

of the ice cream mix with a single or a symbiotic culture starter prior to the whipping-freezing step

(Tamime and Robinson 2007; Soukoulis and Tzia 2008). Direct inoculation into the final ice cream mix

might allow none, partial, or full fermentation depending on the flavor–texture quality characteristics

required (Soukoulis and Tzia 2008). On the other hand, using microencapsulated probiotic bacteria, that

is, in biopolymer cross-linked or spray/freeze-dried matrices facilitates the manufacturing process (no

78

need for cultured milk base preparation). Employing microencapsulated probiotics for the production of

functional ice cream has gained much attention during the last few several years due to the versatility of

the method, the prolonged shelf life attained for both microcapsules and ice cream, and the minimized

impact of the carrier material on the sensory, texture, and structural aspects of the finished product

(Mohammadi and others 2011).

Adding probiotics without any preacidification prior to freezing would not be expected to impact flavor-

taste characteristics of the final product, as probiotic cells would not exert any remarkable metabolic

activity leading to the formation of volatile and nonvolatile flavor compounds. On the other hand,

incorporating probiotics into partially/fully prefermented ice cream base, or immobilized within

biopolymers, can improve the textural and sensory properties of ice cream. Specifically, increase of mix

viscosity, enhancement of melting resistance, development of peculiar organoleptic properties such as

refreshing and pleasantly sour flavor, improved body and controlled iciness, and ice crystal induced

grittiness have been reported.

2.2 Synbiotic Ice cream

Synbiotic are the combination of probiotic and prebiotic. Probiotics such as lactobacilli and

bifidobacteria and prebiotics such as inulin, soybeans, raw oats, unrefined wheat or barley can be used to

produce synbiotic type of ice cream with acceptable quality. Generally, prebiotics are added during the

early stage of preparation of mix. Probiotics are added just before the freezing.

2.3 Ice cream with insoluble fiber

Enrichment of ice cream with insoluble fiber has been investigated in a series of studies to attempt

improving its rheological properties, storage stability, and melting resistance, but also in order to provide

health benefits. The addition of DF (oat, wheat, and apple) with a high content of insoluble matter (45%

to 93% w/w) into ice cream enhanced macroviscosity and induced a significant elevation of the glass

transition and melting point of the frozen systems (Soukoulis and others 2009). The cryoprotective effects

of oat and wheat fiber were attributed to their ability to retain high amounts of water, leading to hindered

mobility of the water molecules in the freeze-concentrated serum.

In a recent study, deMoreas Crizel and others (2013) have investigated the use of orange peel and orange

peel-pulp-seed-isolated fiber (rich in soluble and insoluble fiber and total phenolics) as a potential fat

replacer. The authors reported that incorporating both orange fiber types exerted a fat-mimetic function,

allowing a 70% fat reduction (from 18 to 5 g/100 g in the finished product), with no significant

modification of color, odor, and texture despite a slight impairment of the flavor and overall acceptability

of the low-fat formulation. Dervisoglu (2006) evaluated the feasibility of using food industry by-products

rich in insoluble DF, such as citrus fiber. Citrus fiber as an individual stabilizing agent (0.4% to 1.2%

w/w) did not significantly impact viscosity development and air incorporation. However, increased citrus

fiber content was associated with a remarkable improvement of ice cream melting resistance.

2.4 Ice cream with low GI

Polyols such as maltitol, sorbitol, xylitol, mannitol, and isomalt have been implemented in the

manufacture of low or non-sugar ice creams. Among these maltitol has been identified as a very efficient

ingredient for lowering the GI and the sugar content of ice cream without compromising texture, flavor,

or taste. O¨ zdemir and others (2003) developed an ice cream product suitable for diabetic patients based

exclusively on sorbitol or maltitol. The authors demonstrated that blood glucose concentration was

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reduced from 381 mg/100 g (sucrose-based product) to 104 and 108 mg/100 g in the case of sorbitol- or

maltitol-based analogs, respectively.

2.5 Ice cream with natural antioxidants

Incorporation of fruit based products (such as pomegranate peel extract, grape seed extract, peppermint

essential oil, kiwi fruit juice), agricultural waste product (such as molasses and pomace), herbal or green

tea extract into ice cream may increase the phenolic content (and thereby antioxidant activity) in final

product. Most of these natural products produce ice cream having very similar characteristics to that of

regular ice cream.

2.6 Frozen Yogurt with rice bran oil

Frozen yogurt (FY) is a dessert characterized by having the textural properties of ice cream combined

with the acidic taste of yogurt. It can be fortified with rice bran oil without change in sensorial and

textural properties as compared to regular product. But the melting resistance of such product is more

compared to traditional product.

2.7 Probiotic Ice cream from goat milk

Ice cream manufactured using goat milk is incorporated with Bifidobacterium animalis subsp. Lactis. The

addition of B. animalis decreases the pH (p < 0.05), but it has no effect on physicochemical properties,

including overrun and melting behavior of ice cream from goat’s milk. Morever, the goat’s milk ice

cream with B. animalis has good sensory attributes and satisfactory probiotic viability (6e7 log CFU/g).

2.8 Ice cream with kiwi fruit juice

Ice cream prepared using a substantial amount (49%) of juice from kiwifruit with green, gold or red flesh

has potential consumer appeal, through the combination of kiwifruit's unique color, natural flavor and

health-promoting constituents. The aqueous fractions from purees of kiwifruit with green, gold and red

flesh (AFKWs) can be added to a basic low-fat ice cream mix without any commercial flavoring and

coloring agents. Such ice cream has pleasant color and flavor of the kiwifruit used.

3. Advances in analysis of ice cream characteristics

Although the physico-chemical analyses are promising techniques, they are time consuming, require large

amount of sample and a lot of pollutant reagents, and in some cases, obtain the limited knowledge of

characteristics of product. There is an increasing demand of the food industries and research institutes to

have means of measurement allowing the characterization of foods. Use of alternative techniques such as

rheometry, spectroscopy, X-ray, electro-analytical techniques, ultrasound, and laser can be helpful in

determining the interfacial properties, droplet size distribution, the effect of formulation on ice cream

structure, and thermo-mechanical properties, visualization of the 3D microstructure, control of freezing,

detection of microbial concentration, quantification of different populations of protein in an emulsified

system, and so on.

80

Designing Aspects of Newer Dairy Beverages

Sathish Kumar, M.H, Latha Sabikhi and Gunvantsinh Rathod


1. Introduction

India has witnessed radical shift in consumption of non-alcoholic drinks over the recent past.

Increasing middle class population, rapid urbanisation and rising disposable income are some

of the major factors fuelling this growth. Besides this, growing health consciousness among

India’s young population has brought about a revolution in the Indian non-alcoholic drinks

market. It has been seen that cola and other carbonated drinks sales have fallen dramatically

due to rising health concerns and this seems to have benefited the country’s non-carbonated

drinks market such as fruit juices, fruit drinks and dairy drinks. A report from the Associated

Chambers of Commerce and Industry (ASSOCHAM) in India reveals that current market of

non-alcoholic beverages in India is US$1.2billion and it would grow to US$ 2.3 billion by

2015 at the annual growth rate of 20%. The current consumption of non-alcoholic beverages

stands at 175 million litres and estimated to touch 35 billion litres by 2015. It further revealed

that carbonated drinks captured 30% share among non-alcoholic beverages and accounts for

US$370million, fruit drink segment accounts for US$250million and energy drink market

accounts for US$ 100 million in terms of value.

Figure 1: Classification of Dairy Beverages

Dairy Beverages

Milk based

Pasteurized flvoured milk

Sterilized flvoured milk

Fermented dairy drinks

Yoghurt drinks

Fermented whey drinks

Buttermilk Functional beverages

Minerals and Vitamins

fortified drinks

Herbal bioactives

fortifieddrinks

Probiotic dairy beverages

Composite dairy drinks

Fruit and milk drinks

Fruit & whey drinks

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2. Dairy beverages

Milk provides more satiating nutrients than other beverages. Three cups of milk are

recommended daily by American Dietary Guidelines released in 2005. The interest in

developing dairy beverages is rising day-by-day, driven by the market potential for the health

and well-being of the consumers. Fermented dairy beverages make up an important

contribution to the human diet in many countries because fermentation is an inexpensive

technology, which preserves the food, improves its nutritional value and enhances its sensory

properties.

2.1. Flavoured milk

Flavored milk is popular in traditional flavors such as kesar, elaichi and chocolate, recently

innovative flavors including butterscotch, strawberry, vanilla, and thandai are enter into the

market. Flavoured milk is the most attractive choice for children. It has been considered as

highly palatable and nourishing beverage. Processing and packaging technology adapted for

long life flavoured milk widely in India is a) standardization of milk to desired fat and snf

level, b) homogenization, c) addition of SMP (if required to adjust solids level), d) addition of

sugar, colour and flavour, mixing, filling flavoured milk into glass bottles, capping and

followed by retort sterilization. Recently major dairy giants launched the flavoured milk in

sterilizable (retortable) plastic bottles and few producers launched DHA and EPA fortified

flavoured milk in India. Sterilization was shown to induce lipid oxidation in dairy beverages

but at the same time it produces Maillard Reaction Products (MRPs) that provide antioxidant

properties (Giroux et al., 2008). Recently, it has been shown that pre-heated milk protein-

sugar blends provide MRPs in the early stage of sterilization, which efficiently prevents lipid

oxidation of dairy beverages (Giroux et al., 2010).

Figure 2: Direct Liquid Inoculation System

2.2. Probiotic dairy beverages

The development of probiotics in the last two decades has signalled an important advance in

the food industry. The probiotic microorganisms also have been directly incorporated into the

drinks after the heat treatment. The key to the development of this second generation

1. Flow meter - measures base

product flow

2. Ingredient - in aseptic bag

3. Hose and Pump- ingredient is

transferred from the bag via a

hose and peristaltic pump

4. Injection point-where the

ingredient is dosed into the

product

5. Consumption measurement - the

consumption of the ingredient is

measured by a load cell

6. Control panel - recipe control,

accurate dosing and traceability

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probiotic products is the design of special Direct Liquid Inoculation System(Fig. 2) by Tetra

Pak commercially available as ‘Tetra FlexDos’. It allows food producers to add the probiotic

bacteria directly to the beverages after heat treatment and just before they are filled into the

cartons. The innovation is expected to significantly boost the market for the probiotic

beverages, which have so far been restricted by the delicate nature of the ingredient and

concerns over the contamination.

2.3. Yoghurt drinks

Yogurt is a conventional food known for its therapeutic, nutritional, and sensory properties.

One possible method of enhancing those properties further is by creating yogurt drinks.

Hydrocolloids have been widely used in texture stabilization of fermented milk products.

High methoxyl pectin is preferred in acidic milk beverages as a stabilizer. Pectin molecules

interact with casein through calcium ions and prevent their aggregation, sedimentation and

hence serum separation by ionic and steric stabilization in acidic milk beverages (Luceyet

al.,1999). Yogurt drinks are highly desired by consumers due to their healthy, convenient,

and portable characteristics. They are being marketed and modified successfully to fit the

target populations, separate formulations for kids and adults are in the market with varying

sugar and fat level. Yogurt drinks, mainly the low-fat and non-fat varieties (326 KJ/serving),

are increasingly popular among the adult female population. Adding an indigestible

carbohydrate and a prebiotic like inulin to yogurt drinks, which has been linked to improved

colon health, and increased absorption of calcium and minerals increases the marketability of

such products. High protein yoghurt drink containing higher α-lactalbumin suppressed hunger

significantly in comparison to control(Hurselet al., 2010). In a study by Gonzalez et al.,

(2011) reveals that the whole milk based yogurt beverages were liked over the skim milk

yogurt drink. The whole milk beverage containing the prebiotic (fructooligosaccharide) being

the most acceptable. However, symbiotic yoghurt drink (fructooligosaccharide and

Lactobacillus acidophilus) was not preferred due to higher intensities of sour and yeasty

aroma.

2.4. Fermented whey beverages

These beverages are obtained by fermenting lactose in whey to lactic acid by lactic acid

bacteria (LAB) and can be mainly categorized into whey based fermented drink, whey based

fermented carbonated drink and whey based cultured dairy products.

Product Type Characteristics

Rivella Whey based fermented

carbonated drink

35% deproteinated whey serum + water, sugar

and flavor

Surelli Whey based fermented

carbonated drink

Almost same as rivella

Fauna-fit Soft drink type Approx. 85% sweet whey UF

permeate fermented & after second UF mixed

with fruit juice (mango, pineapple, strawberry)

Whey kwas Alcoholic kefir like drink Deproteinated whey inoculated with

thermophillic starter and then treated with

yeast

Servovit Whey based cultured

dairy products

Carbonated product based on whey and

cultured buttermilk

83

Kumiss like

beverage

Whey based fermented

drink

Whey + buttermilk inoculated with culture

comprised of kumiss yeast L. bulgaricus and L.

acidophillus

Milone Whey based fermented

drink (mildly alcoholic

and sweet sour in flavour)

Whey + 1% lactic acid + equal amount of tea,

copptn, remaining liquid is then incubated with

lactose fermenting yeast (0.8% ethanol)

Alcoholic

whey beer

Beer like beverage Whey beer brewed with hops but without

added malt

Malted

whey beer

Beer like beverage Malt and hops both were added to whey and

fermented with bottom yeast

Whey malt

beer

Beer like beverage 50% whey + malt, hop, sugar and coloring

matter (produced with top fermentation)

Whey

nutrient beer

----------- Whey + hops and nutrient salts ( low alcoholic

product )

Whey

champagne

Alcoholic wine like Deproteinated whey inoculated with 0.1%

fresh baker’s yeast + addition of coloring and

flavouring substance

Whevit Alcoholic soft drink Whey is fermented with yeast culture

(Saccharomyces cerevisiae),can be carbonated

and non carbonated

Acidowhey Non carbonated soft drink

type

Whey fermented with lactic acid bacteria and

free from preservative and synthetic color

2.5. Milk and fruit drinks

Milk and fruit drinks have established themselves well in the European market and have

grown in recent years by around 7%. New market opportunities are arising in Europe and

China. The motivation for using dairy and fruit drinks is not just refreshment, but also issues

such as healthy nutrition, functional ingredients, satisfaction and enjoyment. It is being the

common practice to have milk shakes prepared freshly by blending milk and fruit pulpslike

mango, grape etc in India. Now, technological advances are being made to process these

drinks commercially, and to extend their shelf life. Recently, Coca Cola launched ‘Maaza

Milky Delite’ prepared with mango pulp and milk solids in India (ET Bureau Aug. 18, 2010).

The pH control is the key factor contributing for longer shelf life and addition of stabilizer

keeps the proteins in suspension at acidic pH.

Casein micelles in milk (pH 6.7) are generally assumed to stay in the suspended state as the

result of steric repulsive interactions between the micelles. In acidified milk drinks (AMDs),

pH of the product was adjusted to acidic pH range to provide longer shelf life to the product.

As the pH decreases calcium leaves the micelle. Below pH 5, electrical charges are strongly

modified. Acid groups of the casein micelles are neutralized. An aggregation of sub micelles

occurs due to disappearance of the negative charges. Extensive aggregation of casein micelles

occur as the pH of the product brought down to less than 4.6, which would result in a

macroscopic gel. Therefore there is a need for the addition of a stabiliser to avoid the

flocculation of milk proteins and subsequent macroscopic whey separation. And other

requirement of the product is it should be as homogeneous as milk for better acceptability.

A stabiliser act to increase steric repulsion between the casein micelles and also sometimes

need to slightly increase the viscosity of the medium to prevent flocculation of proteins.

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Pectin addition to AMD results in some part of pectin to adsorb on casein micelles and some

part to remain in serum phase. Pectin-coated protein particles are connected both to each

other with pectin–pectin ‘bonds’ as well as to serum pectin without a direct connection to any

protein particle as shown in diagram 2. The pH must be above pI of pectin and below that of

protein.

Fig. 2: Pectin interaction with casein micelle and serum pectin

Homogenization is preferred to reduce protein-pectin particle size. Pressures of 2000 -2500

PSI are ideal to reduce protein particles size for pectin coating. Ideal temperature of

homogenization is at 75 to 80°C for optimum dispersion and smoother texture. Flow diagram

of fruit juice based drink preparation is given below (Fig. 3).

3. Conclusion

Recent consumer trends such as convenience, health and wellness, value for money and

indulgence created huge potential in dairyc beverage market. Manufacture wish to introduce

products in each category to tap the business potential with their brand value. It helps market

players in product differentiation. It motivates researchers to diverge the innovation path to

keep the momentum in market.

85

Fig. 3: Generalised flow diagram of fruit-juice based acidified milk processing

4. Suggested Reading

Beverage Marketing Corporatio.(2010). Soy Beverages in the U.S.

Dalgard C. Nielsen F. Morrow J D Enghusen-Poulsen H, Jojung T, Horder M and M P M de Matt. (2009)

Supplementation with orange and blackcurrant juice, but not vitamin E, improves inflammatory markers in

patients with peripheral arterial disease. British Journal of Nutrition, 101 263-269.

Giroux H J. Acteau G, Sabik H and Britten M (2008) Influence of dissolved gases and heat treatments on the

oxidative degradation of polyunsaturated fatty acids enriched dairy beverage. Journal of Agricultural and

Food Chemistry 56 5710 - 5716.

Giroux H J, Houde J and Britten M (2010) Use of heated milk protein sugar blends as antioxidant in dairy

beverages enriched with linseed oil. LWT - Food Science and Technology 43 1373 – 1378.

Goodman J W, Asplin J R and Goldfarb D S (2008). Effect of two sports drinks on urinary lithogenicity.

Published online by Springer-Verlag. 10 December 2008.

Graham T E and L L Spriet (1995) Metabolic catecholamine and exercise performance responses to various

doses of caffeine.Journal of Applied Physiology.78 867-874.

Hursel R. Lucie van der Zee and Margriet S (2010).Effects of a breakfast yoghurt, with additional total whey

protein or caseinomacropeptidedepleted αlactalbumin enriched whey protein, on diet induced thermogenesis

and appetite suppression. British Journal of Nutrition, 103 775 - 780.

Lucey J A, Tamehana M, Singh H and Munro P A (1999) Stability of model acid milk beverage: effect of pectin

concentration, storage temperature and milk heat treatment. Journal of Texture Studies, 30 305–318.

Ramanathan V K. Chung S J, Giacomini K M and Brett C M (1997).Taurine transport in cultured choroid

plexus.Pharmacological Reseacrh, 14 406–409.

Worthley M I, Prabhu A, De Sciscio P, Schultz C, Sanders P and Willoughby S R (2010) detrimental effects of

energy drink consumption on platelet and endothelial function. The American Journal of Medicine, 123 184–

187.

Milk

Mixing Pectin

Fruit Pulp

pH adjustment

Homogenization

Heat Processing

Packaging







http://www.sciencedirect.com/science/journal/00029343/123/2

86

Technological Challenges and Design Aspects of Vitamin and Mineral Fortified Milk

Sumit Arora, Chitra Gupta and Apurva Sharma

Dairy Chemistry Division

1. Introduction

Micronutrients are nutrients required by humans and other living creatures in small quantities to

orchestrate a whole range of physiological functions, which the organisms itself cannot produce.

Micronutrient deficiencies such as iron deficiency anaemia (IDA), vitamin A deficiency (VAD) and

iodine deficiency disorders (IDD) continue to be significant public health problems; affecting more than

one third of the world's population. Apart from well established deficiency cases of iron, iodine and

vitamin A, emerging evidences have been reported on low plasma levels of zinc, folic acid and vitamin D

as well. Calcium and vitamin D deficiency have been reported to cause increasing fracture risk in Indian

population as well as an increased rate of deficiencies related to vitamin B1, B2, B12 has emerged in recent

past. Good nutritional status of population contributes to the socio-economic development of a nation. A

significant proportion of the World's population suffers from, or at risk of vitamins and minerals

deficiencies, referred as micronutrients. Adequate intake and availability of these dietary essential

vitamins and minerals are closely related to the survival, physical and mental development, good health

and overall well-being of all individuals and populations. More than 2 billion people in the world, suffer

from micronutrient deficiencies caused largely by dietary deficiency of vitamins and minerals. Vitamin D,

vitamin A, calcium, iron and zinc are needed to be given the first preference. Diet-related micronutrient

deficiencies rarely occur in isolation; deficiencies of calcium and vitamin D are often observed in the

same populations. Multiple micronutrient fortification can be more effective in improving nutritional

status than fortification with single micronutrient; therefore, the multiple fortifications of appropriate food

vectors, including milk, are of interest from the nutritional standpoint.

2. Milk and milk products as a suitable vehicle for fortification:

Milk in its natural form is almost unique as a balanced source of man’s dietary need. The various steps in

processing and storage have a measurable impact on some specific nutrients. Milk also provides

convenient and useful medium for addition of certain nutrients to man’s diet and has following benefits:

Milk is centrally processed hence the quality control can be effectively implemented.

Milk and milk products are widely consumed regularly in predictable amounts by people of all age

groups.

Cost is affordable by target population.

Stability and bioavailability of the added micronutrients to the milk remain high.

Milk is nearly a complete food and all nutrients exist in almost fully available form, the

bioavailability of added nutrients remains high.

Addition of fortificants usually caused minimum change in colour, taste and appearance.

Fortification of milk with different nutrients depends upon various factors including levels of milk

consumption and nutritional status of target population. The effect of added nutrients on the functional or

sensory characteristics of milk and stability of the nutrients during processing and storage of milk are the

important criteria which have to be considered before fortification.

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3. Consideration during fortification of milk and milk products:

3.1 Bioavailability of commercial preparation

Bioavailability of different compounds facilitates the selection of the optimal compound.

3.2 Nutrient-nutrient reaction

Interaction among the nutrients and other food components is a key factor in nutrient addition. For

example, vitamin C will improve iron absorption; however, phytate, oxalate, uronic acid and polyphenols

of certain foods inhibit iron and calcium absorption. On the other hand, fortification iron will accelerate

vitamin degradation. Fortification of calcium in milk may interfere with absorption of iron or zinc.

3.3 Nutrient- matrix reaction

The added nutrient should not react with any component of the milk. For example, iron is a pro-oxidant

and can accelerate the development of fat rancidity, destroy some of the vitamins and form colored

products.

3.4 Shelf life & packaging

Many of the fortified milk and milk products may have limited shelf life and thus may need different

types of packaging which can be either oxygen impermeable or opaque to light. This is particularly true

for the fortification of liquid milk with vitamin A, since vitamin A is less stable to light. All the fortified

products require proper labeling on the pack.

3.5 Process consideration

The stability of all the vitamins is well known during various processing conditions and the same

knowledge can be applied while processing the vitamin fortified milk. Addition of calcium to milk

resulted in a new distribution of ions between milk serum and casein micelles, leading to physicochemical

changes in casein micelles and affects the heat stability of milk.

3.6 Cost factor

Cost may be a crucial factor in the manufacture and marketing of fortified milk and milk products.

3.7 Safety factor

There should be sufficient insurance against excessive intake of the fortificant. Unlike water soluble

vitamins, fat soluble vitamins exhibited toxicity at higher concentrations.

4. Fortification of milk and milk products with minerals

Milk is saturated with calcium; hence excess calcium may precipitate out. Stabilisers and emulsifiers are

generally used to maintain calcium in suspension to improve the mouthfeel and appearance of products.

Overload of calcium on the protein is the determining factor for the heat stability of milk. The ratio of

calcium-to-protein should not exceed 40 mg calcium per gram of protein. Another important processing

step involves the careful control of final pH followed by calcium salt addition. Finally, the use of a

calcium sequestering agent must be a controlled addition, dependent upon the actual calcium load of the

system. Upon addition of calcium salt, there is a marked drop in the pH, which will render the system

unstable unless it is corrected to near neutrality by the addition of a food grade base.

Addition of ferrous chloride (FeCl2) and ferric chloride (FeCl3) to skim milk led to a decrease in pH. This

decrease is related to the acidities of iron solutions and to exchange between iron ions and micellar bound

H+. The iron binding to caseins depends on their nature (as1-, as2-, β- and К-CN) and the nature of the iron

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compounds. The binding of iron to caseins induces a neutralisation of their negative charge and also

induces change in their structures.

5. Choice of calcium fortificant

Selecting the right form of a salt for milk fortification is the first hurdle. Ideal source for fortification of

food should be highly absorbable, inexpensive and safe for human consumption. A wide variety of

calcium salts are having GRAS status viz. calcium carbonate, calcium chloride, calcium citrate, calcium

gluconate, calcium hydroxide, calcium lactate, calcium oxide and calcium phosphates (mono-, di- and

tribasic), calcium glycerophosphate, calcium pantothenate, calcium pyrophosphate and calcium sulfate.

Several commercial calcium salts have been used for calcium enrichment of milk/beverages, e.g. calcium

carbonate, calcium chloride, calcium phosphate, tribasic calcium phosphate, calcium citrate malate,

calcium lactate, calcium gluconate, calcium lactate gluconate and natural milk calcium. In general,

organic salts of calcium are more bioavailable than inorganic salts. The choice of calcium fortification

compounds may be made more on technological grounds, since there is little variation in the

bioavailability of the calcium from different compounds. The selection of an appropriate salt for a specific

application is usually based on the consideration of a number of properties associated with the respective

product such as calcium content, solubility, taste, cost and bioavailability. The best suited salt for

fortification should possess a high nutritional value and low interference with the absorption of other

nutrients besides its cost effectiveness and minimal effects on consistency, mouthfeel and taste of the

product.

6. Choice of iron fortificant

Technically, iron is the most challenging micronutrient to be added to foods. Iron compounds that have

the best bioavailability tend to be those which interact most strongly with food constituents to produce

undesirable organoleptic changes. The ideal fortificant for food fortification is one that supplies highly

bioavailable iron, does not diminish the nutritional value of the food vehicle through nutrient oxidation,

does not alter its sensory properties, can be used to fortify solid and liquid foods, is resistant to processing

treatments and is low in cost so that it can be accessible to the whole population.

The selection of iron compound for fortification is important in order to avoid interactions of iron with

food vehicle or the total meal because a minor change in organoleptic characteristics of the food will

result in consumer rejection. Solubility, chemical reactivity, bioavailability and cost are other important

issues when selecting an iron compound. For instance, ferrous sulphate is a highly bioavailable and

relatively inexpensive compound, however, because of its high reactivity; it produces undesirable changes

in some fortified foods. On the other hand, elemental iron (reduced, electrolytic or carbonyl) is also

inexpensive, however, it has been reported to have a low bioavailability depending on particle size and

the food vehicle to be fortified.

Novel microencapsulation technologies render iron fortificants that are more resistant to interaction with

other components in the food vehicles, thus minimising organoleptic changes, increasing shelf life and

maximising consumer acceptance. Most of the iron compounds can be microencapsulated, however, the

most available products are the microencapsulated form of both ferrous sulphate and ferrous fumarate.

The coatings are usually a mixture of phospholipids, polysaccharides, protein or partially hydrogenated

oil. Microencapsulation has little influence on relative bioavailability and the main advantages are fewer

organoleptic changes and a prolonged shelf life of fortified foods. This is a result of the bilayer coating,

protection against the interaction between iron and absorption factors in the fortified foods.

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7. Vitamin fortification of milk and milk products

Under ambient conditions the water soluble vitamin C and vitamins of the B-complex group such as

thiamine, riboflavin, vitamin B6, niacin, pantothenic acid, folic acid, biotin and vitamin B12 are powdered

and thus relatively easy to work with when producing most dairy products. The fat soluble vitamins

which include vitamins A, D, E and K, however, exist either as oil or as crystals, which may cause

processing difficulties during the production of certain types of dairy products.

One of the problem encountered with the vitamins, is their limited stability in presence of heat, humidity

and oxygen. Among the water soluble vitamins, vitamin C, folic acid, vitamin B6 and vitamin B12 are the

less stable. While in the case of fat soluble vitamins vitamin A, D and E are least stable. In order to

improve the stability of these vitamins, a number of different coating technologies have been developed.

One of the most important methods to protect the fat soluble vitamins is microencapsulation, which

results in a highly sophisticated powder, where the vitamin is kept protected from degradation by the

coating material used for the encapsulation. During microencapsulation, the fat soluble vitamins are

brought from the oil or crystal form to a free flowing powder which makes it suitable for handling and

mixing with other dry ingredients.

8. Conclusion:

Fortification ensures a safest method by which manufacturers can deliver health promoting, nutritionally

dense food products. Milk and milk products provide a convenient and useful vehicle for fortification

with micronutrients and also are a part of the daily diet in almost all countries. Dairy products are also

easily targeted for specific consumer audiences, such as females and infants, allowing for the delivery of

category specific functional ingredients. Micronutrient fortification is found to be a promising tool to

address diseases caused by malnutrition. In some countries, optional and mandatory fortifications are in

place. However, fortification has still not gained a foothold in our country. In developed countries,

regulation of fortification is currently receiving more attention than technologies involved.

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Advances in drying of functional dairy foods

P.S. Minz and P. Barnwal

Dairy Engineering Division

1. Introduction

The increasing consumer demand for foods with health benefits has led the food industry to diversify its

products. Most of the foods containing probiotic bacteria are dairy products, although there is a rapidly

growing demand for incorporating probiotics in other segments of the food industry. Drying processes are

a major cause of loss of viability of probiotics, and in the specific case of freeze-drying, the freezing step

causes additional stress on the bacterial cells, making the lipid fraction of the cell membrane more

susceptible to damage during the process (Guergoletto et al., 2012). Although the exact mechanisms of

cell inactivation during drying processes are not yet fully elucidated, it is known that bacterial cells

consist of 70 to 95% water, and its removal poses serious physiological obstacles to the survival of cells

(Santivarangka et al., 2008).

Culturing conditions and the cellular growth phase have great influence on viability during drying, with

the cells in the stationary phase being more resistant than in any of the other stages. For that reason, this is

the stage of development that has been used in different drying processes, such as: spray drying; fluid bed

drying; vacuum drying; as well as freeze drying. When the water is removed at a fast rate, the

microorganism has not the time it needs to adapt itself through genetic expression or by adjusting its

metabolism. An osmotic pressure response can be induced by stress conditions and leads to accumulation

of compatible solutes, as well as to a cross-response with the effects of the other stress conditions. Pre

adaptation to heat induces the heat-shock response of proteins and enzymes, such as chaperones and

proteases. The first help to stabilize the RNA and repair denatured proteins, while the proteases degrade

denatured proteins, with both reactions leading to heat stress resistance (Fu and Chen, 2011). The recent

advances in drying methods for functional dairy foods are discussed as follows:

2. Foam mat drying

Foam-mat drying is a promising new technique in the field of drying aqueous foods. Foam-mat drying

can be successfully employed for dehydration, under relatively mild conditions of heat sensitive foods or

for the foods that are difficult to dry, sticky and viscous in nature without compromising the quality of

end product. Produced foam can be dried easily at faster drying rates (Meena et al., 2014). Foam mat

drying, originally develop by Morgan, is a process in which the liquid or semi food is converted to form

stable foam by cooperation with foaming agents or stabilizing agents. The foam is then spread into a thin

sheet and dried by using hot air at lower temperature compared to other drying techniques such as spray

drying and steam drying. The dehydrated product can be converted into powder later using grinding

process (Chandak and Chivate 1972; Labelle 1984; Srinivasan 1996). The cooperation of air bubbles into

the foam is important and affects to drying rate. Usually, drying rate of foam-mat drying is relatively high

due to the larger surface area exposed to the drying air, resulting in rapid moisture removal (Brydigyr et

al. 1977). The dehydrated powder or flakes has better quality than that of drum dried and spray dried

products because of its honey comb structure and better reconstitution properties (Krasaekoopt and

Bhatia, 2012). This drying process is comparatively simple and inexpensive; however, the foam stability

during drying is very important. If the foam collapses, cellular broken down occurs, resulting in serious

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impairment of the drying process. The foams are stable when low surface tension and high viscosity

occurred at the air/aqueous interface (Cherry and McWatters 1981). There are a lot of factors affecting the

foam characteristics or foam properties include chemical composition of food, type and concentration of

foaming agent, and mixing time (Hart et al. 1967). Yogurt powder was produced by using foam-mat

drying method using two types of foaming agents as methylcellulose and egg albumin (Krasaekoopt and

Bhatia, 2012).

3. Spray freeze drying

The rate-controlling step of freeze drying is almost always the diffusion of water through the solid from

the interior region to the surface. As drying times vary approximately with the square of the sample

thickness, one way to increase drying rates is to reduce the dimensions of the material. This is the basis of

the spray-freeze-drying technique that involves the atomisation of a liquid stream in a manner similar to

spray drying. The spray is then rapidly frozen and the solidified droplets subsequently freeze dried in the

form of a powder. This process is able to work by maintaining a very low dew point of water within the

system, which allows ice to sublime even in the presence of air at atmospheric pressure. Although the

technique was first developed in the 1950s it does not appear to have had widespread use. The most

recent work in this field has been done by using cold dessicated gas to first freeze and then dry the spray

in an integrated fluidised bed at atmospheric pressure. Due to the small particle sizes produced, drying

times of two hours were achieved. A spray-freeze-drying rig has been developed at Loughborough

University, UK, in which the fluidised bed freeze-drying section is capable of operating at reduced

pressures. The equipment is in two parts: a spray freezing chamber and a separate sub-atmospheric

fluidised bed freeze drying unit. Spray frozen particles are first formed by spraying the concentrate

through a heated hydraulic nozzle into a cooled spray chamber (1.5 m high x 0.8 m diameter). The

chamber is previously purged with nitrogen gas from a cylinder before cooling with liquid nitrogen from

a 500 l dewar. The frozen particles are collected from the outlet of the chamber in polystyrene containers.

The spray frozen particles are then loaded into the fluidised bed freeze dryer, which consists of a

polycarbonate cylinder inside a stainless steel vacuum vessel. The top and bottom of the polycarbonate

cylinder are fitted with a fine mesh and a distributor to allow low temperature gas flow through the bed,

whilst maintaining the particles within the drying chamber. The depth of the bed is typically 1 cm, which

yields a product mass of around 10–20 g. The fluidisation gas is provided from the liquid nitrogen dewar

via a trim gas heater and evacuated via a vacuum pump (Stapley and Rielly, 2007).

4. Zeodration

Zeodration is classical vacuum drying one step further with zeolite adsorbers replacing the classical

vapour condensers. During normal vacuum drying both water and aroma molecules are evaporated from

the product and ultimately discarded. However, zeolite will only adsorb water molecules; aroma

molecules are excluded from entering the zeolite pores due to their size. This improved retention of aroma

molecules provides a better product than freeze dried, with improved flavour, colour and aroma. Our

Zeodration drying process has a major advantage over freeze drying. In traditional freeze drying ice

crystals penetrate the cell wall. With Zeodration this shape change is eliminated due to higher processing

temperatures, resulting in less damage to cell walls and greater product stability (Bucher, 2015).

5. Vacuum band drier

vacuum belt drying plants are designed for the continuous charging of liquid or solid products. The dried

granulate is also being discharged continuously and under clean room conditions (GMP). After the drying

process, the products can be milled and customised. Vacuum belt drying can be regarded as one of the

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most economical and gentle drying methods. Due to the vacuum, very low water evaporation

temperatures can be achieved. Aroma, product color and bulk density can specifically be influenced by

the process. Vacuum belt drying plants find their application in the pharmaceutical, food and chemical

industry. Advantages of vacuum belt drying plants are (SPX, 2015).

Short drying time

Minimal loss of aroma

Little loss of product

No oxidation of product

No mechanical stress

Solvent recovery possible

Low product temperatures

Less energy consumption

Completely closed system

Controllable maillard reaction

Instant properties of the dried product

6. Microwave vacuum belt dryers

Using conventional vacuum dryer the product is heated using contact heating within several heating zone.

Heated plates are used as heat transfer medium using pressure water, steam, oil or electrical sources.

Compared to the above mentioned conventional heating systems microwave is the better alternative.

Microwave vacuum belt dryers are used for the continuous and automatic drying of temperature sensitive

products with low thermal conductivity such as herbal extracts, food, pharmaceutical and chemical

products. The drying process is adapted to the desired dry product quality such as final solids content,

solubility, density and others. The feed of solids is effected by means of a suitably designed feeding

device with attached and product orientated metering and distribution set-up in the dryer. When feeding

wet product a metering pump and an oscillating feeder are allocated. The movement of this feeder is

adjustable during operation. Therefore an easy optimising is possible. Pump able products pass normally

a high viscose and sticky phase during drying. Towards the end of the drying and influenced by the steam

bubbles a dry cake is formed. This cake is brittle and is broken at the end of the belt and, if required,

granulated. The vacuum belt dryer consists of a casing with built in transporting belt. The belts are made

from selected PTFE coated glass fibres. For the continuous and automatic operation of such a plant, a belt

control device that runs reliably for a long period is imperative. For the product quality to be achieved, the

heating temperature during drying is of essential influence. In the tradition vacuum belt dryer normally

the belts run over 3 or 4 heating zones and at the end through a cooling zone. These are fed with hot

water, steam or thermo oil. With using microwave heating careful arranged microwave heater with

infinitely variable power making sure an optimum energy transfer into the product. The temperature

profile may be selected to the needs of the product. The product temperature will be controlled with

infrared thermometers installed on top of the dryer. When the product temperature will climb up in a

critical value, the microwave power will be reduced automatically. Microwave heater usage normally is

1.2/2/3/6kW-2450MHz (Pueschner, 2015).

7. Conclusion

Several drying technologics have reached maturity viz. furher R&D resources are unlikely to yield

appreciable returns on investment. innovative approaches are needed to meet the challenges of global

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competitiveness. efficient energy utilization, environmental impact and product quality. New products

and new processes often demand new drying techniques. Some of the innovations are a result of

“technology push” as opposed to “market-pull“ (Mujumdar, 1996). The product development in the dairy

food segment is mostly targeted towards improving the nutritional profile. Thus innovate drying

technologies need to be developed and evaluated.

8. Selected Reading

Brydigyr A M, Rzepecka M A and McConnell M B (1977) Characterization and drying of tomato paste foam by hot

air and microwave energy. Journal de l'Institut Canadien de Science et Technologie Alimentaire 9 313-319.

Chandak A J and Chivate M R (1972) Recent development in foam-mat drying. Indian Food Packer 26 26-32.

Cherry J P and McWatters K H (1981) Whippability and aeration. In: Protein Functionality in Foods. ACS

Symposium Series 147. Amer. Chem. Soc., Washington DC, 149-179.

Fu N and Chen X D (2011) Towards a maximal cell survival in convective thermal drying processes. Food Research

International, 44 1127-1149.

Guergoletto K B, Tsuruda A Y, Hirooka E Y, Martins E P, de Souza J C B, Sivieri K, Roig S M, Hirooka E Y and

Garcia S (2012) Dried Probiotics for Use in Functional Food Applications. INTECH Open Access Publisher.

Hart M R, Graham R P, Ginnette L F and Morgan A I (1967) Foams for foam-mat drying. Food Technology 17 302-

304.

Krasaekoopt W and Bhatia S (2012) Production of yogurt powder using foam-mat drying. Faculty of Biotechnology,

Assumption University Bangkok, Thailand, 15(3), 166-171.

Labelle R L (1984) Principles of foam mat drying, Journal of Food Technology 20 89-91.

Meena A, Jawake P, Jain S K, Mudgal V D, Saloda M A and Sharma K C (2014) Foam Mat Drying of Papaya.

Journal of Agricultural Engineering 51 9-18.

Mujumdar A S (1996) Innovation in drying. Drying Technology, 14, 1459-1475.

Pueschner (2015) Microwave vacuum drying for advanced process technology.

http://www.pueschner.com/downloads/vacuumdrying.pdf

Santivarangkna C, Higl B and Foerst P (2008) Protection mechanisms of sugars during different stages of

preparation process of dried lactic acid starter cultures. Food Microbiology, 25 429−441.

SPX. (2015) Vacuum and Freeze Drying Technology. http://www.spxflow.com/en/assets/pdf/e&e_vacuum-

tech_300_02_09_2014_GB-web.pdf

Stapley A and Rielly C (2007) Advances in unconventional freeze drying technology. Chemical Engineer, 790, 33-

35.

94

Application of Cryogenics in Processing for Dairy and Food Products

P. Barnwal

Dairy Engineering Division

1. Introduction

Cryogenics is the branch of Physics and Engineering which deals with the production of “freezing

cold” and the study of materials at such low temperatures. There is no clear sharp dividing line where

refrigeration ends and cryogenics starts. The National Bureau of standards, UK has defined cryogenic

temperature as -150oC and below. According to this definition, liquid nitrogen (boiling point -

195.6oC) falls in cryogenic range, whereas carbon dioxide (boiling point -78.5

oC) does not. However,

in general, cryogenics is defined as a branch of engineering specializing in technical operations at

very low temperature, generally below -50oC and to create such a low temperature, cryogenic liquids

are used.

The terminology ‘cryogenics’ is related with Kamerlingh Onnes, a Dutch physicist, who wanted to

produce a gas in his laboratory that could be refrigerated and become a low temperature boiling

liquid. Cryogenics became a buzz word in India only during 1980s when Russia refused to supply

Cryogenic engines to ISRO for its space programme. Cryogenics, however, started in India as early as

1930 when a British company installed an Oxygen Plant.

The word ‘cryogen’ is used in practice to describe a low temperature boiling liquid. Cryogens are

ultra-cold fluids which require proper precautions and safety measures during its handling. An

instantaneous contact between cryogen and exposed skin can produce a painful burn and a splash of

cryogenic liquid to eye may result in loss of vision. Hence, adequate personal protective equipment

such as heavy gloves, face shield, safety goggles, and proper safety mechanisms should be installed

including warning sensors for cryogens handling.

Liquid nitrogen (LN2) and carbon dioxide (CO2), in liquid or solid form, are the two major cryogens

used for food applications. Cryogenic liquids are those which boil at cryogenic temperatures at

atmosphere pressure. Liquid forms of hydrogen, helium, nitrogen, oxygen, inert gases, air, methane,

carbon dioxide, etc are common cryogens. The thermo-physical properties of these two cryogenic

liquids are presented in Table 1. The use of cryogens in industry, defence and space programmes has

simulated the emergence of the new field of cryogenic engineering.

Table 1: Some thermo-physical properties of liquid nitrogen (LN2) and liquid carbon dioxide

(LCO2)

S.No. Properties Liquid nitrogen Liquid Carbon Dioxide

1 Density ( kg/m3) 808 464

2 Boiling point (oC) -195.6 -78.5

3 Thermal conductivity (W/m-K) 0.14 0.19

4 Specific heat of liquid ( kJ/kg-K) 2.05 2.26

5 Latent heat of evaporation (kJ/kg) 199 352

6 Total usable refrigeration effect

(kJ/kg)

690 565

As a refrigerant, liquid nitrogen (LN2) is being used for many decades. In recent years, it is finding

widespread applications in almost every branch of science, vacuum technology, electronics, space

technology, biology, medicine, agriculture, etc. Nevertheless, the scope for application of cryogenics

in the area of food processing is no less important. Cryogenics systems may be used for several unit

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operations e.g. cooling, chilling or freezing for a wide variety of dairy and food products etc.

Cryogenic technology has found applications for production of high value frozen products like,

fruits and vegetables, meat products, marine products, fish, prawn, etc. Cryogenic freezing offers a

wide range of unique benefits including high cooling rates, high throughput, flexibility (acceptability

to different products), low capital cost, low dehydration loss and better quality.

Some applications of cryogenic technology are: shrink-fitting of metals, liquid oxygen is used in

welding, in the manufacture of steel, liquid oxygen in artificial breathing in hospitals & aircrafts, for

the preservation of blood, dead bodies and medicines, for freezing the food for preservation - by spray

of liquid nitrogen, quick healing of wounds, cooling the body parts by anaesthesia, preservation of

bull insemination for better creed, for the manufacture of cryogenic magnets, super conductive

transformers and Super conduction motors, used in separation of gases i.e. air, Coke oven gas, Helium

3 from Helium 4, economic transport of ice cream, superconductivity makes computers compact,

liquid hydrogen is used as a fuel in rockets, spectrum lines are more sharpened at low temperatures.

Cryogenic technology has also limited applications in peeling of fruits and vegetables where these are

immersed in liquid nitrogen for few seconds and then thawed in warm water to loosen the peel.

Packaging and homogenization of biological tissues have also limited applications of cryogenic

technology.

India is one of the largest consumers of foods, quantity–wise because of its large population. The

industries for preservation of food products, particularly those of easily perishable nature, is not well

developed as expected. Against the background of the huge spoilage of foods, the preservation

technology calls for its special attention. The Indian products have to be cost effective by using latest

state of art of technology to produce world-class quality goods.

2. Dairy Production: Cryo Preservation in Animal Husbandary

India has vast animal genetic resources with a wide variety of indigenous farm animals including

various types of cattle. Artificial insemination (AI) of cattle using bull semen preserved at cryogenic

temperature is now a standard commercial practice. The “desi” breed can beatifically inseminated by

the exotic semen and thereby genetic improvement can be made which ultimately leads to enhanced

milk production.

Cryogenics is playing an important role in the field of animal husbandry. Artificial insemination of

cows and buffalos has become a normal method of breeding quality cattle’s using frozen semen from

quality sires. This technique of artificial insemination is particularly useful in a country like India

where the paucity of quality sires has been the main hurdle in the way of cattle improvement

programme. Although the storage of semen for 3 to 4 days may be satisfactory for day to day

requirements of an AI centre, yet difficult to maintain its fertility for long. This causes a serious

wastage of semen from valuable sires. The majority of AI activity today is performed with frozen

semen. Freezing has permitted semen to be collected, processed and used anywhere for years

afterwards. Cryo preservation is the most advanced in the bovine species and has served as a basic

model for other species.

3. Cryogenics in Processing for dairy and food Products

Ice creams, sherbets and other dairy products can be made by directly injecting liquid nitrogen (LN2)

into the mixture while it is being churned. This method of manufacturing not only increases the

storage life of the product but also decreases deterioration during storage and in fact it actually

decreases the cost of manufacturing. Commercial viability of using liquid nitrogen in making ice

cream, sherbets etc has been examined and practices in developed countries.

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The fresh food begins to deteriorate its quality from the moment it is harvested. Preservation of fresh

as well as processed food by conventional means including refrigeration often leads to quality

deterioration viz. discolouration, browning, loss of vitamins as well as aroma and flavour because of

enzymatic and chemical interaction of the products. During the freezing process, the temperature of

food products first falls rapidly from the initial temperature to just bellow 0ºC. The temperature then

falls very slowly until most of the water has changed its state to ice. Once the critical zone is passed,

the temperature drops again quite quickly. Since spoilage continues fairly rapidly at temperature just

below 0ºC, it is important to pass the critical range quickly. The more rapid the freezing, the better

quality of the product obtained.

3.1 Cryo Preservation of Food

Food preservation means destruction of micro-organisms and spores, slowing the rate of chemical

reactions such as oxidation and inactivation of enzymes etc. Food Processing are generally practiced

for destruction of toxins, improving physico-chemical, sensory and aesthetic properties

Most varieties of food items like meat, fruits, vegetables and marine products are perishable in nature.

They deteriorate fast because of bacteriological, enzymatic, oxidative and other chemical reactions.

Since most chemical reactions die down below minus 1200C the shelf life of these products can be

significantly enhanced by Instant Quick Freezing (IQF) Technique. The technique enables to preserve

the taste, aroma, texture or the nutrition value of the food product. Shelf life of the products is

increased dramatically. The cryo preservation of food and marine products for storage and exports has

become an ever growing industry with large market share.

3.1.1 Advantages in Using LN2 as refrigerant

Dehydration loss is less than 1% as against 3 to 10% in conventional freezing; exclusion of O2

minimizes oxidative rancidity during freezing; minimum drip loss, improved quality in terms of

flavour, texture, appearance; reduced floor space equipment; reduced handling losses of the products,

flexibility, high through-put, low investment cost, reduced maintenance cost, minimum man power

requirement and rapid installation and compactness.

3.2 Freezing:

Both refrigeration and freezing involves removal of sensible and latent heat from a food. Freezing

involves the conversion of the aqueous part of a food from water into ice. Thus, freezing makes water

unavailable for microorganisms and for chemical reactions. It lowers the temperature which enables

in prevention of (i) spoilage organisms to grow and (ii) chemical reactions that affect product

degradation, to be slowed or inhibited and helpful in food preservation. Ammonia, carbon dioxide or

liquid nitrogen is generally used for food freezing. Carbon dioxide, in its various forms, is a very

useful food cooling substance. The most commonly and widely used cryogen for cryogenic freezing is

liquid nitrogen. In freezing of dairy and food products, the product must first be cooled to the

transition point of water (0°C). Nitrogen, in its liquid form at -195.6°C, is one of the coldest

substances. Liquid nitrogen is completely inert, colourless, tasteless and odourless. It has no adverse

environmental effects, unlike other refrigerants. This freezing technique exploits features of nitrogen

that make it an ideal natural refrigerant for use in the food industry.

3.2.1 Freezers

3.2.1.1 Primary freezers:

Product is in contact with refrigerant in the form of a cryogenic gas or liquid or solid. Its merits are:

very much suitable for small and thin product, very small ice crystals, insignificant dehydration, color,

flavor, structural changes, drip loss on thawing. Its demerits are: it may induce considerable thermal

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stresses; large product may display severe cracking due to extremely uneven cooling at the surface

and interior.

3.2.1.2 Secondary freezers

Plate freezers refrigerant moves heat by circulation within the plates those are in contact with the

product. Its merits are: in it, different refrigerants are used to cool air. Its demerits are : irregular

shaped product are very difficult to freeze, time of freezing is very high, larger ice crystal are formed.

3.2.1.3 Tertiary freezing

Air blast freezers are circulated over the product. Its merits are: varied and irregular shaped product

can be frozen, wide variety of shapes, sizes are available. Its demerit are: freezer burn, drip loss etc.

3.2.1.4 Individual Quick Freezing (IQF)

It is an established fact that preservation by quick freezing retains the desirable qualities of foods.

Cryogenic technology not only offers the quick-freezing but also the following advantages:

Less Energy requirement, good retention of original quality attributes, less dehydration, low capital

investment, minimal space requirement, maintenance/simplicity of operation, continuous, in-line

freezing, minimum off-stream time, maximum turn-down/up capacity, maximum versatility in

relation to products handled and least cost per unit of food shipped.

3.3 Preservation of Fruits and Vegetables

Although the country is one of the largest producers of fruits and vegetables, it is estimated that a

considerable part of the total production is waste due to poor preservation technique and shortage of

cold storage. Controlled Atmosphere and Modified Atmosphere Packaging of fruits and vegetables for

consumer marketing is currently drawing the attention of researchers and processors.

The quality and shelf life of many foods can be improved by deposing droplets of liquid nitrogen into

their packaging on the production line. The droplets of LN2 vaporize almost instantaneously and as

the vapors expand by nearly 200 folds of the liquid volume; this gives a large dilution effect of air and

displaces most of the air originally present in the pack to create a controlled atmosphere that will not

support any microbial action.

3.4 Cryo-Pulverising or Cryo-Grinding of Spices

Spice grinding is an ancient industry like cereal milling industry with the difference that in spice

grinding there is the additional problem of natural volatile flavouring components and essential oils

getting lost during grinding. Spices are valued for aroma and flavour, these impart to various foods.

The fat content of spices generally poses a problem and is an important consideration in grinding. The

other considerations are particle size, product yield, product uniformity, freedom from contamination,

economy and dust free operation.

During grinding, spices lose a significant fraction of their volatile oils or flavouring components due

to the heat generated. There are considerable losses of oil and moisture from different spices during

normal grinding. For different spices the product temperature ranged between 42 and 95oC during

grinding.

The quality of spices could be retained by the technique of cryogenic grinding. In cryogenic grinding

liquid nitrogen is used to reduce the temperature of the product prior to and during the grinding

operation in order to minimize the loss of flavouring volatile components of the product. Thus the

flavour strength per unit mass of the resultant ground product is significantly higher than that in the

conventionally ground product.

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With cryogenic grinding, the temperature of the product can be as low as -195.6oC. But generally such

low temperatures are not used for all spices. In practice, temperatures are regulated anywhere from -

195.6oC to a few degrees below the ambient temperature.

Cryogenic grinding is accomplished by the controlled injection of liquid nitrogen directly into the

mill’s grinding zone. The instantaneous evaporation of liquid nitrogen quickly chills both the spice

and the mill. It also absorbs the frictional heat of grinding. Thus, the temperatures in the grinding zone

generally are well below -73oC.

The extremely low temperature in the grinder solidifies oils so that the spices become brittle, they

crumble easily permitting grinding to a finer and more consistent size. The high quality ground

product would have domestic as well as international market.

In order to obtain high quality ground spices products, a cryogenic grinding system was designed and

developed to cool the spices before feeding to the grinder and also to maintain the cryogenic

temperature in the grinding zone. The main components of the cryogenic grinding system are (i) pre-

cooling unit (cryogenic pre-cooler) and (ii) grinding unit (commercially available grinder, a pin mill

and a hammer mill).

The function of the cryogenic pre-cooler is to remove the heat from the material before it enters the

grinder. The pre-cooling unit (a cooling device) consists of a screw conveyor assembly, an air

compressor, a liquid nitrogen (LN2) dewar, a power transmission arrangement and control panels. The

cryogenic pre-cooler is made up of a screw conveyor enclosed in a properly insulated barrel and a

system to introduce liquid nitrogen into the barrel, thereby providing refrigeration (liquid and cold

gas) within the system. The particle temperature must be low enough to absorb the heat generated in

the grinder and still fracture. Cryogenic pre-coolers, therefore, must have the ability to reduce the

temperature of the seed below its brittle point as well as the freezing point of its oil, before it enters

the grinder.

There must be provision to control the temperature of the pre-cooler and the feed rate to the grinder

for the obvious purpose of controlling the grinding process. Consumption of liquid nitrogen and the

operating cost are important considerations and matters of concern for a cryogenic pre-cooling

system. The liquid nitrogen losses can be minimized to a great extent by proper consideration of the

design and insulation of the pre-cooler. The design of the pre-cooling unit is to prevent the material

from being heated up during grinding. The unit would pre-cool the material before the actual starting

of the grinding operation. Thus, the pre-cooling unit is being designed to match with a commercially

available grinder (a pin mill and a hammer mill) that could withstand low temperature operations.

4 Conclusion

Cryogenics has tremendous scope for application in Processing for dairy and food products. It gives

ultra-low temperature applications in dairy and food sector. Cryo-preservation is the only viable

method available for long-term preservation of the both plant and animal origin species. Cryogenic

preservation of food offers great promise for the country, both from export and also from domestic

consumptions point of view. It is due to assurance of the food quality and safety using cryogenics.

Most industries employ evaporative air chilling systems, preservation by using cryogenics is not so or

less familiar in this sector. Product shrinkage, toughening and loss of tenderness, products shelf life,

microbial activity, drip loss and dehydration losses etc. are the major quality considerations in

freezing of the food products e.g. meat products. The preservation by using cryogenics will improve

the situation. Before using or selecting the cryogenic methods for processing, proper economic

considerations including payback period and life of the system etc. should be taken into account.

Cryogenic grinding of spices is a new development in India and needs demonstration and promotion

in spice pilot plants. In the dairy and food Industry, use of cryogenics has grown from its inception

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about twelve years ago and most of this involves liquid nitrogen. The availability of indigenous

cryogenic technology for dairy and food processing would ensure production of better quality

products within the country and possibility of further exporting the processed products to different

countries.

5. Suggested References

Anon. 2015a. http://www.cryofoods.com/what-is-cryogenic-the-process.asp

Barnwal P, Singh K K (2012) Cryogenic Grinding Technology for Retention of Spice Quality for Health

Purposes. In: Health Food: Concept, Technology and Scope. Vol.II. (Eds.Gupta R.K., Mangal Manisha,

Bansal Sangita) Biotech Books, New Delhi. ISBN No. 978-7622-267-9. 489-508.

Liu H, Zeng F, Wang Q, Ou S, Tan L, Gu F (2013) The effect of cryogenic grinding and Hammer milling on the

flavour quality of ground pepper (Piper nigrum L.). Food chemistry 141 3402-3408.

Barnwal P, Singh K K, Kumar P, Mohite A K (2012) Cryogenic Spice Grinding for Quality Retention in

Ground Spices. In: Training Manual/Compendium of summer school on “Newer Concepts and

Techniques in Development of Health Foods” (Eds.Gupta R.K., Mangal Manisha, Bansal Sangita). 183-196.

Goswami T K (2010) Role of Cryogenics in Food Processing and Preservation. International Journal of Food

Engineering 6 Issue 1, ISSN (Online) 1556-3758, DOI: 10.2202/1556-3758.1771.

Barnwal P, Singh K K (2010) Application of cryogenic technology for grinding of spices. In: Compendium of

ICAR sponsored Winter School on “Novel techniques in food processing, co-product utilization and quality

assurance” (Eds. Mridula D., Patil , R.T. and Manikantan, M.R.), 26-32.

Khadatkar R M, Kumar S, Pattanayak S C (2004) Cryo-freezing and cryofreezer. Cryogenics, 44 661-678.

Singh K K and Barnwal P (2012) Application of Cryogenics in Grinding of Spices. In: Book on “Food processing

Technologies, Co-product utilization and Quality assurance” (Eds. Mridula D., Patil , R.T. and Manikantan,

M.R.), 373-395.

Barnwal P (2012) Spice Grinding Using Cryogenic Technology. In: Course manual of Training to international

participants (ASEAN delegates) on Production and processing technologies for value addition of

horticultural products” Course director: Dr. R.K.Gupta, HoD, FG & OP Division. 73-79. held during April

30 to May 12, 2012 at CIPHET Ludhiana.

Singh K K and Goswami T K (1999) Design of a cryogenic grinding system for spices. Journal of Food

Engineering 39 359-368.

Mahajan P V and Goswami T K (2007) Use of liquid nitrogen in CA storage: Theoretical analysis and

experimental validation. Journal of food engineering 82 77-83.

100

Oligosaccharides in Formulation of Functional Food

Indumathi K P, Rita


1. Introduction

The use of foods that promote better health and reduction of the risk of diseases are gaining popularity.

One important group is oligosaccharides like inulin or sucrose-derived fructo-oligosaccharides, soy

derived galactosyl-sucroses and galacto-oligosaccharides derived from lactose, xylo-oligosaccharides and

lactulose. Several beneficial effects are claimed on the consumption of non-digestible oligosaccharides.

Such properties include non cariogenicity, a low calorific value and the ability to stimulate the growth of

beneficial bacteria in colon. They are also associated with a lower risk of infections and diarrhea, and an

improvement of the immune system response. Also, due to the decrease in intestinal pH by the

fermentation, NDOs promote reduction of the pathogen and an increased bioavailability of minerals.

Since their average daily ingestion is lower than the level considered as safe (not over 15g/day)

supplementation of non-digestible oligosaccharides could be beneficial.

2. Oligosaccharides

Oligosaccharides are water soluble and typically 0.3-0.6 times as sweet as sucrose. The sweetness

depends on chemical structure, the degree of polymerization of the oligosaccharides present and the levels

of mono- and disaccharides in the mixture. The low sweetness makes them suitable as bulking agents and

as flavor enhancers. The higher their molecular weight, the more viscosity they provide leading to

improved body and mouthfeel. Polydextrose, a carbohydrate-based sugar (bulking agent) and fat replacer,

is soluble up to 80% giving viscous solutions which behave Newtonian. Because of its high Tg (glass

transition temperature, 110 C), it contributes to more stable foods. Polydextrose also functions as a

cryoprotectant, freezing point depressor and gives an over-all cooling effect to the food. Oligosaccharides

do not bind minerals and easy to incorporate into processed foods. Many of them have also shown to be

strong inhibitors of starch retrogradation.

Fat contributes key sensory and physiological benefits to foods, it contributes to the combined perception

of mouthfeel, taste and aroma/odour. Fat also contributes to creaminess, appearance, palatability, texture,

lubrication properties of foods and increases the feeling of satiety during meals. It also can carry

lipophilic flavour compounds, can act as a precursor for flavour development and stabilize flavour. A

very important characteristic of fat is its use for frying. Carbohydrate-based fat substitutes physically and

chemically resemble fats and oils, they are also stable at cooking and frying temperatures. Carbohydrate-

based fat mimetics differ strongly from fats and oils. Generally they adsorb a substantial amount of water

and are therefore not suitable for frying. However, many of them are suitable for baking and retorting.

Since they can only carry water-soluble flavour but not lipid soluble flavour they are less flavourful than

fats and oils. In addition, lowering the fat content of a food by replacing fat with a fat substitute also

affects the vapour pressure of the food which is directly related to flavour intensity. The functionality of

the carbohydrate based fat substitutes is based on their ability to increase viscosity, to form gels, provide

mouthfeel and texture, and to increase water-holding capacity.

Carbohydrate-based fat substitutes are mixtures of sucrose esters formed by chemical transesterification

or interesterification of sucrose with one to eight fatty acids, the class with six to eight fatty acids are

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called sucrose fatty acids polyesters, the class with one to three fatty acids are called sucrose fatty acid

esters (SFE). Unlike sucrose fatty esters polyesters the SFEs are easily hydrolyzed and absorbed by

digestive lipases and are, thus, caloric. SFEs containing upto seven free hydroxyl groups with one to three

fatty acid esters results in hydrophilic and lipophilic properties and, thus, gives them excellent

emulsifying and surface active properties. In addition they are effective lubricants, anticaking agents,

thinning agents and antimicrobials. Other carbohydrates modified to fatty acid esters are sorbitol,

trehalose, raffinose and stachyose. The functionality and potential application of the sucrose fatty acid

polyesters is governed by the type of fatty esters used in the manufacture. The chemical stability of

carbohydrate-based fat mimetics is comparable with the stability of oligosaccharides and depends upon

the type of constituent sugar residues, ring form and anomeric configuration, type of linkages and degree

of branching. It also depends upon their solubility. At low and high pHs and high temperature they are

liable to degradation. Since they are polysaccharides their participation in Maillard reactions is negligible.

Polydextrose, being a mixture of molecules ranging in degree of polymerization of 1 to 100 with a

molecular weight average of 10, shows Maillard reactions unless they have been reduced. For use as a fat

mimetic polydextrose is coated with fat. Polydextrose is only partially fermented by intestinal

microorganisms producing short chain fatty acids.

Some oligosaccharides also function as preservatives. In terms of antimicrobial activity, chitosan seems

superior to chitin since it contains amino groups which could interact with the negatively charged

bacterial cell membranes and then inhibit the bacterial growth. Other mechanisms for antimicrobial

activity of chitosan have also been suggested, as the blockage of RNA transcription by adsorption of

penetrated chitosan to bacterial DNA or the chelating action of chitosan with metal trace elements or

essential nutrients, leading to microbial growth inhibition. Antimicrobial activities of chitosan were also

demonstrated against many different kinds of microorganisms. Accordingly, chitosan was shown to

inhibit food spoilage microorganisms, such as Candida sp., Escherichia coli and Staphylococcus aureus.

However, as the culture media employed poorly represent what really happens in complex food systems,

this polysaccharide has also been tested in food products. Several studies were realized in fruit juices and

emulsified sauces, but also in solid foods such as meat, mayonnaise, tofu, houmous and chilled salads.

Finally, chitosan was also studied as an edible antimicrobial film to cover fresh fruits and vegetables,

pizza and meat.

Current applications of non-digestible oligosaccharides in the industry include desserts such as jellies,

puddings, sherbats, confectionary products such as candy, cookies, biscuits, breakfast cereals; chocolate

and sweets; breads and pastries; table spreads and spreads such as jams and marmalades; meat products

such as fish paste and tofu. The product in which oligosaccharides is to be added determines the nature of

oligosaccharide to be added. Bread for eg is a suitable food for galactooligosaccharides inclusion during

the fermentation with yeast and the baking of bread, they are not broken down, and render bread excellent

in taste and texture. Infant food and food special for old aged or hospitalized people are promising

examples of products for galactooligosaccharides inclusion, since these people are more susceptible to

modifications in the intestinal microflora.

Maltodextrins – oligosaccharides formed by the partial hydrolysis of starch have a low bulk density, are

fully soluble and have a low sweetness. Frozen desserts may use maltodextrin as its water holding

capacity and relatively low molecular weight give it freezing point depressant qualities. It also undergoes

some non-enzymatic (Maillard) browning.

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Soluble fibres such as inulin, fructo-oligosaccharides, and Nutriose® (a chemically modified dextrin from

Roquette) are being used more often to help replace sugar in a range of products. Inulin and

Oligofructose belong to a class of carbohydrates known as fructans.

They are extracted from the chicory plant using hot water and are used as food ingredients to help

formulate healthy products. Their prebiotic effect means they help support a healthy digestive function.

Inulin and oligofructose are available in many commercial forms including powder, granulated, instant,

gel and low sugar, meaning that they can be applied to many different products. The organoleptic

properties of both products are dependent on the level of application in the finished product. Inulin only

has a slightly sweet taste and has no associated aftertaste or off flavours. When added into a high acid

environment, such as some acidic soft drinks, hydrolysis of this product may occur. Due to this,

oligofructose may not be a suitable ingredient for long shelf life products of this nature. Inulin gels are

more resistant to acid hydrolysis however, but hydrolysis can occur. Overall inulin and oligofructose are

beneficial food ingredients that have nutrition claims associated with fibre enrichment.


O’Donnell K (2005) Carbohydrate and intense sweeteners. In: Chemistry and technology of soft drinks and fruit

juices, (P.R Ashurst), 2nd Edition, Oxford: Blackwell Publishing Ltd.

Roberfroid MB, Slavin J (2000) Nondigestible oligosaccharides. Crit Rev Food Sci Nutr.40:461–80.

Quemener B, Thibault J F, Coussement P. (1994) Determination of inulin and oligofructose in food products and

integration in the AOAC method for the measurement of total dietary fibre. Lebensm-Wiss Technol. 27:125–32

Roberfroid M. (2005) Inulin-type fructans as functional food ingredients. Boca Raton, FL: CRC Press.

103

Osteoanabolic Effect of Milk Derived Bioactive Peptides

Suman Kapila, Srinu Reddi and Rajeev Kapila

Animal Biochemistry Division

1. Introduction

In the modern era, due to change in life style and increase in life expectancy, there is an increase in the

incidence of old age diseases like osteoporosis, cardiovascular disease, Alzheimer’s disease and ovarian

cancer. Osteoporosis is characterized by low bone mineral density with micro architectural deterioration

of bone tissue leading to enhanced bone fragility and increased susceptibility to fractures. There are 2

forms of osteoporosis, one related to estrogen deficiency at the menopause and the other to calcium

deficiency and aging of skeleton. Estrogen accelerates osteoclast apoptosis and promotes osteoblast

proliferation hence, estrogen is said to possess both anti-catabolic and anabolic activity with respect to

bone remodeling. Women live on average a third of their life in a postmenopausal stage therefore; they

are at higher risk of developing osteoporosis. On a global basis, Indians have the highest prevalence of

osteopenia. Compared to Caucasians, osteoporotic fractures in the Indian population occur 10-12 years

earlier in age. Osteoporotic fractures are more common in Indian men than in the West. These facts

dictate an urgent need to address issues relevant to the prevention of osteoporosis.

Dairy products have also been shown to increase not only bone mineral density but also bone growth

(Matkovic et al., 2004). In recent years, extensive scientific evidences have been provided for the

existence of biologically active peptides derived from milk proteins that may have beneficial effects on

human health. Bioactive peptides have been defined as peptides with hormone- or drug like activity that

eventually modulate physiological function through binding interactions to specific receptors on target

cells leading to induction of physiological responses. According to their functional properties, bioactive

peptides may be classified as antimicrobial, antithrombotic, antihypertensive, opioid, immunomodulatory,

mineral binding and antioxidative. Among different bioactive peptides ACE inhibitory peptides,

immunomodulating peptides and caseinophosphophopeptides are the most favorite bioactive peptides for

application to foodstuffs formulated to provide specific health benefits. Casein derived peptides have

already found interesting applications as dietary supplements and as pharmaceutical preparations such as

tablets, toothpaste, and dental filling material. Recently, it has been reported that antioxidative and ACE-

inhibitory peptides have anabolic effect on bones thus they have the potential to improve bone health

(Narva et al., 2004; Shimizu, et al., 2008; Hunttunen et al., 2008; Shanmugam, 2013). So, identification

of peptides from milk for their application in treatment of osteoporosis as anabolic therapy for

osteoporosis has become the most desirable therapeutic option. Although drug treatments are efficient in

104

osteoporosis treatment, they are always accompanied by a risk of side effects, such as thromboembolic

disease, gastrointestinal adverse effects or even cancer, and the duration of therapy is often limited.

2. Osteoblastogenesis

The progressive development of the osteoblast is characterized by a definite sequential expression of

tissue specific genes that identifies three distinct periods of osteoblast phenotype development:

proliferation, maturation and extra-cellular matrix synthesis, and matrix mineralization. During the active

proliferation phase, osteoblast-committed progenitor cells (pre-osteoblasts) express genes that support

proliferation and several genes encoding for extracellular matrix proteins, such as type I collagen and

fibronectin. Osteoblasts synthesize type – I collagen–rich matrix that has the unique property of

eventually becoming mineralized (Ducy et al., 2000). Immediately after growth arrest, a developmental

sequence involving the selective expression of specific genes that characterize the differentiated

osteoblast phenotype (alkaline phosphatase (ALP), osteocalcin) occurs. ALP appears in less-differentiated

osteoblasts while osteocalcin appears in a later stage of osteoblast differentiation. The extra-cellular

matrix progresses into the mineralization phase in which osteoblasts synthesize several proteins that are

associated with the mineralized matrix including sialoprotein osteopontin and osteocalcin. Osteoblasts

mineralize the matrix by promoting the seeding of basic calcium phosphate crystals of hydroxyapatite in

the sheltered interior of shed membrane-limited matrix vesicles (MVs) and by propagating hydroxyapatite

mineral onto the collagenous extracellular matrix.

3. Osteogenic peptides

Milk derived CPPs, formed in the gastrointestinal tract or during fermentation enhances calcium

absorption by preventing the formation of insoluble calcium salts in the intestine (Kitts et al., 1992).

Osteoblasts treated with IPP have showed significant decrease in the RANK-L/OPG ratio on Day 17

(Huttunen et al., 2008) and thus decreases the osteoclast activity. IPP and VPP have increased the bone

formation by 4.8 and 5.7 fold at 10-6

and 10-10

M, respectively. While the Lactobacillus helveticus (LBK –

16H) fermented whey increased bone formation by 1.3 – 1.4 times at 10-5

M equivalent of these peptides

but has no effect on osteoclast activity (Narva et al., 2004). Collagen-derived peptides with chemotactic,

angiotensin – I converting enzyme inhibitory activity and osteoblastogeneic activity has protective effect

on the osteoporosis in ovariectomized mice has been reported (Guillerminet et al., 2009). Tryptic

hydrolysates of casein possess osteogenic potential (Behera et al., 2013). A novel peptide having 24

amino-acid sequence is isolated from rat stomach and termed osteoblast activating peptide. This peptide

positively regulates bone formation by augmenting osteoblast differentiation (Fukushima et al., 2010).

Bone forming peptide-1 (BFP-1) derived from BMP-7, have more high activities of osteogenic

differentiation compared with BMP-7 (Kim et al. 2012). The osteogenic growth peptide (OGP) is a

naturally occurring tetradecapeptide, inhibits hOB apoptosis induced by an excess of dexamethasone.

OGP (osteogenic growth peptide) significantly induced osteoblast proliferation by increasing the

expression of cylA & cdk2 genes (Fei et al., 2010).

In our laboratory, for the isolation and characterization of bioactive peptides, casein was isolated from

buffalo milk and hydrolyzed by the digestive enzymes either alone or in combination. The hydrolysates

produced analyzed for ACEI (Angiotensin converting enzyme inhibitory) and antioxidant activity. Pepsin

– Trypsin hydrolysates was subjected to ultrafiltration. The ultrafiltration fraction less than one kDa was

resolved on RP-HPLC. Out of the 16 fractions on RP-HPLC, 11th

fraction of less than one kDa

ultrafiltrate sent for LC-MS/MS analysis followed by custom synthesis. Synthetic peptides were analyzed

for ACEI and antioxidant activity. The peptide showing maximum ACEI and antioxidant activity was

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selected for studying osteoblastogenic potential using rat calvarial cells and for osteoclastogenic activity

using co-culture of calvarial and bone marrow cells.

It was observed that Pepsin–Trypsin hydrolysates at 14.78% degree of hydrolysis showed highest ACEI

and antioxidant activity among other enzymes hydrolysates. Out of the 16 fractions on RP-HPLC, 11th

fraction of less than one kDa ultrafiltrate exhibited highest ACEI and antioxidant activity. LC-MS/MS

analysis of 11th fraction showed the presence of 15 peptides, of which four peptides less than one kDa,

peptide A, B, C and D (coded) deriving from S1-Casein, S2-Casein, -Casein and -Casein, respectively

were selected and synthesized. Synthetic peptide C showed the highest ACEI and antioxidant activity

(Shanmugam et al., 2015) among other peptides. Pepsin–Trypsin hydrolysates of casein and peptide C

enhanced the proliferation of rat calvaria cells significantly at the concentration of 50µg/ml and 30ng/ml,

respectively (p<0.01).Peptide C significantly enhanced the expression of osteoblastic differentiation

marker genes; collagen α1 type I, alkaline phosphatase and osteocalcin. Peptide C significantly

suppressed the expression of osteoclastic marker genes, TRAP and cathepsin K upto 70 % (p<0.01).

Table1: Osteogenic peptides from their respective source of origin

4. Transepitheial transport of peptide

The transepithelial transport of an antioxidative and ACE inhibitory peptide (Peptide C) derived from

casein hydrolysates was investigated along with extensively studied opioid peptide β-casomorphin using a

human intestinal cell (Caco-2) monolayer. The susceptibility to the brush-border peptidases and route of

transepithelial transport were observed to be the primary factors influencing the transport of these

peptides. The apical to basal transport mechanism was studied using bradykinin as control as it shows

resistance to cellular peptidases and its route of transepithelial transport had been established. Peptide C

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(VLPVPQK) and BCM 5 were hydrolysed by cellular peptidases while bradykinin was found intact. The

transport of VLPVPQK (1.0%) was found to be relatively much higher than BCM 5 (0.03%) and

bradykinin (0.1%). Interestingly the effect of some inhibitors on the transport of VLPVPQK suggested

involvement of PepT1 like transporters/SOPT2 while BCM 5, its hydrolytic product and bradykinin were

suggested to be transported mainly via the intracellular transcytosis pathway (Vij et al., 2016).

5. Conclusion

An osteogenic peptide from buffalo casein isolated, identified and its transepithelial transport studied.

But, the molecular mechanism involved in the processes and safety evaluation has to be validated prior to

augmenting peptide VLPVPQK as a potent agent for the therapy/prevention of Osteoporosis.

6. Selected Reading

Behera P, Kumar R, Sandeep I V R, Kapila R, Dang A K and Kapila S (2013) Food Bioscience 2 13-15.

Ducy P, Schinke T and Karsenty G (2000) Science 289 1501–1504.

Fei Q, Guo C, Xu X, Gao J, Zhang J, Chen T and Cui D (2010) Acta Biochimica et Biophysica Sinica 42 801–806.

Fukushima N, Hiraoka K, Shirachi I, Kojima M and Nagata K (2010) Biochemical and Biophysical Research

Communications 400 157–163.

Guillerminet F, Beaupied H, Fabien-Soule V, Tome D, Benhamou C, Roux C and Blais A (2009) Bone 46 827–834.

Haque E, Chand R and Kapila S (2009) Food Reviews International 25 28–43.

Huttunen M M, Pekkinen M, Ahlstrom M E B and Lamberg-Allarddt C J E (2008) Journal of Nutritional

Biochemistry 19 708–715.

Kim H K, Kim J H, Park D S, Park K S, Kang S S, Lee J S, Jeonga M H and Yoon T R (2012) Biomaterials. 33

7057–7063.

Kitts D D, Yuan Y V, Nagasawa T and Moriyama Y (1992) British Journal of Nutrition 68 765–781.

Matkovic V, Landoll J D, Badenhop-Stevens N E, Ha E Y, Crncevic-Orlic Z, Li B and Goel P (2004) Journal of

Nutrition 134 701S–705S

Narva M, Halleen J, Vaananen K and Korpela R (2004) Life Sciences 75 1727–1734.

Shanmugam V P (2013) Ph.D Thesis, NDRI, Karnal.

Shanmugam V P, Kapila S, Sonfack T K and Kapila R (2015) International Dairy Journal 42 1-5.

Shimizu M, Nakagami H, Osako M K, Hanayama R, Kunugiza Y, Kizawa T, Tomita T, Yoshikawa H, Ogihara T

and Morishita R (2008) The FASEB Journal 22 2465–2475.

Vij R, Reddi S, Kapila S and Kapila R (2016). Transepthelial transport of milk derived bioactive peptide

VLPVPQK. Food Chemistry. 190 681-688.

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Newer Detection Tools to Detect Milk Fat Adulteration

Vivek Sharma and Tanmay Hazara


1. Introduction

Milk fat is an important component in the dairy industry, which plays a significant role in economic,

nutrition, physical and chemical properties of milk and milk products. Milk fat is one of the valuable fats

that continue to be a target of unscrupulous traders for the maximization of profits. Methods presently

adopted by food law enforcing agencies to ensure the quality of milk fat are mainly based on the physico-

chemical constants like [Butyro- refractometer reading ( B.R – reading), Reichert-Meissl value (R.M –

value) and Polenske Value (P- value), Phyto- sterol Acetate test ( PA- test) and Baudouin - test]. The said

methods are being used by enforcing agencies to ascertain the quality of milk fat but at the same time

unscrupulous traders are developing such concoctions of adulterant fats so they pass the said tests.

Therefore, in addition to these methods some more methods have been developed by the research

laboratories to tackle the menace of rampant adulteration in ghee. The innovative approaches include

change in the ratios of different fatty acids, sterol analysis, Carbon number profiling of Triglycerides,

Reversed- Phase thin layer chromatography and fractionation of milk fat coupled with other parameters.

In the present article an attempt has been made to compile the information on some recent approaches

developed to detect the adulteration of milk fat. It will be more prudent to focus on the tracer component

based techniques to counter this menace.

2. Modified Holde’s test for liquid paraffin in ghee

Isolate the fat from milk by heat clarification method as described above. Saponify 1 g of fat taken in a

test tube with 5 ml of 0.5 N ethanolic KOH solutions by heating on direct flame, using wire gauge for 5

min. Add about 5 ml of distilled water to the hot saponified solution. Appearance of turbidity indicates

the presence of mineral oil (Kumar, 2005)

3. Rapid color based test for detection of vegetable oils

One ml of clear molten fat was dissolved with 1.5 ml of hexane in a tightly capped test tube. To this was

added 1.0 ml of color developing reagent (distilled water, Sulphuric acid - Sp.gr.1.835 and Nitric acid-

Sp. gr. 1.42 in the ratio of 20:6:14), shaken vigorously and kept undisturbed till it is separated into two

layers. The appearance of a distinct orange tinge in the upper

layer indicates the presence of vegetable oils / fats including

vanaspati (Sharma et al., 2007).

4. Temperature controlled attenuated total reflectance- mid-

infrared (ATR-MIR) spectroscopy:

This is a spectroscopic technique used for the rapid estimation

of butter adulteration. The methodology is typically based on the

infra-red spectroscopic technique (Koca et al., 2010). These

workers collected the Fourier transform infrared spectra of the

samples between 4000 and 650 cm-1

on a FTIR

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spectrophotometer. Here the temperature was controlled, which allowed the stabilization of analysis

temperature at 65± 2°C. The data was analyzed by using statistical tool namely Multivariate data analysis

and calibration models were developed covering all possible adulteration ratios. In this case adulteration

of butter with margarine @ 2.5% could be predicted.

5. FTIR Spectroscopy

The adulteration of butter fat with foreign fat could be detected by observing the FTIR spectra at the

specific wavelength due to the ratios of cis-unsaturations of fatty acid moieties as reported by Sato et al.,

(1990). Nurrulhidayah et al.,(2013) employed FTIR coupled with chemomatrics and PLS analysis and

successfully detected adulteration of milk fat with beef fat and chicken fat at the frequency region of

1500-1000cm-1

(beef fat adulteration) and 1200-1000cm

-1 (chicken fat adulteration

), respectively.

6. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS)

Mass spectrometry is being increasingly used to identify adulterations in food and milk due to its

simplicity and the need for minimum sample preparation (Sanvido et al., 2010). Matrix-assisted laser

desorption/ionisation time of flight mass spectrometry (MALDI-QTOF MS) is a special type of M.S

which demonstrated the information on the basis of m/z(m –mass of a molecule, z- charge of a molecule)

with the help of special matrix , was seen to be a valuable technique for the analysis of oils and fats by

providing characteristic profiles of triglycerides. MALDI-QTOF MS provided rapid and unambiguous

profiles of the TAG composition, and that the TAG are detected mostly as sodiated molecules

[TAG+Na]+ (Saraiva et al., 2009). They reported that in case of pure milk fat the ions of m/z 881.8,

907.8, 909.8 and 911.8 corresponds to [TAG+Na]+ with the POO, OOO, OSO and SSO compositions (P,

palmitic acid; O, oleic acid and S, stearic acid), respectively, which are changed during adulteration with

any foreign fat. Garcia et al.,(2011) reported that during adulteration of milk fat with vegetable oil

MALDI-QTOF MS spectra data changed due to change in the triacylglycerol characteristic, this

phenomena due to the illegal addition of non-milk fat to milk is shown to mostly increase the relative

abundance of the ion clusters centred on charge by mass ratio of triacylglycerol.

7. Tests based on gas liquid chromatography of triglycerides:

European Union has selected this methodology as official reference method to detect non-milk fat in milk

fat by TG analysis using packed or capillary column and low-resolution gas chromatography (GC).

International Organization for Standardization (ISO) and International Dairy Federation (IDF) jointly

specify a method to detect non-milk fat adulterants in milk fats based on TG compositions and in

combination with standardised formulae known as standardised (S) values (S Total, S2, S3, S4 and S5)

for different kinds of adulterants (ISO 2010). Rcently, this approach has been used by Kalla Amrutha

(2013), in detecting the possible adulteration of market samples of ghee. Author has performed the

Triglyceride (TG) analysis using low-resolution Gas chromatography wherein nitrogen was used as

carrier gas. HP-5, capillary column of 2.5 m length (cut from 15 m 9 0.25 mm 9 0.25 lm), column flow

1.20 mL/min of nitrogen carrier gas, with a split ratio of 1:10. The chromatographic conditions were as

follows: the initial oven temperature of 200 °C was increased to 325 °C at the rate of 5 °C/min and held at

the final temperature for 10 min. The injector and detector temperatures were 330 and 360 °C,

respectively. For TG analysis, 1 µl of 5 mg/mL of control and commercial ghee samples prepared in

hexane and Supelco TG standard mix (5 TG mix- 100 mg neat mixture of 99% pure 20% each of C8:0,

C10:0, C12:0, C14:0 and C16:0 acids) from Bellefonte, PA, USA, was injected to GC. ISO/ IDF specified

equations as mentioned below were used to find out the level of adulteration in market samples.

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8. Reversed- phase thin layer chromatographic method:

Recently, a reversed- phase thin layer chromatographic protocol has been developed by Anupma; 2013. The method

is based on the detection of tracer component i.e. ß- sitosterol. The presence of ß- sitosterol band in addition to

cholesterol band on chromatographic plate indicates the presence of vegetable oils in ghee. In this method

unsaponifiable matter is extracted from 0.2 g fat using 5 ml of 5% methanolic KOH in a screw capped test tube. The

tube is incubated in a water bath maintained at 90°C with intermittent shaking after every 5 minutes, for about 20

min. After 20 min of incubation the tube is cooled to room temperature under tap water. One ml water and 5 ml

hexane are added in the tube and tube is vortexed for 1- 2 minute followed by centrifugation at 2000 rpm for about 2

minutes. The upper hexane layer is pipetted out and dried. The unsaponifiable matter is dissolved in 500 µl of

chloroform. 6 µl of the unsaponifiable matter solution is spotted on TLC silica gel 60 RP-18 F 254S plate at a

distance of about 1 cm from the bottom. Plate is developed in a solvent consisting of Petroleum ether: Acetonitrile:

Methanol (20:40:40v/v). Color is developed on the plate by spraying phosphomolybdic acid solution (20%

solution in ethanol) and keeping the plate at 90 - 95°C / 3 min. In adulterated samples additional band corresponding

to ß- sitosterol appears, whereas in pure ghee only one band corresponding to cholesterol is seen.

9. DNA based methodologies

Research has been proved that DNA based methods are more sensitive than the protein or fatty acid based methods

and therefore can be applied reliably for species identification in a wide range of dairy products. This can be the

futuristic tool for detection the adulteration of body fats in ghee. Different workers have used this technique in

identifying the source of milk used for the preparation of cheese etc. PCR method for detection of sheep milk

adulteration in cow milk by using species specific primers have been reported by Bobkova et al., (2009). Similarly,

De et al., (2011) used simplex and duplex PCR for species specific identification of cattle and buffalo milk.

Be proactive than reactive

10. Selected Reading

Aktas N and Kaya M (2001).Detection of beef body fat and margarine in butter fat by differential scanning

calorimetry. Journal of Thermal Analysis and calorimetry.66 795- 801.

Anupma Rani (2013) HPLC profiling of unsaponifiable matter and enriched sterol fraction for the detection of ghee

adulteration with vegetable oils/ fats. M. Tech thesis submitted to NDRI, Karnal (Deemed University).

Bobkova A, Zidek R, Flimelova E, Bobko M, Fiková M (2009) Application of PCR method for milk adulteration

and milk products identification. Potravinárstvo 3 1-3.

Adulterated Samples

Pure ghee

110

De S, Brahma B, Polley S, Mukherjee A, Banerjee D, Gohaina M, et al. (2011) Simplex and duplex PCR assays for

species specific identification of cattle and buffalo milk and cheese. Food Control.22 690–696.

Garcia. Jerusa Simone; Gustavo Braga Sanvido; Sérgio Adriano Saraiva; Jorge Jardim Zacca; Ricardo Guanaes

Cosso; Marcos Nogueira Eberlin ( 2012) Bovine milk powder adulteration with vegetable oils or fats revealed by

MALDI-QTOF MS. Food Chemistry 131 722–726.

ISO (2010) ISO 17678/IDF 202:2010 (E) Determination of Milk fat purity by gas Chromatographic analysis of

triglycerides. International Standard Milk and Milk Products. ISO, IDF. Kalla Amrutha A.L (2013) Detection of

possible adulteration in commercial ghee samples using low-resolution gas chromatography triglyceride profiles.

International Journal of Dairy Technology 66 346-351.

Koca N, Kocaogulu-Vurma N A, Harper W J, Rodriguez-Saona L E (2010) Application of temperature controlled

attenuated total reflectance – mid- infrared (ATR-MIR) spectroscopy for rapid estimation of butter adulteration.

Food Chemistry.121 778- 782.

Kumar A (2008) Detection of adulterants in ghee. Ph.D thesis submitted to NDRI, Karnal (Deemed University).

Kumar A, Ghai D L, Seth R and Sharma V (2009) Apparent solidification time test for detection of foreign oils and

fats adulterated in clarified milk fat, as affected by season and storage. International Journal of Dairy

Technology 62 33 –38.

Kumar A, Lal D, Seth R and Sharma R (2002) Recent trends in detection of adulteration in milk fat – A Review.

Indian Journal of Dairy Science 55 319 - 330.

Kumar A, Lal D, Seth R and Sharma V (2005) Turbidity test for detection of liquid paraffin in ghee. Indian Journal

of Dairy Science 58 298.

Kumar A, Sharma V and Lal D (2008) Development of a thin layer chromatography based method for the detection

of rice bran oil as an adulterant in ghee. Indian Journal of Dairy Science 61 113 – 115.

Nurrulhidayah A F, Rohman A, Amin I, Shuhaimi M and Khatib A (2013) Analysis of chicken fat as adulterant in

butter using fourier transform infrared spectroscopy and chemo metrics. grasas y aceites, 64 julio-septiembre,

349-355

Sanvido G B, Garcia J S, Corilo Y E, Vaz B G, Zacca J J, Cosso R G, et al. (2010). Fast screening and secure

confirmation of milk powder adulteration with maltodextrin via ESI(+)-MS and selective enzymatic hydrolysis.

Journal of Agricultural and Food Chemistry, 58 9407–9412.

Saraiva S A, Catharino R R, Cabral E C and Eberlin M N (2009) Amazonian vegetable oils and fats: Fast

typification and quality control via triacylglycerol (TAG) profiles from dry matrix-assisted laser

desorption/ionization time-of flight (MALDI-TOF) mass spectrometry fingerprinting. Journal of Agricultural

and Food Chemistry, 57, 4030–4034.

Sato T, Kawano S and Iwamoto M (1990). Detection of foreign fat adulteration of milk fat by near infra-red

spectroscopic method. Journal of Dairy Science. 73 3408-3413.

Sharma V, Lal D and Sharma R (2007) Color based platform test for the detection of vegetable oils/fats in ghee.

Indian Journal Dairy Science 60 16 – 18.

111

Production Technologies of Bioactive Peptides from Milk Proteins

Rajesh Kumar Bajaj and Priyanka Singh Rao


1. Introduction:

The discovery of bioactive peptides with potential health benefits has been the subject of growing

commercial interest in the context of health-promoting functional foods (Rowan et al., 2005). The

functional food market abounds in dairy products, the products that provide health benefits in addition to

basic nutrition. Milk proteins remain the major source of bioactive peptides and these peptides display a

wide range of biological activities. Casein and whey proteins are sources of peptides with biological

activity containing 2–50 residues, which are released only after hydrolysis of these proteins in vivo or in

vitro. Their activity depends on both their residue composition and sequence, and some of them are

known to display multi-functional properties. Bioactive peptides can display, inter alia, antioxidant,

hypotensive, immunomodulatory, antithrombotic or opioid properties and, consequently, appear to be

capable to exert beneficial health effects by acting on the nervous, digestive, cardiovascular and immune

systems. Among functional foods, dairy products are considered as a potential source of bioactive

peptides which originate from the hydrolysis of the milk proteins by proteolytic enzymes of starter

bacteria. Thus, the existence of peptides with various bioactivities has been reported in the processed

dairy products such as several cheese varieties and fermented milks like yogurt, sour milk, dahi or kefir.

Several fermented milk drinks containing bioactive peptides added and/or generated during the

fermentation are currently being marketed. As example, milk with hypotensive effects such as “Calpis” or

“Ameal S” (Calpis Co, Japan) and Evolus (Valio Oy, Finland) contains IPP and VPP peptides from β- and

κ-casein. Table 1 lists the occurrence of biological activities of some fermented milk products and

bioactive peptide preparations.

2. Functionality of Bioactive Peptides

There is a growing trend towards the use of these food protein-derived peptides as intervention agents

against chronic human diseases and for maintenance of general well-being. Use of dietary BAPs in

intervention against human diseases offers many advantages, including safety of the natural product, low

health cost, as a source of beneficial and essential amino acids. The incorporation of protein hydrolysates

into foods has been increasingly applied in the food industry. The formulation of new food products by

incorporating biopeptides as ingredients is of great interest for human organisms. The benefit of this

technology is to enhance the daily diets of humans by supplementing biological systems capable of

regulating body functions.

3. Release of bioactive peptides

Although some food proteins are able to elicit their effects by acting directly in their intact form,

generally it is peptides (usually between 3 to 20 amino acids in length) derived from the parent protein

that are of most interest. There are three main mechanisms by which bioactive peptides can be released;

the first two being the result of normal digestive processes: 1) degradation via digestive enzymes 2)

digestion via microbial enzymes primarily in the large intestine or 3) in vitro hydrolysis during food

processing, usually by microbial fermentation or proteolytic digestion 4) Recombinant DNA technology.

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4. Production of bioactive peptides by enzymatic hydrolysis

The enzymatic hydrolysis is the efficient treatment destined for the production of biopeptides from

proteins and it can induce the bioactivity of intact proteins as well. Various proteolytic enzymes including

endoproteinases, such as trypsin, chymotrypsin, pepsin, thermolysin, pancreatin, elastase,

carboxypeptidase and proline-specific endopeptidase, alone or in combination are known to generate

physiological peptides. In addition to this Proteinase preparations sourced from bacteria, fungi, plants

have also been used including Alcalae, neutrase, flavourzyme, papain, bromelain etc can be used to

release bioactive peptides from intact protein molecules. Production of bioactive peptides via normal

digestive processes relies on the active form of the peptide firstly being generated and remaining intact at

its site of absorption or action, which can occur throughout the intestine. In many instances this means

that the protein or the peptide itself must be at least partially resistant to proteolysis, as normally most

food proteins are totally digested during passage through the small intestine. Several proteins such as

lactoferrin and immunoglobulins have been shown to partly resist hydrolysis in the small intestine and

tripeptides with the sequence PP at their C-terminus have also been shown to be resistant to digestion by

peptidases.

5. Production of bioactive peptides with microbial fermentation

The production of biopeptides using microbial fermentation has drawn much of the dairy industry's

attention due to their proteolytic activity as starter cultures. Production of bioactive peptides has been

achieved through fermentation/ripening of protein substrate or precursor using various selected

microorganisms, mainly bacteria. The microbial cells contain proteinases that are responsible for

breakdown of proteins into low mass peptides. Fermentation can be carried out at controlled conditions

for optimum production within a laboratory set up. On the other hand, commercial fermented products

may be used as alternative source of bioactive peptides. This later option is limited due to lack of

uniformity in these products and risk of interfering compounds that could be present in such products.

The other method involves use of suitable proteolytic enzymes to hydrolyse the proteins. This produces

hydrolysates with predictable peptide fractions due to specificity of enzymes in cleaving certain bonds

within the protein chain. Use of combination of both microorganisms and commercial enzymes is

feasible.

Milk products remain the potential materials in generating potent biological peptides by the intervention

of particular microbial proteases. Many examples are elaborated on the potentiality of microbial

fermentation in the production of dairy products such as commercial probiotic bacteria, yogurt bacteria,

and cheese starter bacteria, all these examples being the outcomes of the process of fermentation. Milk

based protein hydrolysates with an antihypertensive activity were produced by using potential dairy yeast

strains belonging to Kluyveromyces marxianus, Kluyveromyces lactis and Debaryomyces hansenii

species. Another study has reported the use of a mixture of commercial starter culture of LAB strains

during fermentation process and demonstrated that the angiotensin-converting enzyme-inhibitory activity

is increased for the hydrolysates after subsequent hydrolysis using microbial proteases. Interesting

observations during secondary proteolysis of cheese ripening have been found to be dependent between

the ripening stage of the cheese and the release of the biopeptides. The beneficial roles of biopeptides

from milk products are considered as promising candidates in promoting various health functions such as

bone functions, heart functions, controlling the stress, digestive systems, improving the immune defense

and reducing the risk of obesity and development of type two diabetes.

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6. Production of bioactive peptides through Recombinant DNA technology

The use of recombinant DNA technology for the production of specific proteins, bioactive peptides or

their precursors by microbial cells constitutes another approach. It permits to increase the production

yield of the specific bioactive peptides. Two types of strategies can be employed: the first consists of

production of the bioactive peptide using a well-known bacterium such as Escherichia coli followed by

peptide addition to the food matrix; the second, to use LAB to synthesize the bioactive peptide directly in

the food product.

7. Separation of Bioactive Peptides

Once the peptides are produced they remain in solution as mixture of different peptides, intact protein and

enzymes hence there is a need for separation techniques. Membranes with different nominal molecular

weight cut-off have been used in micro filtration, ultra-filtration, and nano filtration. The retentate or

reaction mixture is discarded whereas permeate, so called total hydrolysate, and may undergo further

fractionation before use. Membrane separation techniques have provided the best technology available for

the enrichment of peptides with a specific molecular weight range after enzymatic hydrolysis through

conventional batch hydrolysis or continuous hydrolysis. Meanwhile ultra-filtration membrane reactors are

used to improve the efficiency of enzyme-catalysed bioconversion and to increase product yields, and

they can be easily scaled up. Furthermore, ultra-filtration membrane reactors yield a consistently uniform

product with desired molecular mass characteristics. Continuous extraction of bioactive peptides in

membrane reactors has been mainly applied to milk proteins, however, peptide/peptide and

peptide/protein interactions impairs membrane selectivity.

7. Conclusion

The hydrolysis of milk proteins gives rise to a diversity of peptides, some of them displaying remarkable

functionalities relevant to the maintenance of human health. The knowledge about new bioactive peptides

from milk proteins and about their potent functionalities in the milk products is consistently increasing.

Development in the newer separation and enrichment technologies offers novel opportunities in the

development of functional foods and nutraceuticals.

8. Selected Reading

Aleksandra Zambrowicz A, Timmer M, Polanowski A, Lubec G and Trziszka T (2012) Manufacturing of

peptides exhibiting biological activity. Amino acids, DOI 10.1007/s00726-012-1379-7.

Choi J, Sabikhi L, Hassan A and Anand S (2011). Bioactive peptides in dairy products doi: 10.1111/j.1471-

0307.2011.00725.x

Hafeez Z, Cakir-Kiefer Roux E, Perrin C, Miclo L and Dary-Mourot L (2014) Strategies of producing bioactive

peptides from milk proteins to functionalize fermented milk products. Food Research International 63 71-80

Kamau S M, Lu R R, Chen W, liu X M, Tian F W, Shen Y and Gao T (2010) Functional significance of bioactive

peptides derived from milk proteins. Food review International 26 386–401.

Korhonen H and Pihlanto A (2006) Bioactive peptides: production and functionality. International Dairy Journal.

16 945–960.

Mann, B.; Sharma, R and Kumar, R. (2012) “Bioactive Milk Proteins and Peptides” In Health food-concept

Technology and Scope (Eds. Gupta, RK, Bansal, S & Mangal, M.), Biotech books, New Delhi, Chapter 14,

345-369

Rowan AM, Haggarty NW, Ram S (2005). Milk bioactives: discovery and proof of concept, Australian Journal of

Dairy Technology 60 114-20.

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Saadi,S, Saari N, Anwar F, Hamid A A and Ghazali H M (2015) Recent advances in food biopeptides: Production,

biological 3 functionalities and therapeutic applications. Biotechnology Advances. 33 80-116.

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Technology and Applications of Whey Protein Hydrolysate

Priyanka Singh Rao, Athira S. and Richa Singh


1. Introduction

Growing concern over population and environmental control has renewed the pressure on dairy

industries to stop dumping of whey into streams and municipal sewage systems. These legislative

restrictions on whey disposal encouraged a deeper exploration of the widely recognized but less well

understood physical, chemical, nutritional, and biological properties of whey components. So, in the

light of global food storage, the most logical use would be to return whey to the human food chain in

palatable form.

Whey is the liquid remaining after the recovery of cheese. Whey contains more than half of the solids

of original whole milk, including whey protein (20% of total protein) and most of the lactose,

minerals and water-soluble vitamins. Whey represents a rich and varied mixture of secreted proteins

with wide-ranging chemical, physical and functional properties (Smithers et al., 1996). These proteins

not only play an important role in nutrition as an exceptionally rich and balanced source of amino

acids (Regester et al., 1996), but in number of instances also appear to have specific physiological

activity, in vivo. While it has long been recognized that several whey proteins confer non-immune

protection to neonates against disease, and other whey proteins also have putative biological and

physiological effects. Such proteins include α-lactalbumin, β-lactoglobulin, lactoferrin,

lactoperoxidase, immunoglobulins, glycomacropeptides and a variety of growth factors. These

proteins have been implicated in a number of biological effects observed in human and animal studies

ranging from anticancer activity to influence on digestive function.

Whey protein (WP) is now recognized as a value-added food ingredient because of its highly

functional and nutritional properties. The functionality of WPs can be improved by chemical,

enzymatic and physical processes. High pressure treatments are used to enhance the functionality of

WPC, but still remain below the expected levels of the industrial food applications (Kresic et al.,

2006). Enzymatic modifications are highly acceptable and applied in the industry not only for

functionality improvement but also for bioactive enhancement (Athira et al., 2014). These

hydrolysates are also being used as protein supplements for infants, senescent, athletes, and

bodybuilders (Sousa et al., 2004).

2. Whey

Whey is a co-product of cheese-making and casein manufacture in the dairy industry. After the casein

curd separates from milk, following coagulation of the casein proteins through the action of chymosin

(rennet) or mineral/organic acid, the remaining watery and thin liquid is called whey. It has been

reported that about 50% of the milk solids appear in the whey, together with essentially the 100% of

the lactose and some 20% of the protein. The lactose makes up a high proportion (47.5%) of the total

whey solids, and contributes in large part to dairy whey being considered one of the most polluting

food by/co-product streams (biochemical oxygen demand (BOD) 4,35,000 ppm; chemical oxygen

demand (COD) 4,60,000ppm) (Siso, 1996). While the polluting power of whey is well known, this

dairy stream also represents an excellent source of functional proteins and peptides, lipids, vitamins,

minerals and lactose that until relatively recently have been less well recognised. It is these latter

components of whey, notably the proteins and peptides and their properties that have helped transform

whey from a waste material that has often been shunned to a valuable dairy stream containing a

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multitude of components available for exploitation in the agri-food, biotechnology, medical and

related markets.

3. Membrane processes in whey protein recovery

Membrane-based Tangential Flow Filtration (TFF) unit operations are used for clarifying,

concentrating and purifying proteins. Membrane technology is used extensively throughout the dairy

industry to control the protein, minerals and lactose content of a variety of products. These membrane

processes became successful because they can be electively and economically implemented at the

large scale required for most dairy applications. Membrane microfiltration (MF) has been used for the

removal of residual lipids from whey prior to ultrafiltration (Merin et al., 1983). This often involves

heat treatment and/or pH adjustment to aggregate lipids and calcium phosphates (Gesan et al., 1995).

Microfiltration can also be used to remove microorganisms from whey (and milk), thus reducing the

bio burden without need for high temperature pasteurization (Maubois and Ollivier, 1997).

Ultrafiltration (UF) uses polymeric or ceramic membranes which are fully retentive to the whey

proteins to remove lactose and minerals, yielding a retentate stream that can be further processed by

evaporation and spray drying. The net result is a whey protein concentrate (WPC) that is around 60%

protein by weight. The lactose and mineral content in the whey protein concentrate can be further

reduced using a subsequent diafiltration (DF) in which deionized water is continually added to the

retentate while lactose and minerals are simultaneously removed from the filtrate. This combined UF

and DF yields a high value retentate (approximately 85% protein). The main important advantages of

membrane processes are cost reduction, high process speed, the absence of denaturation or protein-

structure modification, and the fact that the protein concentrate is free of salts thereby making it

suitable for all kinds of human foods, even for dietetic or baby-foods (Gardner, 1989).

4. Whey proteins

Whey proteins represent about 20% of the milk proteins. The most abundant of these are β-

lactoglobulin (50%), α-lactalbumin (12%), immunoglobulins (10%), serum albumin (5%) and

proteose peptones (0.23%). From the nutritional point of view, whey proteins have been considered

superior to casein in various aspects. The PER (protein efficiency ratio) value of whey proteins is high

(3.4) compared to standard casein (2.8), and whey proteins have a higher proportion of essential

amino acids than casein (Evans and Gordon, 1980). Their biological value exceeds even that of whole

egg protein. Heating, but to a high temperature results in precipitation of the proteins and reduces the

high nutritive value of whey proteins (Sienkiewicz and Riedel, 1990). The sulphur amino acids

content of whey proteins is higher than that of whole-milk proteins (1.35% versus 0.36%) (Yves,

1979). Lysine content is also higher in whey protein than in total milk-proteins (10.5% versus 7.75%).

Therefore, WPC has become an interesting complement of cereal- based (lysine-deficient) diets and

also for baking products if maillard reactions are required. Whey protein products also offer multiple

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techno-functional benefits including solubility, gelation, aeration, water-binding and emulsification

(Foegeding et al., 2002)..

5. Whey protein hydrolysates (WPHs)

Functionalities of whey proteins can be improved by hydrolysing it through physical, chemical or

enzymatic methods. There are several advantages of using proteases to cause proteolysis instead of

physical or chemical treatments. Acid and alkaline hydrolysis tend to be difficult process to control

and yield products with reduced nutritional qualities. Chemical hydrolysis can form toxic substances

like lysino-alanine (Lahl and Windstaff, 1989). So, for WPs modification, enzymatic hydrolysis is the

most applicable method in food industry. Many food-grade proteases are available for protein

hydrolysis. These proteases which can be classified based on their origin, i.e. animal, plant or

microbial origin, their mode of catalytic action, i.e. endo- or exo-activity or on basis of their catalytic

site. Endoproteases cleave amide bonds within the protein chain, contrary to exoproteases that remove

terminal amino acids from proteins or peptides, either at the C-terminus (carboxypeptidases) or at the

N-terminus (aminopeptidases). The nature of the catalytic site of proteases differs according to the

active group that will form the enzyme/substrate intermediate. The active group can be either an

amino acid, i.e. serine, cysteine or aspartic acid, or a metallo group, most often zinc (Adler-Nissen,

1993). The functional and biological properties of the WPHs depend to a great extent on the type of

enzyme used (specificity and selectivity), hydrolysis conditions (enzyme-to-substrate ratio, incubation

temperature, pH, and time) employed, and the source of the protein, native or denatured, WPI vs.

WPC, membrane or ion-exchange product, etc.

5. 1 Characterization of hydrolysates

Due to protein hydrolysis, molecular properties of proteins changes, like decreased molecular weight,

increased charge, exposure of hydrophobic groups and disclosure of reactive amino acid side-chains

(Nielsen, 1997). These molecular changes can be detected with several analytical methods, which

reflect one or several molecular properties. As a result of the molecular changes, the functional

properties of proteins are affected. Although the term functional property is often only applied to

indicate techno-functional properties of hydrolysates, it should also comprise bio-functional properties

and can be sub divided in nutritional and physiological or biological functionality (Mahmoud, 1994).

Nutritional properties of hydrolysates reflect by their increased digestibility and decreased

allergenicity as compared to the parental proteins. The physiological properties comprise potential

bioactivities of hydrolysates, which originate from the liberation of bioactive peptides. Finally, the

techno-functional properties represent technological functionality, such as solubility, foam and

emulsion properties.

5.1.1 Molecular characterization

The most commonly used parameter describing the result of a hydrolysis process is the degree of

hydrolysis (DH), used as an indicator of the extent of hydrolysis. Another important parameter for

protein hydrolysis is the molecular weight distribution of the peptides in hydrolysates. The molecular

weight distribution is indicated by SDS-PAGE (Galvao et al., 2001) or by size exclusion

chromatography (Madsen et al., 1997). These techniques are most often used to compare hydrolytic

action of various proteases, or to characterize hydrolysates. Philanto-Leppala et al., (2000) reported

that the -lactalbumin was completely decomposed during pepsin treatment and the hydrolysate was

characterized by a high content of polypeptide > 5 kDa. These polypeptides were further degraded

into smaller peptides during trypsin treatment. There was no major difference in the molecular mass

distribution when heated and unheated -lactalbumins were used. They also reported that the heated

- lactoglobulin (~30%) was more readily hydrolyzed by pepsin than unheated -lactoglobulin, more

component < 5 kDa being found.

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5.1.2 Biofunctional properties

The term bioactivity refers to food components that can affect biological processes or substrates and

hence have an impact on body function or condition and ultimately health. The two major risk factors

contributing to the worldwide incidence of cardiovascular disease are hypertension and dyslipidemia.

Whey based peptides have demonstrated activity that may reduce both risk factors (Clare and

Swaisgood, 2000). Recent research has shown that bioactive whey peptides may be involved in these

functions like ACE inhibitory activity, opioid-like activity, antithrombotic activity, cholesterol-

reducing activity (Madureira, et al., 2010). Whey peptides may also have other functions, including

antioxidant activity, which improves overall cardiovascular health. Whey proteins can be broken

down into various bioactive peptides through enzymatic proteolysis. This process can occur during

gastrointestinal digestion, by fermentation of milk, or through controlled reactions in the laboratory or

whey processing facility. Regardless of the method of hydrolysis, in order to exert antihypertensive

activity, the peptides must be absorbed from the intestine in an active form. Relatively high levels of

bioactive peptides could potentially be produced using low amounts of whey. These whey peptides

could enter peripheral blood intact and potentially exert systemic effects.

5.1.3 Techno – functional properties

WPH have a variety of functional applications. The term functional properties are often used in

relation to physico-chemical properties of proteins. It is important to study the functional properties of

hydrolysates because molecular changes occur during hydrolysis may alter techno functional

behaviour of the hydrolysates than to that of intact protein in terms of solubility, viscosity, gelling,

sensory properties, emulsion and foam properties (Panyam and Kilara,1996; Nielsen, 1997)

5.1.3.1 Solubility

Generally, the solubility at the isoelectric point (pI) of proteins increases with hydrolysis, which is

mainly the result of reduction in molecular weight and the increase in the number of polar groups. The

effect of hydrolysis on solubility at other pH values depends on the protein studied. Caseinates for

example, are very soluble at pH values above and below the pI (pH 4-5). Consequently, at these pH

values the solubility of hydrolysates is similar to or slightly lower than that of intact caseinates

(Slattery and Fitzgerald, 1998). Whey protein, which is, except at the pI, slightly less soluble than

casein, shows increased solubility with hydrolysis over the entire pH range (Lieske and Konrad,

1996).

5.1.3.2 Gelling properties

Whey proteins can form gels that range in properties from viscous fluid soft, smooth pastes or curds

to stiff, rubbery gels. These gels vary in hardness, cohesiveness, stickiness, color and mouth feel

(Zirbel and Kinsella; 1998). Some enzymes can induce gelation following whey protein hydrolysis;

others impair gelling properties (Forgeding et al., 2002). Gels confer structure, texture and stability to

food products; they also allow the retention of large quantities of water and other small molecules

inside the food matrix. These aspects are appreciated by processed food manufacturers. The loss of

gelling ability of whey protein hydrolysates was due to the stabilization of -lactoglobulin (the main

whey protein involved in the gelling phenomenon) by a hydrophobic peptide liberated during tryptic

hydrolysis.

5.1.3.3 Emulsion and foaming properties

The functional properties of milk proteins may be improved by limited proteolysis. Hence the limited

proteolysis of whey protein concentrate reduces its emulsifying capacity, increases its specific foam

volume but reduces foam stability and increases heat stability. Emulsion and foam properties of

hydrolysate were similar or inferior to those of the parental proteins. Foam-forming ability of whey

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protein hydrolysates was correlated to the Molecular Weight Distribution (MWD) of the peptides,

showing that especially peptides with MW of 3-5 kDa contributed to foam-forming ability.

Concerning prevention of emulsion instability due to coalescence it was shown that peptides with a

molecular weight larger than 2 kDa are needed. Foam-stabilizing ability of casein hydrolysates also

depended on the MWD of hydrolysates, but higher molecular weight peptides, i.e. larger than 7 kDa,

were needed to obtain good foam stability (Ven et al., 2002).

Reports concerning the altered emulsion and foam properties of both whey proteins and casein

hydrolysate compared to the parental protein. For both whey protein and casein, hydrolysis has been

reported to improve as well as to reduce emulsion-forming properties (Slattery and Fitzgerald, 1998).

The effect of hydrolysis sometimes seems to depend on the enzyme used for hydrolysis. Partial

proteolysis of WPC with trypsin greatly improves thermal stability and emulsifying properties

(Hidalgo and Camper, 1977). These differences might result from differences in the DH of the

hydrolysate or from differences in the methods used to prepare the emulsions and to quantify the

emulsion-forming properties. For example, the Emulsion Activity Index (EAI) measures the turbidity

of an emulsion with a certain oil fraction, whereas the emulsifying capacity (EC) determines the

amount of oil that can be dispersed by a certain amount of protein or hydrolysate. Although both

methods are used to quantify emulsion-forming ability of hydrolysates, they actually do not measure

the same properties.

For β-lactoglobulin, most reported hydrolysates showed improved emulsion stability (Caessens et al.,

1999). Hydrolysates of whey protein isolate (WPI) prepared with pancreatic enzymes (trypsin or

chymotrypsin) or bacterial enzymes (Alcalase or Neutrase) were fractionated over a 10 kDa

membrane, to study the differences in functional properties between total hydrolysates and high and

low molecular weight peptide fractions. Total tryptic and chymotryptic hydrolysates and their

retentate fractions (MW>10 kDa) showed increased emulsion stability relative to WPI. The same

applied for Alcalase or Neutrase hydrolysates with DH levels higher than 6%. With all tested

hydrolysate, the fractions with peptides <10 kDa showed decreased emulsion stability compared to

WPI. These results suggest that high molecular weight peptides have an essential role in emulsion

stabilisation (Mutilangi et al., 1996). The positive effect of high molecular weight peptides on

emulsion stability also appeared from a study concerning different fractions of a plasmin hydrolysate

of β-lactoglobulin. A fraction containing peptides of 7 to 14 kDa showed improved emulsion stability

relative to the parental protein, but an emulsion prepared with a fraction containing peptides <2 kDa

was not able to stabilize the emulsion (Caessens et al., 1999).Hydrolysis of whey protein and casein

generally resulted in increased foam-forming ability of the hydrolysate compared to the parental

proteins (Caessens et al., 1999a). In two studies, WPH were fractionated with ultrafiltration over a 10

kDa membrane (Mutilangi et al., 1996). For almost all hydrolysates, it appeared that the permeate

fraction with peptides having a MW <10 kDa formed a higher foam overrun than the original protein.

The foam-forming behaviour of the retentate fraction decreased for some hydrolysates, although for

trypsin, chymotrypsin and alcalase. The effect of hydrolysis on foam stability seems to depend on

enzyme specificity and degree of hydrolysis (Slattery and Fitzgerald, 1998). In case of tryptic WPH, it

was shown that the fraction with peptides having MW <10 kDa had better foam-stabilizing properties

than the peptides with MW >10 kDa. Other hydrolysate fractionated over a 10 kDa membrane,

showed for both the high and low molecular weight fractions either increased or decreased foam

stability relative to WPI (Althouse et al., 1995; Mutilangi et al., 1996).

Differences in foam-stabilizing behaviour of permeate relative to the retentate fraction depended on

the enzyme used for hydrolysis (Althouse et al., 1995). According to a study with a papain

hydrolysate of whey protein, fractionated with a 1 kDa cut-off membrane, it was shown that the low-

MW fraction (<1kDa) was essential for foam stabilization, though further emulsion and foam

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properties, no contribution of this fraction was detected (Lieske and Konrad, 1996). Finally, for a

plasmin hydrolysate of β-lactoglobulin it was shown that the fraction containing peptides <2 kDa was

not able to stabilize the foam, whereas the hydrolysate fraction containing larger peptides did form a

stable foam (Caessens et al., 1999a).

6. Conclusion

Whey protein provides a plethora of bioactive ingredients for incorporation into functional food

products. This has come at a time when consumers want more from food than just basic nutrition, but

rather a capacity to alleviate the onset of lifestyle diseases through diet. WP products are used in food

applications considering their functional benefits over the other proteins in the industry. The major

functional activities of the protein depend on their hydration, gelation, interfacial, aggregation, and

sensorial properties. These properties are better enhanced via enzymatic hydrolysis than via chemical

or technical means. WPHs enhance functionality due to their ability to expose the globular protein

structure, reduce the average molecular weight and, increase the ionic strength, molecular charges,

and protein-to-protein interactions, properties which are lacking in non-hydrolyzed WPCs and WPIs.

The optimum functional ability of WPHs would be achieved by using proper hydrolysis conditions

(enzyme-to-substrate ratio, temperature, pH, and time), the type of enzyme, and environmental

conditions in food industry. So Whey components, particularly the proteins and peptides, will

increasingly be preferred as ingredients for functional foods and nutraceuticals, and as active

medicinal agents, built upon the strong consumer trend for health and wellbeing, and continuing

discovery and substantiation of the biological functionality of whey constituents.


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Technochemical and Biological Properties of Glycomacropeptide (GMP) and its

Application in Functional Foods

Richa Singh


1. Introduction

Milk proteins are broadly classified into two groups; caseins and whey proteins. Casein is the principal

milk proteins and whey proteins only account 20% (w/w) of the total milk protein. Whey is a portion of

skim milk that left out after removal of casein, mainly by following two processes:

Enzymatic coagulation, resulting in casein coagulates, which are used for cheese production and

results into sweet whey;

Acid precipitation at the isoelectric point (pH=4.6), resulting in isoelectric casein, which is

transformed into caseinate and acid whey.

Whey has high biological oxygen demand (BOD) in range of 30,000 to 50,000 mg oxygen per liter. It is

not a polluting agent for itself, but when disposed in watercourses, it causes great polluting effects (Porto

et al., 2005). When whey is disposed without treatment it poses about 100 times more BOD than

domestic sewage. An industry with an average production of 10,000 liters of whey per day pollutes as

much as a population of 5,000 inhabitants. Whey is the main byproduct of cheese industries. Therefore,

its disposal can exert a potential threat to environment but at the same time this whey could also be

utilized with great economical interest as a protein source for the food industry. Whey proteins have

number of functional properties such as solubility, viscosity, emulsification and gelling that can be

potentially applied to food industry.

The main protein components in whey are α-lactalbumin, ß-lactoglobulin, bovine serum albumin,

immunoglobulin, glycomacropeptide (GMP; only present in sweet whey), lactoferrin and lactoperoxidase

(Doultani et al., 2004).

2. Glycomacropeptide (GMP):

GMP is a part of sweet whey only and is released when bovine k-casein is treated with chymosin (rennet)

enzyme during cheese making. The protein is hydrolysed on the peptide bond Phe105-Met106 into para-k-

casein (residues 1-105), which remains with the curd, and residues 106-169, which is removed with the

whey. This peptide is usually known as caseinomacropeptide (CMP) or casein-derived peptide (CDP) or

Glycomacropeptide (GMP). Usually GMP refers to the glycosylated form, due to its high carbohydrate

content, and CMP to the peptide´s non-glycosylated form. The glycosylated form represents 50 to 60% of

total CMP and is composed of mainly galactose (Gal), N-acetylgalactosamine (GalNAc) and

acetylneuraminic acid (NeuAc) (Thomä et al., 2006). Smaller concentrations of GMP also exist in bovine

milk. However, GMP released from casein is almost ten times higher than free GMP in mature milk

(Furlanetti and Prata 2003). GMP constitutes 20–25% of total proteins in whey products viz., whey

powder, whey protein isolates (WPI), and whey protein concentrates (WPC) etc., manufactured from

cheese whey (Farías et al., 2010). It is recognized as a bioactive peptide and is thought to be an ingredient

with a potential use in functional foods. The composition of GMP is variable and depends on the

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particular whey source and the fractionation technology employed in its isolation (Martín–Diana et al.

2006).

2.1 Chemical properties of GMP

2.1.1 Amino acid composition

GMP is made up of 64 amino acid; significantly rich in Pro, Glu, Ser, and Thr but lacks aromatic amino

acids; phenylalanine, tryptophan, tyrosine (Oliva et al., 2002) and also in Cys (Fig. 1). It is rich in

branched-chain amino acids. Of the three branched chain amino acids viz., Leu, Ile, and Val, it is rich in

Ile and Val.

Fig. 1 Primary structure of bovine GMP variant A. The enclosed amino acids represent the sites

corresponding to B variant (Eigel et al. 1984)

2.1.2 Glycosylation

GMP (106-169 residue) is highly glycosylated because, in κ-CN carbohydrate moiety is attached at

Thr131, 133, 135, 142 and Ser141 positions through O-glycosylation linkages (Eigel et al., 1984). In

GMP the distribution of monosaccharide, disaccharide, trisaccharide (straight and branched), and

tetrasaccharide chains is found to be 0.8%, 6.3%, 18.4%, 18.5%, and 56.0%, respectively (Saito et al.

1991). It has been established that following five saccharides are found in mature cow milk:

1. Monosaccharide GalNAc - O - R

2. Disaccharide Gal β1 -> 3 GalNAc - O – R

3. Trisaccharide NeuAc α2 -> 3 Gal β1 -> 3 GalNAc -O - R

4. Trisaccharide Gal β1 -> 3 (NeuAc α2 –> 6) GalNAc - O - R

5. Tetrasaccharide NeuAc α2 ->3 Gal β1 - 3(NeuAc α2 –> 6) GalNAc - O - R

Where Gal=galactose; GalNAc =N-acetylgalactosamine, and NeuAc=sialic acid

Fucose (Fuc) has also been identified as constituents in these saccharide in GMP isolated from cow

colostrum (Fiat & Jolles, 1989);

The most predominant carbohydrate in GMP is NeuAc, known as sialic acid. GMP purified to 90% is

highly glycosylated with 7 to 8% sialic acid (Martín-Diana et al., 2003). On the basis of glycosylation,

GMP can be classified into two major fractions (Kreuß et al., 2009):

1. Glycosylated and phosphorylated glyco-peptide (gGMP),

2. Non-glycosylated but phosphorylated aglyco-peptide (aGMP)

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Heating of the milk or whey prior to isolation of GMP has an influence on the extent of glycosylation in

isolated GMP; more severe heating produces GMP with less glycosylation (Taylor and Woonton 2009).

GMP is present in milk whey in concentrations of 1.2-1.5 g/L.

2.1.3 Molecular weight

The molecular weight of GMP is between 7 and 8 kDa. Its low molecular weight of 8 kDa makes it

difficult to visualize with Coomassie Blue stain in sodium dodecyl sulphate (SDS)-PAGE. Quantification

procedures for GMP require trichloroacetic acid precipitation of the other whey proteins leaving only the

GMP in solution (van Hooydonk & Olieman, 1982; Sharma et al. 1993). Literature suggests that GMP

has the ability of associating and dissociating under selected pH conditions. Kawasaki et al. (1993a)

proposed that the monomer of k-casein GMP of molecular weight 9 kDa is obtained at pH≤4 and the

polymer of k-casein GMP of molecular weight between 45-50 kDa is obtained at pH higher than 4.

2.1.4 Isoelectric point (pI)

GMP is an acidic peptide with pI of around 4, highly soluble and heat stable (Thomä et al. 2006). This

relatively acidic pI is related to the high amount of acidic amino acid side chains (Glu and Asp). GMP

does not have a single pI because of heterogeneity in glycosylation and phosphorylation (Kreu et al. 2008;

Lieske et al. 2004). The pI of 64 amino acids is around 4, but the sialate and phosphate have relatively

low pKa values of 2.6 and 2.0, respectively. The pI of GMP depends upon sialic acid and phosphate

content present in it. In addition, GMP retains a net negative charge, even at pH 3, so it is not collected on

cation exchangers, nor does it move with the rest of the proteins in native polyacrylamide gel

electrophoresis (PAGE).

2.1.5 UV absorption

As GMP lacks aromatic amino acids (Phe, Trp, and Tyr) therefore, it has no absorption at 280 nm. It is

known that GMP is only detected in the range of 205-226 nm, and differences in the absorption at 210

and 280 nm are frequently used to characterize GMP (Oliva et al. 2002); this has been the most

commonly used tool to perceive the presence of GMP during HPLC.

2.2 Functional properties of GMP

The functional properties of GMP include its emulsification, foaming, and gel formation ability.

Functional properties of GMP are affected by the glycosylation of GMP.

2.2.1 Emulsification and foaming properties

Emulsifying and foaming properties are among the most important functional properties of milk proteins,

and the characteristics of most dairy products depend on how the milk protein fraction is organized at fat–

serum and air–serum interfaces (Dickinson 2003). GMP present in WPC also contributes to the

emulsification (Chobert et al. 1989). When compared with WPC, the emulsifying activity index of GMP

was shown to be significantly lower (36 m2 g−1 for GMP and 185 m2 g−1 for WPC), but GMP had more

stable emulsifying activity index with respect to pH than WPC, thus it can be possibly utilized as

emulsifier in foods which undergo large pH variations during processing, i.e., fermented dairy products

(Martin-Diana et al. 2005). Like emulsification, foaming ability also depends upon glycosylation. GMP is

a good foam-building peptide as it efficiently reduces surface tension and assures high gas content during

foaming but makes unstable foams compared to foams made with WPI (Thomä-Worringer et al. 2007).

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2.2.2 Gelling ability:

Gel is an intermediary form between solid and liquid. Food processing and the development of new

products require ingredients such as the gelling agents, which build up a structural matrix that supplies

food its desirable texture. It has been proposed that GMP has the capability to form gel and this is because

of strong hydrophobic links between the monomer molecules. At neutral pH, most of the hydrophobic

domains (containing Val, Pro, Ile, Ala, and Met) cannot interact as these are masked by strong charge

density of negatively charged amino acids (Glu and Asp). Above pH 6.5, Glu and Asp get deprotonated

which produces a strong negative shield thereby preventing GMP self-association. Below pH 6.5, the

protonation of these acidic amino acids increases which decreases their shielding effect, allowing the

hydrophobic domains to interact causing self-assembly. GMP solutions (3–10%, w/ w) below pH 4.5

show time-dependent self-assembly at room temperature leading to gelation. A GMP self-assembly model

(Fig. 2b) has been proposed according to which self-assembly occurs in two different stages—a first stage

of hydrophobic interaction to form dimers and in the second stage the dimers further interact through

electrostatic bonds to form gels. In the second stage, the presence of charged glycosidic side chain plays

an important role as it contributes to the negative charge at low pH (Farías et al. 2010).

2.2.3 Biological properties of GMP

GMP provide protection against toxins, bacteria, virus and immune system regulation Brody (2000). It

has also been reported by Oliva et al (2002) that GMP has an effect on gastrointestinal mobility,

inhibition of the binding of the cholera toxin to its receptor, inhibition of the influenza virus

hemagglutination, effect on the growth of lactic acid bacteria, effect on digestion, promotion of growth of

bifidobacteria and antithrombotic activity. Kawasaki et al. (1992) reported inhibitory effect of GMP upon

the cholera toxin and related it to the terminal sialic acid. Aimutis (2004) reported GMP also inhibited the

growth of the cariogenic bacteria Streptococcus mutans and of other species. It may be due to glycosidic

structures bond to GMP and these are important for the biological activity including anticariogenic

activity. GMP showed to inhibit dental enamel demineralization and also promoted remineralization.

Korhonen & Pihlanto, 2006 reported that GMP inhibits gastric secretion and stomach contractions as

GMP stimulates cholecystokinine release and this hormone is involved in the control of food ingestion

and digestion in the duodenum of animals and humans.

3. Nutritional Importance of GMP

GMP has special nutritional properties. It is rich in branched-chain amino acids and poor in methionine,

thus it can be used as an ingredient in diets for hepatic patients (Thoma et al., 2006). Because it lacks

aromatic amino acids (phenylalanine, tyrosine and tryptophan) CMP is suggested as an ingredient in the

diet of phenylketonuric patients (Oliva et al., 2002). GMP is also recognized for enhancing the absorption

of minerals such as calcium, iron and zinc. Kelleher et al. (2003) verified in their studies that GMP

supplementation enhanced zinc absorption, allowing a reduction of this mineral in infant formulas. GMP

is also rich source of sialic acid which is important in neuronal and cognitive development

4. Application

GMP, found only in sweet whey, is a biologically active compound. It possess various biological and

nutritional properties that make it a potential ingredient for functional foods. Glycomacropeptide (GMP)

is a highly bioactive whey protein with superior purity. Isolated from fresh cheese whey, this product is

appears as a light colored, homogeneous, and free flowing powder with a clean, bland flavor. GMP has

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many unique characteristics compared to other whey proteins. It has low levels of aromatic amino acids

and relatively high amounts of branched chain amino acids, making it an ideal ingredient in nutritional

formulations for people suffering from hepatic diseases. It is also good ingredient for formulations

designed for people with PKU, since it contributes a minimal amount of phenylalanine.

Glycomacropeptide facilitates the production of conventional food products, such as protein bars, desserts

and UHT beverages and makes them an outstanding protein source for people with phenylketonuria

(PKU). GMP is a rich source of protein-bound sialic acid, an essential nutrient for infant brain

development and cognition. In infant formula, it is a natural substitute for the sialic acid present in human

milk. dietary sialic acid using GMP as a source of sialic acid improves learning and memory during early

development. Some of the added benefits of using GMP are linked to weight management, bone health,

compliance and better pH stabilization.

5. Selected Reading

Neelima, Sharma R, Rajput Y S and Mann B (2013) Chemical and functional properties of Glycomacropeptide

(GMP) and its role in the detection of cheese whey adulteration in milk: a review. Dairy Science and Technology

93 21–43.

Ceppa (2007) Review: isolation and purification of milk whey Glycomacropeptide. Curitiba. 25 121-132.

Ernest P B (2000) Biological activities of bovine Glycomacropeptide. British Journal of Nutrition. 84 39-46.

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Cholesterol Reduction Technologies for Milk and Milk Products

Vivek Sharma


1. Introduction

Milk is considered to be a complete food as it contains almost all essential nutrients required for human

health and growth. Lipids, the most important constituent of milk, play significant role in the nutrition,

flavour and physico-chemical properties of milk and milk products. They are also rich source of fat-

soluble vitamins (A, D, E & K) and essential fatty acids, apart from having pleasant sensory attributes.

Milk fat is easily digestible than other oils and fats. It contains number of components which show

anticarcinogenic activity, e.g. sphingomyeline, conjugated linoleic acid, -carotene etc. So one (especially

vegetarians) cannot avoid it in one’s diet. But recent trend, in the society, is against fat-rich dairy

products due to the presence of saturated fat & cholesterol as these are known to increase the incidence of

coronary heart disease (CHD). CHD is one of the common causes of heart attack. Through a period of

time, many researchers have shown that dietary cholesterol, serum cholesterol and occurrence of coronary

heart disease (CHD) have positive correlation. Milk fat contains about 0.25 to 0.40% cholesterol.

Consumption of ghee and other fat-rich dairy products makes appreciable contribution to cholesterol

intake. Furthermore, some cholesterol oxidation products (COPs) have been reported to be more harmful

than cholesterol itself as they are cytotoxic, atherogenic, mutagenic and carcinogenic. In recent years,

demand of cholesterol-free foods has increased tremendously. This has led to increase in market of

margarine, vegetable fat filled dairy products, milk fat replaced dairy products, etc. Owing to the adverse

affects of cholesterol on human health, various physical, chemical and biological methods have been

developed for reducing cholesterol in foods including milk.

2. Cholesterol content in milk

Animal food products like milk and milk products, meat and meat products and eggs are the major

sources of cholesterol in our diet. Among these, chicken egg contains highest amount (about 215 mg/egg)

of cholesterol. Cholesterol accounts for 0.25-0.45% of the total lipids in milk. Cholesterol concentrates in

the milk fat globule membrane (MFGM). In milk, 80% of the cholesterol is associated with the milk fat

globules and the remaining 20% is partitioned into the skim milk phase where it is associated with

fragments of cell membrane (Patton & Jensen, 1975).

Pantulu and Murthy (1982) observed 8-10 times higher content of cholesterol in whey than in whole milk.

Srinivasan (1984) reported the average cholesterol content of cow and buffalo milk as 2.8 and 1.9 mg/g

fat, respectively. Bindal and Jain (1972) estimated free and esterified cholesterol in Desi ghee, using TLC

method and reported their contents as 0.288 and 0.038% and 0.214 and 0.056 % in cow and buffalo ghee,

respectively. Prasad and Pandita (1987) observed cholesterol content of ghee prepared from milk of

Haryana, Sahiwal and Sahiwal X Friesian cows and from Murrah buffaloes, to be 303, 310, 328 and 240

mg/100 g fat, respectively.

3. Approaches to reduce cholesterol in milk

Since dairy products contain significant amounts of cholesterol, a number of processes for removal of

cholesterol have been developed to produce low-cholesterol dairy products. These include steam

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stripping, molecular distillation, solvent or super-critical extraction, reaction with cyclic anhydride,

enzymatic method and treatments with adsorbents like saponin, activated charcoal and cyclodextrin.

These are briefly discussed below.

3.1 Steam Stripping

This process is similar to that used in the deodorization of vegetable oils and removal of unsaponifiable

matter. To remove cholesterol by steam stripping, the fat is first deareted under vacuum after which it is

heated with steam upto 2320C and then subjected to steam at low pressure in cylindrical tall chamber. The

anhydrous milk fat (AMF) passing over a series of plates is spread in many thin layers, which increases

the stripping efficiency. The steam rises and carries with it the evaporated cholesterol to be condensed

and collected with other volatiles. This process can remove upto 93% of cholesterol though with 5% fat

losses. The major disadvantage to the process is that it removes flavouring compounds also (Schlimme &

Kiel, 1989).

3.2 Molecular Distillation

In this process, AMF is molecularly distilled at temperature 190 and 210oC at a vacuum of 10

-4 Torr.

Fractions distilled at 190 and 210oC represented 3.43 and 3.99 % of the initial mass and contained more

than 93 % of the total cholesterol (Lanzani et al, 1994 and Sharma et al., 1999). Arul et al. (1988)

fractionated AMF into four fractions at temperatures of 245 and 2650C and pressure of 220 and 100 mm

Hg. Two low melting point fractions were blended together to yield a total of three fractions (liquid,

intermediate and solid). About 78% of the total cholesterol was found in the liquid fraction while the

remaining was found in the intermediate (18%) and solid (4%) fractions in the esterified form. But,

because of the high heat used in the process, the quality of the end product is adversely affected.

3.3 Solvent Extraction

In this process butter oil is mixed with propane and ethanol in the mixing vessel. The low viscous mixture

of butter fat, ethanol and propane is fed into the extraction column. A mixture of ethanol and water,

containing a small amount of propane is used as extractant. The extract, a solution of cholesterol and

butter fat in a mixture of ethanol, water and some propane is withdrawn at the bottom of the extraction

column, which is splitted into two phases. The upper phase consists of fat and cholesterol, which are

subsequently separated, in a further processing step. Around 90 to 95% of the cholesterol is extracted in

this counter-current procedure operated at 30oC and 10 bar (Czech et al., 1993).

3.4 Supercritical Carbon Dioxide Extraction

Some studies have shown that supercritical carbon dioxide (SC-CO2) can be used to fractionate AMF

with evidence that cholesterol can be concentrated into selected fractions. Kaufmann et al., (1982)

obtained two fractions of milk fat by SC-CO2 extraction at a pressure of 200 bars and temperature of

80oC. In this process, the liquid fractions were enriched in total cholesterol. However, Huber et al. (1996)

observed that direct supercritical extraction of cholesterol from AMF is not feasible because of the low

selectivity of cholesterol and poor solubility of AMF. Moreover, under these conditions, important milk

flavours also get separated with the cholesterol. Therefore, they proposed another process for cholesterol

removal from AMF, dissolved in SC-CO2 under high solubility conditions for AMF (40 MPa at 70oC) to

achieve rapid extraction. In this process, the dissolved AMF in SC-CO2 is passed isobarically and

isothermally through a high-pressure column, filled with a suitable adsorbent (e.g. silica gel) to eliminate

cholesterol. Finally, the supercritical mixture is fractionated by either descending or ascending

temperature profile in separators connected in series. Karkare and Alkio (1993) found that over 99% of

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cholesterol from milk fat could be removed using an SC-CO2 extraction system equipped with a silica gel

column.

3.5 Reaction with Cyclic Anhydride

Gu et al. (1994) developed a method for cholesterol removal from milk fat based on the reaction between

the hydroxyl group of cholesterol and a cyclic anhydride such as succinic anhydride. The conversion of

cholesterol into an acid derivative makes it possible to remove these from fats by extraction with aqueous

alkali. Addition of acetic acid increases the rate of reaction and prevents the distillation of cyclic

anhydride from reaction mixture. They removed 50% cholesterol from animal fats but alongwith it -

tocopherol (50%), - and - lactones also get removed.

3.6. Enzymatic Method

McDonald et al. (1983) have described an enzymatic process using cholesterol reductase for conversion

of cholesterol to biologically inactive, e.g., non-toxic, non-absorbable products like coprosterol, which is

either not or is only poorly adsorbed by the body. This approach, which is theoretically suitable for

reducing the cholesterol content of milk fat, has been verified biologically at least in part, by the finding

that a portion of the intestinal cholesterol is reduced to coprosterol by intestinal bacteria and subsequently

eliminated.

3.7 Adsorption Methods

Cholesterol can be removed by its adsorption on certain material. Adsorbents, which are used to remove

cholesterol, are activated charcoal, saponins and -cyclodextrin.

3.7.1 Activated charcoal

Bindal et al., (1994) could remove half of the cholesterol present in milk fat through treatment of liquid

fat with activated charcoal. Another activated charcoal method claimed 95% of cholesterol removal from

AMF but many other compounds including yellow pigments were also removed simultaneously (Sharma

et al., 1999).

3.7.2 Saponins

Saponins are naturally occurring plant compounds that can be used to selectively bind to cholesterol and

precipitate it out. 80% and 90% cholesterol reduction in cream and anhydrous milk fat was obtained by

using this method (Riccomini et al., 1990). Oh et al. (1998) found 70.5% of the cholesterol removal when

milk was treated with 1.5% saponin at 45oC for 30 min. Further, addition of 0.25% celite increased

cholesterol removal to 72%. However, the methods using activated charcoal or saponins are relatively

non-selective and remove flavour and nutritional components also when cholesterol is removed (Lee et

al., 1999; Sharma et al., 1999).

3.7.3 -cyclodextrin

Beta cyclodextrin, one of the well known members of cyclodextrin family, is a cyclic oligosaccharide of

seven glucose units joined ‘head to tail’ by -1, 4 linkage and is produced by the action of enzyme

cyclodextrin glycosyl transferase (CGT) on hydrolyzed starch syrup. Beta cyclodextrin has torus like

structure (Figure B). The central cavity is hydrophobic, giving the molecule its affinity for non-polar

molecules such as cholesterol (Szejtli, 2004). The radius of the cavity can accommodate a cholesterol

molecule almost exactly, explaining the highly specific nature of -cyclodextrin’s ability to form an

inclusion complex with cholesterol (Hettinga, 1996).

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Seth and Singh (1994) observed that buffalo milk samples on treatment with beta-cyclodextrin at 1, 2 and

4%, the cholesterol level was reduced from 36 mg to 25 mg, 19.5 mg and 8 mg per 100 ml, respectively,

corresponding to 30.5, 45.8 and 77.6% reduction, respectively. Lee et al., (1999) studied the cholesterol

removal from homogenized milk and reported that 92 – 95% cholesterol removal from milk by using -

cyclodextrin at 0.5-1.5% level. Ahn and Kwak (1999) used -cyclodextrin for cholesterol removal in

cream. Average cholesterol removal increased as -cyclodextrin concentration increased, regardless of

other factors. At 10% of -cyclodextrin level, the effect of stirring speed was more noticeable than that of

stirring time. When stirring was 1600 rpm and stirring was 20 min, the cholesterol was reduced by

97.99% using 15% -cyclodextrin. Recently NDRI has developed a process to remove cholesterol from

ghee, where removal of about 90% cholesterol has been reported by using -cyclodextrin in the range of 1

to 7%.

4. Selected Reading

Ahn J and Kwak H S (1999) Optimizing cholesterol removal in cream using beta-cyclodextrin and response surface

methodology. Journal of Food Science 64 629-632.

Bindal M P, Wadhwa B K, Lal D, Rai T and Aggarwal P K (1994) Removal of cholesterol from milk and milk

products: Application of biotechnical processes. NDRI Annual Report. 98-99.

Gu Y F, Chen Y and Hammond E G (1994) Use of cyclic anhydrides to remove cholesterol and other hydroxy

compounds from animal fats and oils. Journal of American oil chemists society 71 1205-1207.

Hettinga D (1996) Butter. in Bailey’s industrial oil and fat products, Vol. 3, 5th

edn, (eds Y. H. Hui), John Wiley &

Sons, INC. New York, 1-23.

Huber W, Molero A, Pereyra C and Martinez de la Ossa E (1996) Dynamic supercritical carbon dioxide extraction

for removal of cholesterol from anhydrous milk fat. International Journal of Food Science and Technology 31

143-151.

Lanzani A, Bondioli P, Mariai C, Folegatti L, Venturii S, Fedeli E and Barreteau P (1994) A new short-path

distillation system applied to the reduction of cholesterol in butter and lard. Journal of American

oil chemists society 71 609-614.

Lee D K, Ahn J and Kwak H S (1999) Cholesterol removal from homogenized milk with beta-cyclodextrin. Journal

of Dairy Science 82 2327-2330.

Oh H I, Chang E J and Kwak H S (1998) Conditions of the removal of cholesterol from milk by treatment with

saponin. Korean Journal Dairy Science 20 253-260.

Pantulu P C and Murthy M K R (1982) Lipid composition of skimmed milk and whey. Asian Journal of Dairy

Research 1 17-20.

Riccomini M A, Wick C, Peterson A, Jimenez-Flores R and Richardson T (1990) Cholesterol removal from cream

and anhydrous milk fat using saponins. Journal of Dairy Science 73 107.

Seth R and Singh A (1994-95) Removal of cholesterol from milk using -cyclodextrin and preparation of milk

products from such treated milk. NDRI Annual Report. 95.

Sharma R, Nath B S and Lal D (1999) Approaches for cholesterol removal from milk fat: An overview. Indian

Journal of Dairy and Biosciences 10 138-146.

Szejtli J (2004) Cyclodextrins. In Chemical and Functional Properties of Food Saccharides (eds Piotr Tomasik),

CRC Press, Boca Raton, 271-289.

https://www.google.co.in/search?biw=1517&bih=714&q=journal+of+american+oil+chemists+society&sa=X&ved=0CBkQ7xYoAGoVChMIsN292-fLyAIV0AmOCh3pzAze



131

Rapid Diagnostic Tests for Detection of Milk Adulterants – Current Status

Rajan Sharma, Bimlesh Mann, Satya K and Dhiraj Kumar Nanda


1. Introduction

The menace of adulteration has taken serious proportion as highlighted by many media reports as well as

by the report of Food Safety and Standards Authority of India (FSSAI). During festival as well as summer

season when there is short supply of milk, the electronic media is flooded with reports of adulteration of

milk and milk products. Although, India is largest milk producer in the World with 140 Million MT

(2013-14) of milk production per annum, the scarcity of milk is felt during lean season and festival days.

Compositional differences in milk are exploited by unscrupulous persons to adulterate milk. Perhaps,

addition of water to buffalo milk is most commonly practiced. The image of milk has been considerably

deteriorated due to the reports of its adulteration with harmful chemicals such as fertilizers, ammonium

salts, potassium sulphate, causitc soda, detergents, urea etc. Media reports indicate the adulteration of

milk with ‘milk-like-preparation’ – the so called synthetic milk. Table 1 indicates the type of common

adulterants of milk and motive behind their addition to milk. The problem of milk adulteration has also

been noticed in many other parts of the world. Table 2 present a data of type of adulterants used in other

countries.

Table 1. Common adulterants reported in milk and their purpose

Nature of

chemical/adulterants

Name of adulterants Purpose

Carbohydrate Sucrose, Glucose, Starch,

Maltodextrin To falsely increase the total solids

To mask the addition of water

Salts and fertilizers Urea, Ammonium sulphate,

NaCl etc. To falsely increase the total solids

To mask the addition of water

Neutralizers NaOH, Na2CO3, NaHCO3 etc. To mask the increase in acidity and to

prevent coagulation of milk during

heating

Preservatives Hydrogen peroxide, Formalin,

Boric acid etc To fraudulently elongate the shelf-life

of milk

Detergents Liquid detergents, Washing

powders etc To emulsify the extraneously added

fat/oil

Water Water/Pond water/ To increase the volume of milk

Extraneous fat/oil Vegetable fats/oils/Refined oil To increase/substitute fat content of

milk

Miscellaneous Soya milk, Cheese whey,

Synthetic milk To substitute milk with cheaper milk-

like ingredients

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Table 2. Adulterants reported in milk of other countries

Name country/region Type of adulterant reported

Pakistan, Bangladesh, Sri

Lanka

Water, Urea, detergent, formalin, H2O2, Neutralizers, Starch, Flour,

vegetable oil, formalin, quaternary ammonium (QAC) compounds,

Cane sugar, sorbitol, salt, boric acid, hypochlorite

China Melamine

Brazil

Water, Neutralizers, Sodium citrate, non-acid cheese whey, synthetic

milk

Europe

Cheese Whey , Milk fat adulteration

Species adulteration of Milk

2. Methods for detection of adulterants in milk and milk products

The rapid tests for the detection of adulterants in milk are required as these tests will act as deterrent for

unscrupulous persons involved in unscrupulous activities as well as a tool in the hands of FSSAI officials

to eradicate the menace of adulteration. At present, the methods which are being used for the detection of

adulteration in milk are mainly drawn from those listed in Bureau of Indian Standards (IS: 1479 (Part 1-

1960, Reaffirmed 2003) or other publications being brought by National Dairy Research Institute (NDRI),

Karnal. Many of these methods have been modified by researchers, quality control personnel working in

dairy laboratories and other commercial laboratories. Many organizations including NDRI, Karnal,

National Dairy Development Board (NDDB), Anand and other private firms have come up with easy-to-

carry kits for detection of adulterants in milk. In all these commercially available kits, the methodology

used has been mainly adopted from BIS or NDRI Publications. It is also true that, unscrupulous people

are finding more innovative ways to adulterate milk with cheaper ingredients. In the last decade, the

adulteration of milk with milk-like-preparation popularly known as ‘synthetic milk’ has surfaced.

NDRI is working proactively for developing various analytical techniques and simpler methodology for

the detection of adulteration in milk and milk products. A kit developed in the Division of Dairy

Chemistry for the detection of various adulterants in milk is in high demand among dairy professionals

and public analysts across the country. The kit contains reagents for detection of 12 adulterants in milk

viz., neutralizers, urea, pond water, starch, sugar, glucose, maltodextrin, salt, formalin, ammonium

compounds, hydrogen peroxide, hydrogenated vegetable oil, etc. In the past, a rapid method developed, at

the Institute, for the detection of addition of vegetable/refined oil in milk has been validated and adapted

by Bureau of Indian Standards (BIS). Similarly, the qualitative and quantitative methods developed for

the presence of added urea in milk has been adapted by BIS. A simple test developed for the detection of

the presence of detergent in milk has been included in the analytical methods recommended by FSSAI.

Recently, a new colour based test has been developed for the rapid detection of detergent in milk. NDRI

is also working in the development of strip based tests for detection of various adulterants in milk and a

significant development has occurred in this direction.

3. Detection of common adulterants in milk – recent development

3.1 Chemical methods

3.1.1 Strip based tests for rapid detection in of adulteration milk

Five different paper based strip methods have been developed for the rapid detection of neutralizers, urea,

glucose, hydrogen peroxide and maltodextrin in milk using the concept of dry chemistry. These strip

based method involves either dipping the strip (neutralizer and urea) in milk sample or applying a drop of

milk on the developed strip (glucose, hydrogen peroxide and maltodextrin) followed by visualization of

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colour. The colour of the strip changes to deep red in milk containing neutralizers (immediately) and urea

(after 2 min) while in pure milk samples, the strip retained its original yellow colour (Patent application

No. 3472/DEL/2013). In case of glucose and hydrogen peroxide detection in milk, the strip colour

changes to deep pink in adulterated milk samples either immediately (hydrogen peroxide) or after 5 min

(glucose) while the strip retained its original white colour in case of pure milk. Maltodextrin detection

strip (Patent application No. 2097/DEL/2014) changes to yellowish green colour after 5 min in case of

adulterated milk samples while in case of pure milk strip remained white in colour. The strip based tests

have been validated at third party laboratory as well under filed conditions. The sensitivity of the strips

has been established and limit of detection of the developed strip in milk was 0.04% for NaOH (0.06%

Na2CO3, 0.1% NaHCO3); 0.08% for urea (total urea); 0.03% for glucose; 0.005% for H2O2 and 0.05% for

maltodextrin. The developed strips were easy to use and require just one step wherein either they are

dipped in milk sample or a drop of milk sample is applied to the prepared strip followed by visualization

of change in colour. In all cases, sensitivity of the prepared strips was found better than the existing wet

chemistry based tests. The shelf-life of strips is 8 months at room temperature for neutralizer, 4 months at

refrigeration temperature for urea, 2 months for hydrogen peroxide, glucose and maltodextrin. The

technology of strip based tests is available for commercialization.

3.1.2 Detection of Detergent in Milk

Detergents are considered as the essential component of the formulation being used for the preparation of

synthetic milk. Because of ease in availability of anionic detergent, these are being used for

emulsification of added fat of non-milk origin. The other ingredients being used for synthetic milk

formulation are urea, salt, soda, sucrose, vegetable oils, skim milk powder, water etc. The liquid thus

formed has the appearance of genuine milk (i.e. colour, consistency) and it is reported to be used for the

adulteration of dairy milk from 5 to 10%. The detection of detergent in milk is therefore essential for

checking the adulteration of milk with synthetic milk. A qualitative rapid and sensitive test for detection

of detergent in milk has been developed. The method is primarily based on the ionic interaction between

the anionic detergent and cationic dye. Anionic detergents have a property to form a complex with

cationic dyes. The solubility of dye and dye-detergent complex differs significantly as dye-detergent

complex is relatively less polar in comparison to dye alone. Formation of dye-detergent complex between

cationic dye and anionic detergents and subsequently its extraction into the hydrophobic solvent is the

major principle behind the developed method. The method is sensitive to detection 20 mg of detergent in

100 ml of milk. The test can detect all brands of detergent available in the market and sensitivity varies

with different brands of commercial detergent. The method has been validated at Punjab Biotechnology

Incubator, Mohali – a NABL accredited laboratory. The test can detect all brands of commercial

detergents available in the market. The test is nearly five times more sensitive than paper

chromatographic method. The results are available in 2 min. The test does not require any equipment and

the cost of ingredient used for preparation of test reagent is very low. The technology of this test (Patent

Application No. 3677/DEL/2011) is available from NDRI, Karnal.

3.2 Instrument based methods

In recent times two different types of equipments have been launched for the rapid detection of

adulterants in milk. In the first type of equipment named MilkoScreenTM

, the detection is based on the

concept of FT-IR (Fourier Transform infrared spectroscopy) has been used. The machine has been

launched by IndiFoss Analytical Ltd. Ahmadabad (www.indifoss.com) which is part of FOSS, Denmark.

The machine has been claimed to detect urea (≥ 0.25%), melamine (≥ 0.1%), ammonium sulphate (≥

http://www.indifoss.com/

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0.1%), sucrose (≥ 0.7%) and water (≥ 20%). In the second type of equipment launched by Rajasthan

Electronics and Instruments Ltd. Jaipur, named “EMAT (Electronic Milk Adulteration Tester)”, the

principal of detection is based on identification of specific ions of adulterants in milk sample. The

machine has been claimed to detect urea (≥ 0.1%), salt (≥ 0.2%), liquid soap (≥ 0.2%), detergent (≥

0.3%), caustic soda (≥ 0.3%) and hydrogen peroxide (≥ 0.3%).

4. Adulteration of milk products

Among the milk products, ghee is the most common milk product being adulterated. Because of high cost

of milk fat and availability of look-alike cheaper substitutes (vegetable oils, hydrogenated vegetable oils),

ghee is frequently adulterated. The other milk products which are being adulterated in this country are

khoa, khoa based sweets, paneer etc. Table 3 indicates the types of adulterants reported to be added in

various milk products.

Table 3. Adulterants added in various types of milk products

Milk product Adulterant (s) reported to be added

Ghee Vegetable oil hydrogenated vegetable oil, animal body fat, designer

oil, mineral oil etc.

Paneer Edible and non-edible oil, starch etc.

Khoa and khoa based

sweets

Edible and non-edible oil, starch etc.

Milk Powder Maltodextrin, whey powder

The tests has also been developed for the detection of adulteration of milk products such as ghee, khoa

and paneer etc. The adulteration detection tests developed for the milk either has been validated or

modified for their application in products like khoa and panerr. For the detection of vegetable oil in ghee,

a simple chemical method has also been developed. Due to the natural variation in the composition of

milk fat and also due to the range of available cheap adulterants (body fats as well as vegetable oils), a

combination of tests needs to be applied for confirming the purity of milk fat. Apart from the

conventional tests (e.g. Reichert Meissl value and Butyro-refractometer reading), new generation of tests

such as Apparent Solidification Time (AST), Opacity Test, TLC, HPLC etc. have been developed at this

Institute. In recent times, using the concept of solvent fractionation technique have been developed for

combating the menace of adulteration wherein after enriching the adulterant in a particular fraction,

conventional tests are applied for ascertaining the presence of adulterant.

5. Conclusion

Milk and milk products, since time immemorial, have formed an important part of our diet. Milk is

naturally designed as a nutrient dense food source that nourishes and provides immunological protection

for mammalian offspring. The media reports about adulteration of milk and milk products certainly would

discourage the consumption of milk and thus would deprive people of such a valuable healthy

commodity. Although, methods exist for detection of common adulteration in milk, every dairy industry

in India is not using these methods at reception dock. The reason may be a long list of adulterants and

problem is further aggravated by entry of new adulterants. The industry at times finds it difficult to screen

every tanker/can of milk for all the listed adulterants as most of the methods involve chemists as well as

investment in the form of reagents/glassware. Consumers in the country are also demanding simpler tests

with which they can ascertain the quality of milk they buy. The existing milk adulteration detection

methods are based on the wet chemistry which essentially requires mixing of milk sample with liquid

reagents and at times boiling. For avoiding hassles of purchase of chemicals and preparation of reagents,

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kits for detection of adulterants are now manufactured and are commercially available. Even these kits are

not to be used at house-hold level. The development of such strip based tests is need of the hour as use of

such kits will provide results in short duration and results can be interpreted by simply observing colour

change of strip by naked eye. Also there will not be requirement of skilled person of applying the test at

house-hold level. Research organizations and private industry should work together to develops such

types of tests.

6. Selected Reading

BIS (1960) IS: 1479. (1960), Reaffirmed 2003. Methods of test for dairy industry. Part I. Rapid examination of milk.

Bureau of Indian Standards, New Delhi.

Patent Application No. 3677/DEL/2011. Barui, A. K., Sharma, R. and Rajput, Y.S. (2011). A qualitative and

quantitative test for detection of anionic detergent in milk and like. Indian Patent Office, New Delhi.

Patent Application No. 3472/DEL/2013. Sharma, R., Panchal, B.K. and Rajput, Y.S. (2013). A strip for detection of

added urea in milk and process for the same. Indian Patent Office, New Delhi.

Patent Application No. 2097/DEL/2014. Sharma, R., Rajput, Y.S., Narula, P., Thakur, R. and Kumar, B. (2014). A

strip for detection of maltodextrin in milk and process for the same. Indian Patent Office, New Delhi.

136

Spores / Enzymes Based Sensors–An Innovative Approach for Monitoring Food Safety

Naresh Kumar, Raghu H V, Pradip Kumar Sharma, Ankita Kalyan, Nimisha Tehri, Birjesh Kumar

and Spurthi, M

Dairy Microbiology Division

1. Introduction

Being a major constituent of the diet, quality Assurance of milk is considered essential to the health

and welfare of a community. However, the area of interest of developing nations is to provide enough

food to the people rather than quality and hygiene of the food (Ellis & Sumberg 1998). They rely on

general practices such as pasteurization and refrigeration to check microbial proliferation in milk and

sometimes pass over these practices also due to lack of infrastructure and funds (Angulo et al. 2009;

Lues, et al. 2003). Milk is a balanced food stuff with a very low microbial load at the time milking but

various contaminants such as pathogenic organisms, antibiotics, pesticides, mycotoxins etc. enter

during various stages of production and processing (Angulo et al. 2009). The diseases spread through

contaminants is well known and the epidemiological impact of such diseases is considerable. The

testing of milk and milk products for presence of these contaminating agents has become a mandatory

practice for dairy industry before dispatching the products into the global market. Food safety

standards based on risk analysis have become bench mark in global trade in view of enhanced

consumers awareness linked with diseases and public health. The current conventional tools for

contaminants monitoring are time consuming and need innovative interventions for their surveillance

during production and processing stages in supply chain. These conventional methods, that generally

involve isolation and confirmation procedures for the detection of the microbes and other platform

tests that are carried to determine non-microbial contaminants of the milk which often are time

consuming and laborious to perform. The other associated problem with such tests is that the product

needs to be hold till the results come. To overcome this unmanageable situation dairy industry is

looking for alternatives to conventional methods. The methods that are rapid, cost effective, and easy

to perform and significantly validated with approved standard methods, are the need of the hour. Our

laboratory is playing a prominent role in this field by developing spore based detection systems for

monitoring microbial and non-microbial contaminants in milk and milk products.

2. Biosensors

Biosensors are devices that can combine a biochemical molecule with a physical signal that can be

translated into an indication of safety or quality of the food. Biosensors help carrying out the

procedures that are sensitive, selective, rapid, cost effective and portable. These devices are evolving

as excellent substitutes for the existing conventional techniques. But still operation of biosensors is a

challenging task for their utility owing to the cost and shelf life of bio-recognition molecule (Kumar et

al. 2013). The members of genera Clostridium and Bacillus have the aptitude to form endospores

during stress and starvation conditions. The dormant spore state is very much stable allowing them to

survive even for millions of years. The typical structure of spore is responsible for its survival ability

even millions of years in dormant state (Desnous et al. 2010) as shown in Fig.1.

137

Fig. 1 (a) Schematic diagram of structure of bacterial spores (b) Dormant spores of Bacillus

megaterium as observed under Phase contrast microscopy

The dormant bacterial spores having unique ability to sense environmental changes in response to

specific ‘‘germinant’’ and their transformation into growing vegetative cells in real time have

enormous scope for their application in bio-sensing of contaminants in food products. The spore

germination concepts involved the release of DPA / or marker enzymes and their action on specific

chromogenic / or fluorogenic substrates as a mean for detection of contaminants in dairy foods has

been explored successful to target antibiotic residues (Indian Patent No. 264145/09/12/2014),

Aflatoxin M1 (3064/DEL/2010) and pesticide residues in milk (patent filing under process). Spore–

enzyme-sensors were also developed for Enterococci (119/DEL/2012), L. monocytogenes

(1357/DEL/2013) and E. coli (2214 /DEL / 2014) in milk after pre-enrichment in selective liquid

medium. The developed spore based analytical devices are cost effective, robust and few such

products like antibiotic residues and other kits for listeria /enterococci were commercialized for its

application at farm levels/or manufacturing stage for screening of contaminants in dairy supply chain

in our country .

3. Spore based sensor for detection of antibiotic residues in milk: The developed technology is

working on principle of spore germination and its inhibition in presence of antibiotic residues in milk.

In case when antibiotic residues are absent in milk, the calcium dipicolinic acid (DPA) is released

during germination which change the color of the bromocresol purple (BCP) indicator from purple to

yellow (Indian Patent no. 264145). However, in presence of antibiotic residues in milk the spore

germination process is inhibited at ≥ MRL level of contaminants and no change in color of BCP

indicates the presence of drug residues in milk when incubated at 64oC for 2.50-3.00 hrs as shown in

Fig.2 (Kumar et al. 2014).

Fig. 2. Microbial drug residues (MDR) test kit for detection of antibiotic residues

138

3.1 Novel Features

o The Cost effective (Rs. 39.5 per test)

o Semi-quantitative detection at Codex MRL

o Validated with AOAC approved Charm 6602 Assay

o Minimal false positive / negative results

o No interference of inhibitors other than antibiotic residues

o Stability of test kits up to 12 months under refrigeration storage

o Test kit can perform at dairy farm, milk collection center, dairy reception dock and R&D

institutions

o Kit can be used for routine monitoring of milk as well as for regulatory compliance

4. Spore based sensor for detection of pesticides: Pesticides are well known carcinogen and their

impact on human beings and presence in different food products including milk are well known in the

prior art. In current innovation, spore based assay(s) which exploit the principle of “spore germination

and enzyme inhibition” for detection of pesticide residues was successfully developed and

miniaturized for their application under field conditions. Three step assay protocol which involves

spore germination; pesticide exposure; enzyme-substrate reaction was developed (Fig.3). Using this

protocol all marker enzymes were screened for their inhibition by pesticide .The optimized 3 step

assay protocol was evaluated for LOD after spiking pesticides in acetonitrile and LOD for

organophosphorous (OP), carbamate (CM) and organochlorine (OC) were observed at 10 ppb-10

ppm, 1-10 ppm and 100 ppm respectively. This protocol was also transformed in a colorimetric 96

well plate assay with LOD in a range of 1-100 ppb, 100 ppb and 50 ppm for OP, CM and OC group.

The protocol developed in tube was further attempted to miniaturize on paper strip for exploring its

potential for field application. Nine different types of paper were screened for optimal enzyme

substrate reaction and assay showed sensitivity in the range of 10ppb-1ppm for OP, 10ppm for CM

and 100ppm for OC pesticides (Patent filing under process). The specificity of developed assay was

evaluated by checking for cross reactivity with other inhibitors, however, marker enzyme activity was

found unaffected by antibiotics, aflatoxin, detergents, heavy metals, preservatives and sanitizers.

Subsequently, a protocol for extraction of pesticides from food sample i.e. spiked reconstituted milk

samples was optimized. Three step Spore based assay" miniaturised on 96 well plate /paper strip was

found useful in detection of OP and CM group of pesticides employing spores as an inexpensive

source of marker enzyme(s) alternative to acetylcholinesterase enzyme which is present only in

eukaryotic system. Developed spore based assay can be of immense use for food industry , R & D

institutions and can also be applied under field condition since it does not require much expertise and

is cost effective when compared with existing prior-art .

139

Fig. 3 Spore based sensor for detection of pesticides

5. Two stage enzyme assay for detection of L. monocytogenes: The two-stage assay was developed

for detection of L. monocytogenes based on the principle of targeting “enzyme-substrate reaction for

the specific marker enzyme (s) to release free chromogen that can be visually detected by color

change. The assay can confirm the presence of L. monocytogenes within real time of 4.30±0.10h after

initial pre-enrichment of food samples in novel selective medium, i.e., LSEM for 18/or 24 as against

5-7 days protocol following conventional method .The detailed test procedure is depicted in Fig.4.

Fig 4: Two stage enzyme assay for detection of L. monocytogenes

5.1 Observations

o Colour change from yellow to black indicates the presumptive detection of Listeria spp. i.e.

24±2 hour at 1.4 log cfu levels for 25g / or 23.15±1.0 hour per g of the milk sample

o Appearance of green color in T-1 indicates the confirmation of L. monocytogenes and yellow

color in T-2 indicates Listeria spp. (Balhara et al. 2014)

5.2 Novel Features

o Cost effective enzyme assay (Rs. (75) per test against Rs. (762.00) in conventional method)

o Rapid detection within one working day as against 5-7 days protocol in conventional method

o Novel LSEM medium with selective inhibition of contaminants like E. faecalis, S. aureus, B.

cereus, Lactobacilli and members of Enterobacteriaceae family

140

o Two stage assay can be used for regulatory compliance of food samples including dairy

products as specified in FSS Act. 2011

o Internal Lab Validation with conventional method (ISO: 11290 Part-1: 1996)

o Third party validation at M/s. SGS India Pvt. Ltd. Gurgaon (certificate no. SGS GG12-

009772.001 dated 09-11-2012).

o Technology was licensed to M/s. Neugen Diagnostic Pvt. Ltd. Hyderabad with a Non-exclusive

license.

6. Enzyme based assay for Detection of E. coli / Coliforms in Milk

Two-stage test was developed for detection of E. coli based on the principle of targeting “enzyme

substrate reaction for specific marker enzyme (s) to release free chromogen in stage-1, which can be

visually detected by a color change after 12.0±1.0h of incubation in E. coli selective medium

(ECSM). In stage-2 using specific enzyme substrate mixture, confirmation of E. coli can be achieved

within 3.0±0.15 h as shown in Fig.5. The developed test can be used in dairy industry for routine

detection of E. coli in milk and milk products for regulatory compliance (Kumar et al. 2014). In

another media called as Coliform selective media (CSM), 0.1 ml of spiked milk or natural milk in 0.9

ml of lyophilized CSM (reconstituted with distil water pH 7.0) is inoculated and incubated at 37±1oC

for 12±1.0 hour. Appearance of yellow color confirms presence of coliforms as depicted in Fig.5.

Fig. 5. Enzyme assay for detection of E. coli / Coliforms in Milk

141

6.1 Novel Features

o The developed enzyme assay for E. coli can confirm <1.0 log cfu/ mL within 12.00±1.0 hour of

incubation as against 3-4 days protocol following conventional method (IS: 5887 Part-1: 1976)

o Selective inhibition of contaminants like Salmonella, Shigella, Yersinia, Proteus, Serratia,

Citrobacter, Enterobacter, L. monocytogenes, B. cereus, S. aureus , L. casei other than E. coli spp.,

hence no chance of false positive results.

o The developed enzyme assay for coliforms can confirm <1.0 log cfu/ml within 12.15 ± 0.30 h of

incubation as against 2-3 days protocol following conventional method (IS: 5401 Part-2: 2002)

o Selective inhibition of non-coliforms like Salmonella, Shigella, Yersinia and Proteus

o Wide scope of application to raw, pasteurized and dried milks for routine as well as for regulatory

standard compliance

o Lab Validation of developed kit with IS: 5887 Part-1:1976 using raw, pasteurized and dried milk.

7. Enzyme based assay for detection of enterococci in milk

The two stage enzyme assay for detection of Enterococci in milk involves application of specific

enzymatic reaction in selective medium. The marker enzyme which participates in unique biochemical

pathways of specific genera or strain hydrolyze chromogenic substrate complex and release free

chromogen which can be detected visually by color change (Fig.6). The developed technology for

detection of Enterococci is a two stage enzyme assay wherein the appearance of black color in stage-1

gives presumptive identification of Enterococci in developed selective enrichment medium i.e.

Esculin Based Sodium Azide Medium (EBSAM) In stage-2 using a specific Enzyme Substrate

Mixture (ESM), appearance of yellow and orange red color in tube-1 & tube-2 respectively indicates

confirmation of Enterococci in milk samples. Currently, commercially available media like citrate

azide agar need an incubation period of 72-96 h for detection of Enterococci in milk. Development of

this new “Two stage Micro Technique” is to facilitate its use in R &D institutions and dairy industry

for rapid detection of Enterococci. The technology has potential to replace the existing medium for

Enterococci for being cost effective (Rs. 98.3 per litre as against Citrate azide agar (CAA) available @

Rs. 262.5 per Litre, Bile Esculin azide agar available @ Rs. 493.5per litre.

142

Fig. 6 Enzyme based assay for detection of enterococci in milk

7.1 Observations

o Presumptive detection of enterococci in milk. Stage 1: Appearance of black color in stage-1 gives

presumptive identification of Enterococci in developed selective enrichment medium i.e.

EBSAM.

o Confirmatory detection of enterococci in milk. Stage 2: In presence of specific ESM, appearance

of yellow and orange red color in tube-1 (T1) & tube-2 (T2) respectively, indicates confirmation

of Enterococci in milk sample. (Kumar et al. 2012).

7.2 Novel features:

o The EBSAM medium is specific for growth of Enterococcus spp. and appearance of black color

after 18.45 ± 0.15 and 24.0 h indicates Enterococci counts >1000 and 100 cfu/mL respectively.

o The working performance of enzyme based bio-assay was compared with IS: 5887 Part-2 (3 days

protocol) and results were found in accordance with the claims of the developed enzyme based

assay.

8. Conclusions

The living phase of bacterial spores revolves around two phases i.e., the dormant state and the

metabolically active vegetative state. This conversion from one phase to another phase is completed

only if spore senses favorable conditions in the environment and presence or absence of microbial or

non-microbial contaminants directly or indirectly affect this conversion. This phenomenon can

therefore be targeted to sense the presence of contaminants in milk and hence develop spore based

biosensor systems. A number of spore based sensing system have been developed to detect aflatoxins,

antibiotics and microbial pathogens in milk. These biosensing systems are superior over existing

methods in terms of better sensitivity, low cost and help on site rapid analysis of milk and milk

products. The spore based biosensor is a novel strategy being exploited to ensure safe and healthy

milk to each consumers.

9. Selected Reading

Angulo F J, LeJeune J T and Rajala-Schultz PJ (2009) Unpasteurized Milk: A Continued Public Health Threat,

Clinical Infectious Disease 48 93-100.

Balhara M, Kumar N, Thakur G, Raghu H V, Singh V K, Lawania R and Khan A Shabnam (2014) Novel

enzyme-substrate based bio-assay for real time detection of L. monocytogenes” in milk system”. Indian

143

Patent reg. No. 1357/DEL/2014. Published in Indian patent Journal Publication no. 50/2014 date 12th

Dec

2014.

Desnous C, Guillaume D and Clivio P (2010) Spore photoproduct: a key to bacterial eternal life. Chemical

Reviews 110 1213-1232.

Ellis F and Sumberg J (1998) Food production, urban areas and policy responses, World Development, 26 213-

225.

Kumar N, Kaur G, Thakur G, Raghu H V, Singh N A, Raghav N and Singh V K (2012). Real-time detection of

Enterococci in dairy foods using spore germination based bioassay. Indian patent publication no. 19/2015

Date: 08/05/2015 (119/DEL/2012).

Kumar N, Lawaniya R, Avinash Arora B, Raghu H V, Balhara M, Kadyan S and Singh V K (2014) Spore-

sensor for rapid detection of E. coli in milk employing marker enzymes and marker sugar as germinants.

Patent: Reg. IPR 2214/DEL/2014.

Kumar N, Patil G R, Rane S, Malik R K (2014) A Novel Process of Sporulation, Activation and Germination in

Thermophilic Bacteria for Rapid Detection of Antibiotic Residues in Milk. Indian Patent No. 264145

dated 9.12.2014.

Kumar N, Thakur G, Raghu H V, Singh N, Sharma P K, Singh V K, Khan A, Balhara M, Avinash Lawaniya R,

Kouser S, Tehri N, Gopaul R and Arora S. (2013) Bacterial Spore Based Biosensor for Detection of

Contaminants in Milk. Journal of Food Processing Technology 4 277.

Lues J, Venter P and Westhuizen V (2003) Enumeration of potential microbiological hazards in milk from a

marginal urban settlement in central South Africa, Food Microbiology 20 321-326.

Kumar N, Tehri N, Gopaul R, Sharma P K, Kumar B, Morab S, Raghu H.V. Rapid spores-enzyme based

miniaturised assay (s) for detection of pesticide residues. Patent filing under process.

Prandini A, Tansini G, Sigolo S, Filippi L, Laporta M and Piva G (2009) On the occurrence of Aflatoxin M1 in

milk and dairy products. Food. Chemical Toxicology. 47 984-991.

Shundo L, Navas S A, Lamardo L C A, Ruvieri V and Sabino M (2009) Estimate of Aflatoxin M1 exposure in

milk and occurrence in Brazil. Food Control 20 655-657.

144

Bio-Functional Applications of Lactic Acid Bacteria

Shilpa Vij and Jagrani Minj


1. Introduction

Lactic acid bacteria (LAB) are the main group of microorganisms that has been used successfully for

decades for the production of fermented milks as they are producing different biofunctional

components such as organic acids, exopolysaccharides, bioactive peptides, folate, oligosaccharides,

dietary sugars, vitamins etc. These organisms belong to the genera of Lactococcus, Leuconostoc,

Pediococcus, Streptococcus and Lactobacillus. LAB are industrially important microbes that are used

all over the world in a wide variety of industrial food fermentations. They are excellent ambassadors

for an often maligned microbial world. Now a day functional lactic acid bacteria are recognizing as

probiotic bacteria. As probiotic bacteria are live microorganisms which when administered in

adequate amounts confer a health benefits to the host. The health benefits may be treatment of lactose

intolerance, antioxidant, antidiarrheal, antimicrobial, immunomodulatory, antidiabetic, anticancer,

antiallergic, toxin neutralizer, antiobesity, hypocholesterolemic, blood pressure lowering etc.

Sometimes even one lactic acid bacteria exhibits more than two functional aspects simultaneously

such as antimicrobial and immune stimulatory activity. In the same way, food derived bioactive

peptides which are produced by proteolytic lactic acid bacteria exhibit many functional role.

Peptidomics study reveals that specific peptide sequences have a unique functional property. Peptide

sequences show antimicrobial activity, antioxidant activity and ACE-inhibitory activity. It is well

known that ACE is a multifunctional enzyme that plays an important role in the regulation of blood

pressure and Coronary heart diseases. High blood pressure and heart related diseases are life style

related diseases which is increasing day by day in western countries as well as in India. ACE peptides

were also found in fermented milk products made with Lactobacillus helveticus.

2. Antimicrobial compounds

2.1 Organic Acids

Organic acids occurring in foods are additives or end-products of carbohydrate metabolism of LAB.

Lactic and acetic acids are the main products of the fermentation of carbohydrates by LAB. These

acids, generally recognised as safe agents for the preservation of foods diffuse through the membrane

of the target organisms and ultimately inhibit their growth.

2.2 Bacteriocins

Bacteriocins are bacterial ribosomally synthesized peptides or proteins with antimicrobial activity and

kill very closely related bacteria upon binding to the inner membrane or other cytosolic targets.

Nowadays, the term bacteriocin is mostly used to describe the small, heat-stable cationic peptides

synthesized by Gram positive bacteria, namely lactic acid bacteria (LAB), which display a wider

spectrum of inhibition. LAB produce different types of bacteriocins against their closely related

organisms. Based on their cationic and their hydrophobic nature, most of these peptides act as

membrane permeabilizers. Pore formation leads to the total or partial dissipation of the proton motive

force, ultimately causing cell death. Bacteriocin pore formation seems to be target mediated. Nisin

and other lantibiotics use the cell wall precursor lipid II as a docking molecule. Thereby, two modes

of action, i.e. inhibition of cell wall biosynthesis and pore formation, are combined within one

molecule for potent antimicrobial activity.

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3. Antifungal compounds

Antifungal compounds are produced by LAB such as other organic acids, acetoin, carbon dioxide,

diacetyl, hydrogen peroxide, caproicacid, 3-hydroxy fatty acids, phenyllactic acid, cyclic dipeptides,

reuterin, fungicins and other proteinaceous compounds exhibiting antifungal potential.

3.1 Reuterin

Reuterin is produced from glycerol by starving cells under anaerobic conditions, and the active

reuterin is an equilibrium mixture of monomeric, hydrated monomeric and cyclic dimeric forms of 3-

HPA (3-hydroxypropionaldehyde). Reuterin is active against Gram-positive and Gram-negative

bacteria, yeast and fungi. Antifungal activity has been shown against species of Candida, Torulopsis,

Saccharomyces, Aspergillus and Fusarium. The production of reuterin (3-HPA) has also been

reported from L. brevis and L. buchneri, L. collinoides and L. coryniformis. L. reuteri produce the

antibiotic reutericyclin, a tetramic acid active against many Gram-positive bacteria, including

common sourdough LAB, but lacking activity against yeast.

4. Bioactive Peptides

Bioactive peptides are described as food-derived components (genuine or generated) that, in addition

to their nutritional value, exert a physiological effect in the body. Biological activities associated with

such peptides include immunomodulatory, antibacterial, anti-hypertensive and opioid-like properties.

Milk proteins are recognized as a primary source of bioactive peptides, which can be encrypted

withinthe amino acid sequence of dairy proteins, requiring proteolysis for release and activation.

Fermentation of milk proteins using the proteolytic systems of lactic acid bacteria (LAB) is an

attractive approach for generation of functional foods enriched in bioactive peptides given the low

cost and positive nutritional image associated with fermented milk drinks and yoghurt.Thus,

fermentation of milk and milk proteins by proteolytic lactic acid bacteria can lead to development of

functional foods conferring specific health benefits to the consumer beyond basic nutrition. The

starter culture applied in the manufacture of `Festivo‟ cheese, a novel bioactive cheese is a mixture of

commercial starter cultures containing 12 different strains of the following genera or species:

Lactococcus sp. and Leuconostoc sp. (BD type cultures), Propionibacterium sp., Lactobacillus sp. as

well as Lactobacillus acidophilus and Bifidobacterium.

5. Exopolysaccharides (EPS)

Many bacteria are known to produce cell-surface polysaccharides involved in a wide variety of

biological functions including prevention of desiccation, protection from environmental stresses,

adherence to surfaces, pathogenesis and symbiosis. Cell-surface polysaccharides comprise O-antigens

lipopolysaccharides (LPSs), lipoteichoic acids (LTAs), capsular polysaccharides (CPSs) and

exopolysaccharides (EPSs). LAB produce exopolysaccharides (EPS), which are homopolysaccharide

consisting of α-D-glucans such as dextrans mainly composed of α-1,6-linked residues with variable

(strain specific) degrees of branching and alternans composed of α-1,3 and α-1,6 linkages. EPS

produced by LAB have health benefits, such as protection against gastric ulcers, cholesterol lowering

activity and a capability to modulate the immune system and anti tu moural effect

6. Oligosaccharides

6.1 Mannitol

Mannitol (D-) Mannitol is a naturally occurring six-carbon sugar alcohol or polyol. Mannitol is a low-

calorie sugar that could replace sucrose, lactose, glucose or fructose in food products. The mannitol

production in lactic acid bacteria is strongly dependent on the pathway of carbohydrate fermentation:

LAB possess either a homofermentative or a heterofermentative pathway. It is metabolized

independently of insulin and is also applicable in diabetic food products. L. pseudomesenteroides and

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L. mesenteroides are known for their ability to produce mannitol in the fermentation of fructose.

Mannitol is only partially metabolized by humans and it does not induce hyperglycemia, which makes

it useful for diabetics. Mannitol is applied as a food additive (E421) as a sweet tasting bodying and

texturing agent and it is used as a sweet builder in sugar free chewing gum and in pharmaceutical

preparations. Mannitol has some laxative properties and the daily intake of mannitol should therefore

not exceed 20 g. LAB are found to produce small amounts of mannitol intracellularly e.g.

Streptococcus mutans, L. Leichmanii.

7. LAB as cell factory for nutraceutical compounds

Besides their lactic acid forming capacity, LAB also have the ability to contribute to the production of

several important nutraceuticals through fermentation. The term nutraceutical is defined as “any

substance that may be considered a food or part of a food and provides medical or health benefits,

including the prevention and treatment of disease”. Among the bioactive compounds, phenolic acid,

primarily located on the out layers (aleurone, pericarp) of cereals can act as antioxidants through a

number of different mechanisms. The so-called chain breaking mechanisms, which include hydrogen

donation and radical acceptor (i.e., radical scavenging activity), are the most likely means by which

phenolic acids act as antioxidants. LAB cereal fermentation represents an efficient means to increase

the level of phenolic compounds if appropriate fermentation conditions are applied. Even though most

LAB are auxotrophic for several vitamins,it is known that certain Lactobacillus strains have the

capability to enrich fermented cereal products synthesizing water-soluble vitamins such as vitamin C

and those included in the B-group (folates, riboflavin, and vitamin B12) among others. LAB to

improve the folate content of fermented cornmeal. An increase in the folate level to almost threefold

after 4 days of fermentation at 30◦C. Starter cultures consisting of Lb. plantarum strains produce

lysine in situ during cereal fermentation.

7.1 B vitamins

The B vitamins folate (B11), riboflavin (B2) and cobalamin (B12) are essential in the human diet.

Vitamin deficiency is encountered all over the world including specific population groups, such as the

elderly and adolescents, in well-developed countries. Several food-grade microorganisms produce

excess amounts of these vitamins offering the possibility to fortify raw food material by bacterial

fermentation. Current studies related to riboflavin production in L. lactis focus on obtaining variant

strains that display increased riboflavin production by selection for resistance against purine and

riboflavin analogues, such as roseoflavin, leading to deregulation of riboflavin production.

7.2 Ameliorate nutritional value LAB

Fermentation does not just have a preservation function in fermented cereal products. It can also have

multiple effects on the nutritional value of food through decreasing anti-nutritional biomolecules, thus

amassing bioavailability of suppressed nutrients, and more directly by hydrolyzing carbohydrates and

nondigestible oligosaccharides into functional compounds. LAB fermentation impacts the nutritional

characteristics of various cereal beverages increase digestibility. LAB and yeasts produce well

digestible macro- and micronutrients. Certain oligosaccharides, such as raffinose, stachyose, and

verbascose, which are abundant in cereals, can cause flatulence, diarrhoea, and indigestion. These

oligosaccharides are often resistant to cooking but can be enzymatically hydrolyzed by LAB during

fermentation, thus increasing product digestibility. This oligosaccharide hydrolysis improved the

nutritional value of the fermented cereal beverage by increasing the sugar content and reducing

digestive discomfort.

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8. Biofunctional mechanisms of LAB in health

Various mechanisms are given for all the functional health benefits of fermented milk and milk

products which are made by lactic acid bacteria as well as probiotic bacteria.

8.1 Anti-cancer

Natural Killer activity (NK) is strongly associated with the cancer. The effect of intake of L. casei

strain Shirota containing fermented milk improves the NK cell activity. Fermented product Yakult

helps in immunomodulation by inhancing the host immune system in bladder cancer via stimulation

of macrophages to produce IL-12, which would the stimulate helper T-cells to differentiate, produce

IFN-ϒ and promote cellular immunity against tumour cells. In case of colon cancer development

some lactobacilli and bifidobacteria lowers the level of faecal enzymes those are implicated to

carcinogenesis and they have ability to degrade nitroso-compounds. Enzymes which are involved in

the conversion of procarcinogens to carcinogens are neutralized by the faecal enzyme activity of

urease, nitroreductase, azoreductase, glycocholic acid reductase and β-glucuronidase.

8.2 Inflammatory bowel diseases and Diarrhoeal disease

In case of inflammatory bowel diseases like ulcerative colitis, pouchitis and Chron’s disease are

implicated with the acute inflammation of the intestinal mucosa and is usually associated with

diarrhoea and rectal bleeding with excess production of mucus. Probiotics reduces the incidence of

IBD. Diarrhoeal disease like antibiotic associated diarrhoea is casued by species Candida albicans

and Clostridium difficle. Regular intake of probiotics reduces the occurrence of antibiotic associated

diarrhoea and their complications.

8.3 Anti tumor

Oral administration of L. casei Shirota strain has inhibitory properties on chemically induced tumors

in rat as animal model. The efficacy of probiotic lactic acid bacteria have been associated with their

antimutagenic properties and ability to modulate immune parameters. Probiotic bacteria hinder the T-

cell, NK-cell and macrophages activity which are associated with the tumor development. Probiotic

bacteria help to make the balance between Th1 cells and Th 2 cells or we can say it anti-inflammatory

cytokines like IL-2 and INF α and suppress the tumor inducing pro-inflammatory cytokines like

transforming growth factor-β (TGF-β), IL-4, IL-10. Lactobacillus kefiranofaciens M1 has strong

potential to induce production of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6 and

IL-12.

8.4 Immunomodulatory

Application of probiotic bacteria in our daily diet can enhances the immune system by making proper

balances in pro and anti-inflammatory cytokines. It exhibits the suppression effects on pro-

inflammatory cytokines via production of anti-inflammatory components. Thus improves the immune

system in immune-compromised people. Strong immunity depends upon the resistance of body

against foreign invaders or any type of body abnormalities. Antibodies are the major components of

human body immune systems. Lactic acid bacteria are able to produce antibody IgA and immunity

against Staphylococcus aureus infection.

8.5 Anti allergic

Lactic acid bacteria also play a vital role in minimizing allergic responses. As it is well known that Ig

E is involved in the immediate type hypersensitivity reactions has been control by Lactobacillus

citreum that regulates the serum IgE generation.

8.6 Immunostimulatory

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Various vitamins and bioactive compounds such as conjugated linoleic acid (CLA) have been

produced by yoghurt bacteria in yoghurt and other products. This compound CLA have been reported

for their immunostimulatory and anti-carcinogenic properties.

8.7 Neurological disorders

Production of ϒ-Aminobutyric acid (GABA) is also produced by lactic acid bacteria from L-

glutamate through glutamate decarboxylase activity. GABA has been reported for their improvement

in neurological disorders and improvement in relaxation and sleep. Gammaaminobutyric acid

(GABA) is a major neurotransmitter widely distributed throughout the central nervous system. Low

GABA levels or decreased GABA function in the brain is associated with several psychiatric and

neurological disorders, including anxiety, depression, insomnia, and epilepsy and studies indicate that

GABA can improve relaxation and sleep.

8.8 Anti-nutritional factor

On the other side, lactic acid bacteria through the action of phytase enzyme degrades phytate into

myo-inositol and phosphate during fermentation which is the major anti-nutritional factor that blocks

the availability of minerals.

9. Modulation of gut microbial balance

A deranged gut environment has increasingly been recognized as a source of allergic and autoimmune

disease, as well as acute and chronic infection particularly in patients undergoing advanced medical

and surgical treatment and in patients suffering from various acute diseases. Probiotics can re-

establish the lost balance by adhesion to epithelial cells, competing for nutrients, modifying pH and

producing antimicrobial substances.

10. Conclusions

Lactic acid bacteria traditionally used for carrying fermentation process are now being diversified for

value addition of fermented dairy foods. LAB is also one of the major inhabitants of the human

gastrointestinal tract and is commonly used in fermentation of various kinds of food products.

Furthermore there is mounting scientific evidence that LAB fermented food products positively

influence the health of humans. Functional lactic acid bacteria can offer several organoleptic,

technological, nutritional, or health advantages and have great potential in food fermentation industry.

Therefore, consumption of fermented food products could result into improved health in many low

income communities in the developing world. They may further contribute to the development and

product diversification of food companies for the production of functional foods and health foods.

149

Preparation and Evaluation of DVS Starter Cultures

Surajit Mandal


1. Introduction

A starter culture can be defined as a microbial preparation of large numbers of cells of at least one

microorganism to be added to a raw material to produce a fermented food by accelerating and steering

its fermentation process. The group of lactic acid bacteria (LAB) occupies a central role in these

processes, and has a long and safe history of application and consumption in the production of

fermented foods and beverages. They cause rapid acidification of the raw material through the

production of organic acids, mainly lactic acid. Also, their production of acetic acid, ethanol, aroma

compounds, bacteriocins, exopolysaccharides, and several enzymes is of importance. In this way they

enhance shelf life and microbial safety, improve texture, and contribute to the pleasant sensory profile

of the end product. Lactic acid bacteria, propionibacteria, surface-ripening bacteria, yeasts, and molds

are used as starter cultures for manufacturing of various fermented milk products. Starter cultures

have a multifunctional role in dairy fermentations. The production of lactic acid, by fermenting

lactose is the major role of dairy starters. The acid is responsible for development of characteristic

body and texture of the fermented milk products, contributes to the overall flavour of the products,

and enhances preservation. Diacetyl, acetaldehyde, acetic acid, also produced by the lactic starter

cultures, contribute to flavor and aroma of the final product. Carbon-di-oxide produced by some

hetero-fermentative lactic acid bacteria involves in very characteristics texturization in some

fermented dairy products, viz. “eye” formation in cheeses. Development of flavor and changes in

texture during ripening of cheeses is associated with enzymes originating from bacterial and fungal

cultures, depending on the cheese variety. Dairy starters are also having some direct or indirect

functional health promoting attributes, such as live probiotics, prebiotic exopolysaccharides and

oligosaccharides, bioactive peptides and lipids, etc. Most of the cultured dairy products are produced

using commercial starter cultures that have been selected for a variety of desirable properties in

addition to rapid acid production. These may include flavor production, lack of associated off flavors,

bacteriophage tolerance, ability to produce flavor during cheese ripening, salt tolerance, exo-

polysaccharide production, bacteriocin production, temperature sensitivity, etc.

2. Types of Starter Cultures

In the dairy industry, single or multiple strains of cultures of one or more microorganism are used as

starter cultures. These are belongs to genus Lactococcus (Lactococcus lactis subsp. cremoris, L. lactis

subsp. lactis, L. lactis subsp. lactis biovar diacetylactis), Lactobacillus (L. delbrueckii subsp. lactis,

Lactobaillus acidophilus, Lactobacillus casei), Streptococcus (S. thermophilus), Leuconostoc,

Pediococcus etc. There are two main types of lactic starters:

1) Mesophilic lactic starters (optimum growth temperature: 30°C)

2) Thermophilic lactic starters (optimum growth temperature: 42°C)

Mesophilic cultures usually contain L. cremoris and L. lactis as acid producers and L. diacetylactis

and Leuconostocs as aroma and CO2 producers. Thermophilic starters include strains of S.

thermophilus, and, depending on the product, Lactobacillus bulgaricus, L. helveticus, or L. lactis.

Often, some fermented milks made with thermophilic starters also contain Lactobacillus acidophilus,

L. bulgaricus and bifidobacteria for their healthful and therapeutic properties. Table 1 lists the

common starter cultures and their applications in cheese and fermented dairy products.

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The lactic starter cultures are also subdivided into two groups:

1) Defined cultures; 2) Mixed cultures.

Defined cultures constitute starters in which the number of strains is known. The application of

defined cultures did control the open texture problem, however, and they were prone to slow acid

production due to their susceptibility to bacteriophage. The use of pairs of phage-unrelated strains and

culture rotation to prevent build up of phage in the cheese factory was practiced to minimize the

potential for phage problems. Eventually, the use of multiple strain starter and factory-derived phage-

resistant strains was made to control the phage problem. Mixed cultures, the type and number of

bacteria is unknown.

Table 1: Lactic starter cultures and applications in dairy products

Lactic Acid Bacteria Associated Microorganisms Products

Mesophilic

Lactococcus lactis,

Lactococcus cremosis,

Lactococcus lactis var. diacetylactis,

Leuconostoc cremosis

Lactococcus lactis var.

diacetylactis,

Penicillium camemberti,

P. roqueforti, P. caseicolum,

Brevibacterium linens

Cheddar, Colby Cottage

cheese, Cream cheese,

Camembert and Roquefort

cheese

Thermophilic

Streptococcus thermophilus,

Lactobacillus bulgaricus,

L. lactis, L. casei, L. helveticus,

L. plantarum, Enterococcus faecium

Candida kefyr, Torulopsis,

spp., L. brevis,

Bifidobacterium bifidum,

Propionibacterium

fureudenreichii, P. shermanii

Parmesan, Romano, Grana

Kefir, Koumiss yogurt,

Yakult, Therapeutic cultured

milks, Swiss, Emmenthal,

Gruyere

Mixed starters

Lactococcus lactis, S. thermophilus,

E. faecium, L. helveticus,

L. bulgaricus

… Modified Cheddar, Italian,

Mozzarella, Pasta Filata,

Pizza cheese

Table 2: Starter cultures for fermented dairy products

1. Hard cheeses without

eyes

Lactococcus lactis ssp. lactis, L. lactis ssp. cremoris

2. Cheeses with small eyes Lactococcus lactis ssp. lactis, Lactococcus lactis ssp. lactis var.

diacetylactis, L. lactis ssp. cremoris, Leuconostoc mesenteroides ssp.

cremoris

3. Swiss and Italian type

cheeses

Lactobacillus delbrueckii ssp. lactis, L. helveticus, L. casei, L.

delbrueckii ssp. bulgaricus, Streptococcus thermophilus

4. Butter and buttermilk Lactococcus lactis ssp. lactis, Lactococcus lactis ssp. lactis var.

diacetylactis, L. lactis ssp. cremoris, Leuconostoc mesenteroides ssp.

cremoris

5. Yoghurt L. delbrueckii ssp. bulgaricus, Streptococcus thermophilus

6. Fermented, probiotic

milk

L. casei, L. acidophilus, L. rhamnosus, L. johnsonii, Bifidobacterium

lactis, B. bifidum, B. brevis

7. Kefir L. kefir, L. kefiranofacies, L. brevis (mixed culture)

3. Technology of starter cultures

Lactic starter cultures are generally available from commercial manufacturers in spray-dried, freeze-

dried (lyophilized), or frozen form. Spray-dried and lyophilized cultures need to be inoculated into

milk or other suitable medium and propagated to the bulk volumes required for inoculating a cheese

vat as follows:

151

Stock

culture

(Freze

dried,

frozen,

spray

dried)

Mother

culture

Intermediate

culture

Bulk

culture

Process Milk

(Multipurpose

vat/Cheese

Vat/Lassi tank)

However, the repeated sub-culturing of certain strains of starter bacteria may loss the plasmids and

consequently can affect the characteristics (i.e., phage-resistant becomes phagesensitive, lack of

lactose utilization etc). The yogurt starter cultures (S. thermophilus and L. delbrueckii ssp. bulgaricus)

are normally used the ratio of cocci : rods as 1 : 1. Starter culture activity is affected by the rate of

cooling after incubation, level of acidity at the end of the incubation period, and the temperature and

duration of storage. Many larger dairy plants develop their own cultures. However, preparing and

maintaining bulk cultures requires specialized facilities and equipment. Much research and

development in the starter culture technology has been aimed at designing specialized growth media

for starters, protecting the starter cultures from sub-lethal stress and injury during freezing, and

minimizing the threat of bacteriophage during starter culture preparations. Therefore, the use of

concentrated direct vat starters is gaining much importance in preparation of fermented milks. The

Direct Vat Starters (DVS) cultures are highly concentrated cultures that are made of mixtures of

defined strains in predetermined proportions. The advantages of DVS are imporved quality, high

yield, less rejection of batches, ease of use and reliability.

4. Starter concentrates

Traditionally 'bulk starter' in liquid form was used to inoculate the milk used in the manufacture of

cheese, yoghurt, buttermilk and other fermented products. The use of starter cell concentrates

designated as either Direct Vat Set (DVS) or Direct Vat Inoculation (DVI) cultures have increasing

being used, particularly in small plants, to replace bulk starter in fermented dairy product

manufacture. Starter concentrates used in DVI cultures are concentrated cell preparations containing

in the order of 1011

-1013

CFU/g. They are available as frozen pellets (fig. 1) or in freeze-dried granular

form (fig. 2).

4.1 DVS frozen culture

Under normal conditions starter growth in milk results in a cell concentration of about 109 cfu/ml.

Growth of starters in milk is limited by a number of factors including the accumulation of lactic acid.

Concentrates can be produced by neutralisation (traditional fermentation technology) or removal of

the lactic acid (using diffusion culture), recovering the cells by centrifugation, and by maintaining

starter activity by freeze drying or freezing. Freeze-dried concentrates can be stored for some months

at 4° C. Frozen concentrates are usually stored at -45°C or lower. Some suppliers recommend that

their frozen DVI cultures are stored at -18°C.

4.2 Production of starter concentrates

Commercial starter cultures currently available for direct addition to production vats contain billions

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of viable bacteria per gram, preserved in a form that could be readily and rapidly activated in the

product mix to perform the functions necessary to transform the product mix to the desired cultured

product. To attain that, the selected starter bacteria need to be grown in a suitable medium to high

numbers and to concentrate the cells. The generally used ingredients in media formulations include

non-fat milk, whey, hydrolysates of milk and whey proteins, soy isolates, soy protein hydrolysates,

meat hydrolysates and extracts, egg proteins, com steep liquor, malt extracts, potato infusions, yeast

extracts/yeast autolysates, sugars such as lactose, glucose, high-fructose corn syrup, com sugar,

sucrose, and minerals such as magnesium, manganese, calcium, iron, phosphates, salt, etc. For some

fastidious bacteria, amino acids and vitamins may be included. The phosphates are added to provide

mineral requirements as well as for buffering. For some bacteria, which need unsaturated fatty acids

to protect cell membranes, trace quantities of polysorbates (Tweens) are added. To control foaming,

food grade anti foam ingredients may be incorporated.

The medium is then either sterilized by heating at 121°C for a minimum of 15 minutes or heat-treated

at 85-95°C for 45 minutes or subjected to ultrahigh temperature treatment (UHT) for a few seconds.

After heat treatment, the medium is cooled to the incubation temperature. After the addition of the in-

oculum, the medium is incubated until the predetermined endpoint is reached. During incubation, the

pH is maintained at a predetermined level (constant neutralization to maintain pH). Generally, the

endpoint coincides with the exhaustion of sugar reflected by the trace of the neutralization curve. The

frequency of neutralization reflects the activity of the culture in the fermenter, and when the

frequency decreases, it indicates the near depletion of the sugar. Samples are usually taken to

microscopically examine the fermentate for cell morphology, for any gross contamination, for a rough

estimation of cell numbers, and for quantitative measurement of sugar content. After ascertaining

these, the fermenter is cooled. The cells are harvested either by centrifugation or by ultrafiltration.

The cell concentrate is obtained in the form of a thick liquid of the consistency of cream and is

weighed and rapidly cooled. Sterile preparations of cryoprotectants (glycerol, nonfat milk,

monosodium glutamate, sugars, etc.) are added, and uniformly mixed with the cell concentrate. The

concentrate may be filled as such into cans and frozen or frozen in droplet form in liquid nitrogen

(pellets), retrieved, and packaged. The concentrate as such or in pellet form may also be lyophilized in

industrial scale freeze dryers.

There are two main reasons for using pH control systems in propagating bulk starter cultures:

1. To minimize daily fluctuations in acid development and thereby prevent "over-ripening" of the

starter.

2. To prevent the cellular injury that may occur to some starters when the pH of the medium drops

below 5.0.

In the pH control systems, the acid produced by the starter culture is neutralized to maintain the pH at

around 6.0. The external pH control system, uses whey based medium fortified with phosphates and

yeast extract. The pH is maintained at around 6.0, by intermittent injection of anhydrous or aqueous

ammonia, or sodium hydroxide. The internal pH control system, developed uses a whey based

medium containing encapsulated citrate-phosphate buffers that maintain the pH at around 5.2. Unlike

in the external pH control system, no addition of ammonia or NaOH is necessary.

Higher cell densities (greater than 1010

CFU/g) can be obtained by harvesting the cells from the

fermenter medium by centrifugation, to give a starter population of 1011

- 1012

CFU/ml. Even higher

cell densities can be obtained by freeze drying the 'sludge' obtained by centrifugation. Unfortunately,

the increase in cell population for some strains does not necessarily parallel the increase in the ability

of the concentrated culture to produce acid. These strains are susceptible to damage during the

fermentation, centrifugation and freeze-drying/freezing and storage stages.

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Table 3: Storage conditions and shelf lives of some concentrated cultures

Type of cultures Storage Shelf-life (Months)

1. Freeze dried (Direct Vat) -18°C 12

2. Deep frozen (Direct vat) -45°C 12

3. Freeze dried (Master culture) +5°C 12

4.3 Quality control of commercial cultures (DVS/DVI)

1) Viable cell numbers

2) Absence of contaminants, pathogens, and extraneous matter

3) Acid-producing and other functional activities

4) Package integrity, accuracy of label information on the package

5) Shelf life of the product according to specification

Starter organism 10

10

-1012

cfu/g

Coliforms Absence in 1 g

Enterococci Less than 20 cfu/g

Yeasts and molds Absence in 1 g

Staphylococci (coagulase-positive) Absence in 10 g

Listeria Absence in 25 g

Salmonella Absence in 25 g

5. Conclusions

Commercial starter culture production is a highly demanding operation. It requires specialized knowl-

edge of microbiology, microbial physiology, process engineering, and cryobiology. In addition to

production knowledge, a full-fledged quality control program is necessary to test incoming raw

materials, design and maintain plant sanitation, test sterility of production contact surfaces, monitor

plant environment quality, and test every product lot for the prescribed quality standards. The quality

control section is also required to train and update plant personnel on the importance of sanitation and

strict adherence to process control protocols.


Cogan T M and Hill C (1993) Cheese starter cultures, Ch.6 in: P.F. Fox, ed., Cheese: Chemistry, Physics and

Microbiology, Vol. 1, General Aspects, 2nd

ed., Chapman and Hall, London. 193-206.

Lewis J E (1987) The Lewis method, in: Cheese Starters, Development and Application of the Lewis System,

196-200.

Tamime A Y and Robinson R K (1999) Preservation and production of starter cultures, In: Yoghurt, Science and

Technology, CRC Press, New York and Woodhead Pub. Ltd., Cambridge, UK. 486- 514

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Novel Starters for Value Added Fermented Dairy Products

Rajani C. S., Hitesh Kumar, Sudhir Kumar Tomar


1. Introduction

Lactic acid bacteria are among the powerhouses of the food industry, colonize the surfaces of plants and

animals, and contribute to our health and well-being. LAB are gram-positive usually non-motile, non-

spore-forming rods and cocci. These are gaining importance due to their “generally recognized as safe”

status, long-term use in food and beneficial, probiotic properties. Genera that comprise the LAB are

Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Enterococcus, Tetragenococcus,

Carnobacterium, and Weisella (Table 1). Besides their technological properties in food production, LAB

can confer beneficial properties to their hosts, as probiotics. Beyond being probiotic, certain strains of

LAB are able to produce specific beneficial compounds in foods such as vitamins, low calorie sweeteners,

antioxidants, polysaccharides and minerals.

Table 1. Characteristic of Lactic Acid Bacteria

Genus

Characteristic

Lactobacill

us

Enterococcu

s

Lactococcu

s

Leuconosto

c

Pediococcu

s

Streptococcu

s

Morphology rods cocci cocci cocci cocci in

tetrads

Cocci

CO2 from

glucose*

± − − + − −

Growth

at 10°C ± + + + ± −

at 45°C ± + − − ± ±

in 6.5% NaCl ± + − ± ± −

at pH 4.4 ± + ± ± + −

at pH 9.6 − + − − − −

Lactic acid

configuration

D, L, DL L L D L, DL L

http://textbookofbacteriology.net/lactics.html

2. B Vitamin Production by Lactic Acid Bacteria

The vitamins are disparate group of compound; they have little in common either chemically or in their

metabolic functions. Nutritionally, they form cohesive group of biomolecules that are required in the diet

http://inst.bact.wisc.edu/inst/index.php?module=Book&func=displayglossary#gram

http://inst.bact.wisc.edu/inst/index.php?module=Book&func=displayglossary#spore

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in small amounts for maintenance of normal health and metabolic integrity that contributes to healthy

lifestyle.

2.1 Riboflavin

Riboflavin (vitamin B2) plays an essential role in cellular metabolism, being the precursor of the

coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) both acting as

hydrogen carriers in biological redox reactions involving enzymes such as nicotinamide

adeninedinucleotide (NADH) dehydrogenase. Deficiency of riboflavin leads to loss of hair, inflammation

of the skin, sore throat, hyperaemia edema of oral and mucous membranes, cheilosis, glossitis, cataract

development, migrane prophylaxix and decrease in haemoglobin status. Several studies report a link

between homocysteine levels and cardiovascular diseases. Riboflavin has been traditionally synthesized

for food and feed fortification by chemicals means but past decade has witnessed emerging information

about commercial competive biotechnological processes. A study has described the screening of

riboflavin-producing strains from different fermented milk products obtained in the Vellore region of

India. The selection of spontaneous roseoflavin-resistant mutants was found to be a reliable method to

obtain natural riboflavin-overproducing strains of a number of species commonly used in the food

industry. The toxic riboflavin analogue roseoflavin was used to isolate natural riboflavin-overproducing

variants of the food-grade micro-organisms Lactococcus lactis Lactobacillus plantarum, Leuconosctoc

mesenteroides and Propionibacterium freudenreichii. Recently, LAB were obtained from durum wheat

flour samples and screened for roseoflavin-resistant variants to isolate natural riboflavin-overproducing

strains. Two riboflavin-overproducing strains of Lb. plantarum were isolated and used for the preparation

of bread (by means of sourdough fermentation) and pasta (using a pre-fermentation step) to enhance their

vitamin B2 content. The applied approaches resulted in a considerable increase in vitamin B2 content

(about a two and threefold increase in pasta and bread, respectively), thus representing a convenient and

efficient food-grade biotechnological application for the production of vitamin B2-enriched bread and

pasta. The bioavailability of the riboflavin produced by this strain was similar to that of pure riboflavin

demonstrating the usefulness of this strain for the development of riboflavin-enriched fermented foods.

These this strain could be further exploited for the enhanced production of riboflavin using various strain

improvement strategies to develop a better starter culture for the fermented food industry.

2.2 Folate

Folate, an important B-group vitamin, participates in many metabolic pathways such as DNA and RNA

biosynthesis and amino acid inter-conversions. Health of multi-cellular organisms such as humans starts

at the individual cell level: if our cells are healthy so are we. Healthy cells, in turn, depend on the

continued, faultless replication of our DNA. Folates possess antioxidant properties that protect the

genome by inhibiting free radical attack of DNA in addition to their role in DNA repair and replication

mechanisms. Folate deficiency has been implicated in a wide variety of disorders from Alzheimer’s to

coronary heart diseases; osteoporosis, increased risk of breast and colorectal cancer , poor cognitive

performance , hearing loss , and of course, neural tube defects .The proper selection and use of folate

producing microorganisms is a novel strategy to increase “natural” folate levels in foods. Numerous

researchers have reported that LAB, such as the industrial starter bacteria Lactococcus lactis, S.

thermophilus, and Leuconostoc species have the ability to synthesize folate. Whereas many Lactobacilli

species happen to consume folate. The ability to produce folate can differ remarkably between different

LAB (2 to 214 μg/L folate). It is now known that not only the yogurt starter cultures and L. lactis have the

ability to produce folate but also this important property exists in other LAB species as Lb. acidophilus,

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Leuconostoc lactis, Bifidobacterium longum, and some strains of Propionibacteria, well-known vitamin

B12 producer, can also produce large amounts of folate . Majority of folate produced by L. lactis and

Leuconostoc spp. being intracellular is not excreted into the milk and hence is lesser bioavailable while S.

thermophilus produces folate extracellularly during milk fermentation. Similarly, probiotic

microorganisms as Propionibacteria spp. and Bifidobacteria spp. are also well-known folate producer

but majority of folate produced is intracellular and in the case of Propionibacteria it also consumes folate

from the medium. Moreover, the major limitations of use of these organisms for biofortification of folate

include their requirement of strict anaerobic conditions for folate production and possibilities of folate

utilization by co-cultures when used as adjunct starter. S. thermophilus is known to produce folic acid

during growth in milk, which is a functional attribute. S. thermophilus has a strain-specific ability of

folate production and has been reported to produce higher quantity of folate in comparison to other LAB,

majority of which is excreted into milk and has been reported to be the dominant producer, elevating

folate levels in skim milk, while lactobacilli have been found to deplete the available folate in the skim

milk. Yet fermentations with mixed cultures showed that folate production and utilization by the cultures

is additive. Tomar and others (2009) found culture NCDC177 (35 μg/mL) to be the best folate producer

among the S. thermophilus cultures available at Natl. Collection of Dairy Cultures, Natl. Dairy Research

Inst., Karnal (Haryana, India).

2.3 Vitamin B12

The term vitamin B12 is generally used to describe a type of cobalt corrinoid, particularly of the

cobalamin (cbl) group. In strict terms, vitamin B12 is the form of the vitamin obtained during industrial

production and which does not exist naturally. Animals, plants and fungi are incapable of producing

cobalamin; it is the only vitamin that is exclusively produced by micro-organisms, particularly by

anaerobes. It was shown that Lb. reuteri CRL1098 was able to metabolize glycerol in a B12-free medium;

this being the first hint that a LAB might be able to produce cobalamin. The chromatographic analysis of

the intracellular bacterial extract of Lb. reuteri CRL 1098 confirmed that this strain was able to produce a

cobalamin- like compound with an absorption spectrum closely resembling that of standard cobalamin but

with a different elution time, while cobalamin production was confirmed using different bioassays.

Genetic evidence of cobalamin biosynthesis by Lb. reuteri CRL 1098 was then obtained through the use

of different molecular biology techniques, and it was shown that at least 30 genes are involved in the de

novo synthesis of the vitamin. Recently, a reuterin producing strain of Lactobacillus coryniformis isolated

from goat milk was characterized and was shown to produce a cobalamin-type compound. Vitamin B12

deficiency and depletion are common in underdeveloped/ developing countries, particularly among the

elderly and are most prevalent in poorer populations of lower socioeconomic status around the world.

Food-cobalamin malabsorption is responsible for about 60%-70% of the cases among elderly patients,

and pernicious anemia accounts for 15%-20% of the cases while other causes (nutritional deficiencies,

hereditary disorders and malabsorption) collectively claims for <10%. Researchers have found that H.

pylori gastritis, resulting in to food-cobalamin malabsorption, is found as a major cause of vitamin B12

deficiency in a wide range of age groups. Hence B12 supplementation alone can never be an effective

approach in the treatment of vitamin B12 deficiency as long as H. pylori associated gastritis is intact to the

patient. Nowadays, there is a considerable interest in search of alternative/ adjunctive therapies against H.

pylori which can reduce the antibiotic associated outcomes and increase compliance to patient. In this

odyssey, probiotics have been found as a ‘‘possible tool’’ for eradication of the infection and an

appreciable no. of reports have come out with their positive anti H. pylori effect. After taking in to

consideration these two points, probiotics can be thought of using selectively against H. pylori.

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Microorganisms most commonly used in clinical practice are lactic acid-producing bacteria

(Lactobacillus spp) and Non-lactic acid bacteria (Bifidobacterium and Bacillus). After taking into

consideration of both vitamin B12 deficiency and removal of H. pylori, one of its potent causes,

Lactobacilli can be looked upon as a biological companion of pharmaceutical therapies, which are mostly

accompanied with unfavorable side effects. Lactobacilli can produce vitamin B12 de-novo and show

improvement in B12 related hematological and physical and biochemical markers.

3. Low - Calorie Sugars Produced by Lactic Acid Bacteria

Firstly, food-grade microorganisms and their products are directly applicable in food products, without

any restriction. Secondly, there is no need for a careful separation of products and microorganisms, which

would be the case if microorganisms are not of food grade. Thirdly, some lactic acid bacteria are claimed

as beneficial in the gastrointestinal tract. Mannitol production by those bacteria may strengthen their

health-promoting ability. Low-calorie sugars have been a recent addition and have attracted a great deal

of interest of researchers, manufacturers, and consumers for varied reasons. These sweeteners also getting

popularized as low-carb sugars have been granted generally recommended as safe (GRAS) status and

include both sugars and sugar alcohols (polyols) which in addition to their technological attributes (sugar

replacer, bulking agent, texturiser, humectant, cryoprotectant) have been observed to exert a number of

health benefits (lowcalories, lowglycemic index, anticariogenic, osmotic diuretics, obesity control,

prebiotic). Some of these sweeteners successfully produced by lactic acid bacteria include mannitol,

sorbitol, tagatose, and trehalose and there is a potential to further enhance their production with the help

of metabolic engineering. These safe sweeteners can be exploited as vital food ingredients for

development of low-calorie foods with added functional values especially for children, diabetic patients,

and weight watchers. Mannitol, sorbitol, and erythritol are naturally occurring sugar alcohols. Mannitol is

produced by bacteria, yeasts, fungi, algae, and several plants. This polyol might help these organisms to

cope with different environmental stresses such as osmotic and oxidative stress. Sorbitol is produced by a

variety of both plants and microorganisms. Erythritol production is usually associated with yeasts but has

also been reported for some lactic acid bacteria (LAB). All these polyols - mannitol, sorbitol, and

erythritol - display properties that are beneficial to human health as they are non - metabolizable, insulin -

independent sweeteners, or low - calorie sugars, which make them applicable in dietetic and diabetic food

products. In addition, mannitol is used in the pharmaceutical industry as a powerful osmotic diuretic agent

and as an osmotic agent for decreasing brain and cellular edema. Mannitol biosynthesis through bacterial

fermentation has become an interesting alternative to existing chemical production. Furthermore, the

capability of certain LAB, belonging to both homofermentative and heterofermentative species, to

synthesize mannitol offers the possibility of in situ production in foods. For this reason, different

fermentation technology- based strategies for improving mannitol production by LAB have been reported.

To date, 93-97 mol% mannitol yields are reached using a bioprocess with a heterofermentative LAB

strain.

3.1 Tagatose

Tagatose is one such rare natural monosaccharide, ketose sugar and is naturally present in heat-treated

dairy products. It has the potential for use as a sugar substitute in food since it has a taste and sweetness

similar to sucrose. It has 92 % of the sweetness but only 38 % of the calories of sucrose and does not

show any significant side tastes. D-Tagatose has gained the generally recognized as safe (GRAS) status

from the U.S. Food and Drug Administration; therefore, it can be used as a low calories bulk sweetener

for human consumption. D-Tagatose also caramelizes during cooking, which leads to the desired

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browning. It is found naturally in some dairy products, but is commercially manufactured from lactose

and has many health benefits. As a dietary supplement, it relieves type II diabetes symptoms, causing

weight loss and increase in high-density lipoprotein cholesterol, and functions as an anti-hyperglycemic

agent. It also prevents dental caries and supports healthy intestinal flora. However, the use of D-tagatose

remains limited by its production cost. The precursor of D-tagatose, D-galactose, can be produced by

hydrolyzing lactose. Hence, there has been a growing interest in D-tagatose production by L-Arabinose

isomerases using D-galactose as the starting material. Essentially it is metabolized differently, has a

minimal effect on blood glucose and insulin level and furthermore provides a prebiotic effect. It is

especially suitable as a flavor enhancer and as a low carbohydrate sweetener. As synthetic sweetener is

low calories sweetener, resulting in less energy and friendly in dental health. Besides, the one properties

of this sugar that makes it interesting is prebiotic. The D-tagatose can be absorbed only 15-20% in the

small intestine and not-digestible or fermented in the colon by microflora resulting in production of short-

chain fatty acid (SCFA) and butyrate. Due to the minimal absorption of tagatose in the small intestine,

researchers have found tagatose offers the body fewer calories than typical carbohydrates, including

sucrose. Tagatose is estimated to provide < 1.5 kcal/g, whereas sucrose provides 4 kcal/g. Maximum

levels of tagatose allowed in specific products were outlined by the FDA: 1% in carbonated beverages,

1% in ready-to-drink teas pre-sweetened with low calorie sweeteners, 60% in chewing gum, 30% in icing

or glazes used on baked goods, 15% in hard candies, 10% in dietetic soft candies, 3 grams per serving in

ready-to-eat cereals, 5 grams per serving in powdered products prepared with milk, 10% in low fat,

reduced fat, diet, energy, or nutrient fortified bars, and 3% in light ice cream, frozen milk desserts, low-fat

and non-fat frozen yogurt and related frozen novelties. Nutrilab, a subsidiary of the Belgian company

Damhert, has recently begun using tagatose in chocolate, spreads, cookies, and jams. In addition, they are

selling tagatose as a home sugar replacer under the name Tagatesse.

3.2 Trehalose

Trehalose is a disaccharide made up of two glucose molecules joined together by 1,1 glycosidic linkage

that joins the two hexose rings has low energy (1 kcal/mol), which makes it a very stable structure in

comparison with sucrose (27 kcal/mol). Although there are three possible isomers of trehalose i.e. α,α

(trehalose), α,β (neotrehalose) and β,β (isotrehalose), only α,α trehalose has been isolated and synthesized

by living beings and is commonly referred to as trehalose (α,α-trehalose, α-D-glucopyranosyl α-D-

glucopyranoside, mushroom sugar, mycose and tremalose). Earlier, trehalose was considered a rare sugar

because it was could only be extracted from resurrection plant and trehala manna. Trehalose possesses

high thermo stability and wide pH stability range. It can be hydrolysed only in the presence of trehalase

that can be found in cellular cytoplasm. Being a non-reducing sugar, trehalose does not participate in the

Maillard reactions that cause food browning and has also got low hygroscopic profile. It possesses 45%

sweetness and 2.5 times higher perceived sweetness in comparison to sucrose. All these exclusive features

along its natural functions make trehalose an enormously attractive molecule and thus lend themselves to

several possible applications in the health, food, cosmetic and pharmaceutical industries. It suppresses

dental caries in rats and reduces significant acidification in plaques. It has been found to prevent protein

aggregation, thus benefitting in pathological forms associated with protein aggregation like Alzheimer’s

and Huntington’s disease. Trehalose also protects the activities of key antioxidant enzymes, including

superoxide dismutases, ascorbate catalases and ascorbate peroxidases. It also suppresses osteoporosis and

trehalose has been accepted as a novel food ingredient under the GRAS terms in the United States and

European Union. Trehalose has found large commercial applications as a food ingredient because of its

multifaceted effects such as its inherent mild sweet flavor, its preservative properties that maintain the

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quality of three main nutrients, its powerful water retention capability that preserves the texture of food

by protecting them from drying out of freezing and its ability to suppress bitterness, stringency, harsh

flavor and stench of raw foods, meat and packaged foods. The uses for trehalose span a broad spectrum

that cannot be found in other sugars, the primary one being its use in processed foods. Trehalose is most

commonly used as an additive in western and Japanese confectionary products. Besides the confectionary,

trehalose is used in a variety of processed foods such as dinners, bread, processed vegetables and fruits,

processed seafood, baked goods, beverages, animal-derived deli foods, pouch-packed foods, frozen foods

and refrigerated items as well as foods for lunches, eating out or prepared at home.

4. Glutathione

LAB have taken centre stage in perspectives of modern fermented food industry and probiotic based

therapeutics. These bacteria encounter various stress conditions during industrial processing or in the

gastrointestinal environment. Such conditions are overcome by complex molecular assemblies capable of

synthesizing and/or metabolizing molecules that play a specific role in stress adaptation. Thiols are

important class of molecules which contribute towards stress management in cell. Glutathione, a low

molecular weight thiol antioxidant distributed widely in eukaryotes and Gram negative organisms, is

present sporadically in Gram positive bacteria. However, new insights on its occurrence and role in the

latter group are coming to light. Some LAB and closely related Gram positive organisms are proposed to

possess glutathione synthesis and/or utilization machinery. Also, supplementation of glutathione in food

grade LAB is gaining attention for its role in stress protection and as a nutrient and sulfur source. Owing

to the immense benefits of glutathione, its release by probiotic bacteria could also find important

applications in health improvement. Different species of LAB have evolved specialized mechanisms to

deal with the normally encountered stress conditions in particular niches. These mechanisms essentially

involve intricate maneuvering and interplay of various pathways and biomolecules which support the

growth of the organism in their respective transient environment. Thiols, distributed widely in biological

systems, are one such important class of compounds engaged in stress protection. Important thiol

compounds are glutathione, γ-glutamylcysteine, bacillithiol, mycothiol etc. Glutathione, a tripeptide, is

ubiquitous in eukaryotic system, found widely in Gram negative bacteria but was known to be scarcely

present in Gram positive bacteria.

5. Exopolysaccharides (EPS)

The role of microbes in producing fermented dairy products has evolved from a chance discovery to a

highly elaborated process involving the production of specialized “starter” of bacteria that function

consistently in large cultures. The primary function of almost all starter cultures is to develop acid in the

product. The secondary effects of acid production include coagulation, expulsion of moisture, texture

formation and initiation of flavor production. Starters also help in imparting pleasant acid taste, conferring

protection against potential pathogens and providing a longer shelf life to the product. The food industry

uses polysaccharides as thickeners, emulsifiers, gelling agents and stabilizers. The demand for these

ingredients is mostly met by alginates, carrageenan, cellulose, pectins, starches etc. There is a growing

interest for all-natural, healthy food products. Moreover, in various countries the amount of stabilizers

being used is regulated. In this respect the lactic acid bacteria (LAB) have great potential, as many of its

representatives are known to produce exopolysaccharides (EPS). EPS from LAB are an alternative class

of biothickeners, having potential for development and exploitation as functional food ingredients with

both health and economic benefits. Consumer demand for products with low fat or sugar content and low

levels of additives, as well as cost factors, make EPS a promising and viable alternative as these

160

contribute to texture, mouth-feel, taste perception and stability of the final product. A large variety of EPS

can be produced by LAB employed for production of fermented dairy products. In particular for the

production of yoghurt, drinking yoghurt, cheese, fermented cream, milk-based desserts, EPS producing

LAB play a significant role. They play a major role in the production of fermented dairy products in

Northern Europe, Eastern Europe and Asia. EPS producing lactic cultures have also been successfully

used for the manufacture of Nordic ropy milks. Scandinavian fermented milk drinks display a firm thick,

slimy consistency and these rely on the souring capacity of mesophillic ropy strains of Lactococcus lactis

subsp. lactis and ssp. cremoris and concomitant production of heterotype EPS for texture.

6. Bioproduction/Biotransformation of Trace Elements

6.1 Lactic Acid Bacteria as an Organic Source of Selenium

Selenium is a trace element which is essential for normal functioning of both humans and animals.It

happens to be the only mineral that qualifies for a Food and Drug Administration (FDA)-approved

qualified health claim for general cancer reduction incidence. The Se residue is essential for catalytic

activity as it takes part in catalysis. Thus, Se enhances immunity, growth, reproductive performance, and

inhibition of pathognes. Se deficiency has been associated with the decreased activity of glutathione

peroxidase. Food is the main source of Se for the human population. Se levels in foodstuffs such as

cereals, grains, fruits and vegetables are relatively low and cannot meet people’s daily dietary

requirement. Presence of selenium in food is generally reflected by its levels in soil. It has been reported

that Se bound in organo-metallic complex are much better absorbed by the body than if they are taken in

the inorganic form. In a recent publication, biotransformation of Se (IV) has been studied, when the

process of lactic fermentation was carried out with bacteria Lactobacillus in the presence of increasing

amounts of Se (IV) to produce Se-enriched yogurt. The main species found were selenocystine (SeCys2)

and methylselenocysteine (MeSeCys). It was found that various Lactobacillus species could concentrate

Se intracellular as seleno-cysteine in biomass and suggested that Se-enriched lactobacilli could provide a

means of concentrating selenoproteins and can be used as an organic selenium source for dietary

supplementation. A number of studies have been conducted and resulted in the production of selenium

enriched biomasses. Lactic Acid Bacteria have been recognized to have the ability to synthesize

biomolecules containing Se. It has been reported that Seleno-Lactobacillus could be used as an organic

selenium source. In a US patent application another genera, Pediocccous pentosaceus has been reported

to produce both organic and inorganic fractions of selenium and feeding of such selenium enriched

bacteria to animals showed higher levels of glutathione peroxidase activity in tissues indicating an

increased absorption of and retention of selenium. A US patent have been rewarded for the technology of

food preparation, food supplement and nutraceutical product comprising selenium enriched biomass of

viable Lactobacilli isolated from faecal samples. Advantageously, the microorganism used in the process

of the technology may be selected from the group consisting of the following species: Lactobacillus

bulgaricus, Lactobacillus acidophillus, Bifidobacterium bifidum, Streptococcus thermophilus,

Lactobacillus casei, Lactobacillus rhamnosus and Bifidobacterium longum. The selenium source is the

sodium hydrogen selenite (NaHSeO3) as well, but in powder form. For the inoculation one or the mixture

of the following strains: Lactobacillus acidophillus, Streptococcus thermophilus and Lactobacillus casei

were added. After mixing all components in medium, cans or buckets were placed into the shaking

incubator for 36-48 hours at 37⁰C. In the end of the fermentation process, dense, selenium rich pink or red

yoghurt was prepared.

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6.2 Lactic Acid Bacteria as an Organic Source of Zinc

Zinc (Zn) is one of the essential metal ions to life. After iron, it is the second most abundant transition

metal ion in living organisms, including humans. In order to develop biotechnological sources of trace

elements for diet supplementation, microorganisms (e.g., yeast, lactobacilli, and Spirulina strains) were

proposed as organic matrixes for incorporation of minerals (Mazo et al., 2007; Qin et al., 2007; Slavik et

al., 2008). The addition of inorganic salts into cultivation media enables the biosorption of the mineral

ions by the microbial biomass. As a consequence, the biomass becomes enriched with organic forms of

trace elements, which are present as complexes with amino acids, proteins, lipids, and polysaccharides

(Mazo et al., 2007). Lactic acid bacteria (LAB) are nonpathogenic, food safe microorganisms, which are

commonly applied in food processing. LAB enriched with zinc could be a valuable source of this element

in food, because zinc organic compounds (metalloproteins or bioplexes) are the best form for absorption

by humans. In that form, their absorption rates are higher than in inorganic compounds (Stanley et al.,

2012). Microelements bound in the form of protein complexes are absorbed in the small intestine in a

manner typical of peptides and proteins that enables penetration of microelements in the intestinal wall

(Mrvcic et al., 2009). To increase the daily intake of these mineral, numerous food supplements

containing different inorganic and organic forms of Zn is commercially available. At any rate, it is quite

well known that inorganic salts have a very low bioavailability. Organic salts, commonly based on

gluconate, orotate, citrate, or other molecules, are characterized by a higher systemic effect. The

innovative opportunity of using certain species of LAB enriched with the zinc could represent an

interesting alternative to these preparations. A spectrum of clinical manifestations ranging from mild to

severe degree have now been recognized in human zinc deficiency states so the supplementation of zinc

deficient groups with zinc-enriched LAB may be a new promising application of LAB in addition to their

probiotic activity. Diet integration with bacteria able to internalize Zn may embody a new application of

probiotics LAB. This approach for zinc supplementation could be of interest if zinc complexes are

released from enriched biomass upon passing through the upper gastrointestinal tract, counteracting the

effects of antinutritional factors. If biosorbed zinc is fully bioavailable, zinc enriched biomass of

Lactobacilli could provide the host with high amount of bioavailable zinc that would fulfil the

requirement of the recommended daily intake.

6.3 Metal Nanoparticle Synthesis by Lactic Acid Bacteria

Biological methods of nanoparticle synthesis using microorganisms have offered a reliable, eco-friendly

alternative to chemical and physical methods. LAB are prokaryotes in terms of cellular organization and

they are gram positive (a thick peptidoglycan cell wall) bacteria showing facultative anaerobic properties,

which probably make them suitable candidate microorganism for biosynthesis of metal as well as oxide

nanoparticle. Like most of the bacteria, they have a negative electro-kinetic potential; which readily

attracts the cations and this step probably acts as a trigger of the procedure of biosynthesis (Jha and

Prasad, 2009 & 2010; Prasad & Jha 2009). Some genera of LAB like Lactobacilli, Bifidobacteria, etc. per

se have been recognised to be endowed with the ability to bind, uptake and biotransform metal ions from

the medium (Mrvčić et al., 2012). This property provides important physiological, technological and

nutritional implications for both LAB and humans. The enrichment with selected heavy metals alters their

physicochemical properties and can be used for potentiating their probiotic and health promoting

attributes (Bomba et al., 2002). Biologically synthesized metal NPs have found wide spectrum

applications which include targeted drug delivery, cancer treatment, gene therapy and DNA analysis,

biosensors, antibacterial agents and food packaging.

162

7. GOS (Prebiotic)

Beta-galactosidases are glycoside hydrolases and are categorized within the glycoside hydrolase families

(GH) 1, 2, 35 and 42. GH2 b-galactosidases catalyze the hydrolysis of lactose into glucose and galactose,

and the transfer of the galactosyl-moiety to suitable acceptors.In the presence of high concentrations of

lactose, GH2 b-galactosidases produce galactooligosaccharides (GOSs) (Hsu, GOSs are commercially

employed in infant formula and have shown therapeutic properties reducing the adherence of

enteropathogenic E. coli. One of the main differences between probiotics and prebiotics is that probiotics

are viable food components whereas prebiotics are nonviable food component. Use of probiotics is a way

to replenish bacteria levels in the gut with external microorganisms. Food products having probiotic

components may contain bacteria that are not necessarily indigenous to the human gut so once in the gut

they have to compete to find a place among established, colonized bacteria. Probiotics can also be

destroyed by the contents of the gastrointestinal tract. So probiotics cannot be used in wide range of food

products because of their viability issue. On the other hand, prebiotics are nondigestible, remain intact

through the digestive system and act as food for already established microflora. They are added to dairy

products, table spreads, baked goods, and breads, breakfast cereals and bars, salad dressings, meat

products, and some confectionery items. So prebiotics overcome many of the traditional limitations of

introducing probiotic bacteria in to the GI tract. Therefore, using prebiotics is arguably a more practical

and efficient way to manipulate the gut microflora. The beta-galactosidases (beta-Gals) of Lb. reuteri

L103 and L461 proved to be suitable biocatalysts for the production of prebiotic galacto-oligosaccharides

(GOS) from lactose. Another study employed b-galactosidases present in disrupted crude cell extracts

(CCEs) and whole cells of LAB and bifidobacteria for formation of galactooligosaccharides (GOSs) and

heterooligosaccharides (HeOSs) from lactose and the acceptor carbohydrates mannose, fucose, N-

acetylglucosamine (GlcNAc) and sialic acid. CCEs and whole cells successfully produced up to three

HeOSs with mannose, fucose or GlcNAc in addition to GOS, but did not utilize sialic acid as acceptor.

Heterologously expressed b-galactosidases of S. thermophilus and Lactobacillus plantarum hydrolysed

the novel HeOSs and confirmed, for the first time, fucose as an acceptor carbohydrate. LAB CCEs and

bifidobacteria CCEs and whole cells are suitable sources of b-galactosidases that can be used to

synthesize novel HeOSs with potentially expanded functionality in addition to GOSs.

8. Antifungal potential of Lactobacillus species

Milk is a rich medium and supports growth of most microorganisms. Fermented milk products have high

acidic conditions that favor the growth of yeast and molds. Bio preservation is the extension of shelf-life

and food safety by the use of natural or controlled microbiota and their antimicrobial compounds. Lactic

acid bacteria (LAB) are GRAS status microorganisms and many LAB strains have been reported to have

antagonistic properties which make them particularly useful as biopreservatives. Antifungal activity of

lactobacillus strains has been extensively studied among lactic acid bacteria. Lactobacillus sp. having

antifungal activity has been identified from different environments like silage, vegetables, fermented

products etc. Antagonistic compounds of Lactobacillus sp. have been classified into proteinaceous and

non proteinaceous (Fig 1.). Table 2 enlists a number of lactobacilli which are commercially used as

bioprotective cultures.

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Table 2. Commercial Use of Lactobacilli as bioprotective cultures

Commercial

Bio protective

cultures

Microorganisms Products Target organism Company

Proteria™ AF Lactobacillus paracasei,

Propionibacterium

freudenreichii subsp

shermani

Yogurt and Yogurt

based drinks

Yeasts and Molds Handary

BefreshTM AF L. paracasei,

Pr. freudenreichii subsp

shermani

Yogurt, sour

cream, sours milk,

fresh cheese and

Cottage cheese.

Yeasts and Molds Handary

Holdbac™ YM

B plus

L. rhamnosus and

Pr. Freudenreichii

subsp. shermanii

Fresh fermented

milks, white cheese

Yeasts, molds and

certain hetero-

fermentative lactic

acid bacteria

Danisco

Holdbac™ YM

C plus

L. paracasei,

Pr. Freudenreichii subsp.

shermanii

Fresh fermented

milks, white cheese

Yeasts, molds and

certain hetero-

fermentative lactic

acid bacteria

Danisco

Fig 1. Antifungal Substance (AFS) produce by Lactobacillus sp.

164

FreshQ®

LAB (undisclosed) Yogurt, sour

cream, quark,

Tvarog and white

brined cheese

Yeasts and molds Chr Hansens

Pal Bioprotect D L. rhamnosus,

Pr. freudenreichii

subspecies shermani

Fermented milk,

Mozzarella, Feta

cheese

Yeasts, molds and

heterofermentative

lactobacilli

PAL Lab.

STANDA

Lyofast LPRA Contains probiotic

bacteria L. rhamnosus, L.

plantarum

Fermented milks,

Yogurt, cheese,

Yeasts, molds and

unwanted bacteria

Curds &

Whey

Lyofast BG112 L. plantarum Fermented milk Unwanted bacteria,

yeasts and molds

Sacco Clerici

Lyofast FPR 2 Enterococcus faecium,

L. plantarum, L.

rhamnosus

Fermented milk

products and

cheese

Unwanted bacteria,

yeasts and molds

Sacco Clerici

Lyofast LR B Lactobacillus rhamnosus Fermented milk

products and

cheese

Yeasts and molds Sacco Clerici

Lyofast LPR A L. rhamnosus , L.

plantarum

Fresh cheese, soft

cheese, semi-hard

cheese, and hard

cheese

Unwanted bacteria,

yeasts and molds

Sacco Clerici

9. Conclusions

LAB have been extensively used for centuries as starter cultures to carry out food fermentations and are

looked upon as burgeoning “cell factories” for production of host of functional biomolecules and food

ingredients. As such abilities are recognised to be strain specific, it can be an attractive strategy to

bioprospect prolific metabolite producing strains from their diversified natural niche and enhance their

ability by microbiological and biotechnological interventions. Such strains can be used for microbial

synthesis of biomolecules as an effective and attractive strategy for their delivery as a functional bio-

ingredient through foods to meet the daily recommended intake of human population. This information

opens the way to increase metabolites concentrations in fermented foods through judicious selection of

the microbial species and cultivation conditions. The food industry has taken the steps to use this

information for selecting various health promoting metabolites producing strains as part of their starter

cultures to produce fermented products with elevated levels of these essential compounds. Such products

would provide economic benefits to food manufacturers as increased ‘natural’ metabolite concentrations

would be an important value-added effect without increasing production costs. Consumers would

165

obviously benefit from such products as they could increase their functional food ingredients intake while

consuming these as part of their normal diet.

10. Suggested Reading:

Thakur K, Tomar S K and S. (2016). Lactic acid bacteria as a cell factory for riboflavin production. Microbial

Biotechnology (In Press)

Pophaly S, Singh P, Kumar H, Tomar S K and Singh R (2014) Selenium enrichment of lactic acid bacteria and

bifidobacteria: A functional food perspective. Trends in Food Science & Technology 39 135-145.

Poonam, Pophaly S, Tomar S K, De S. and Singh R (2012). Multifaceted attributes of dairy propionibacteria: a

review. World Journal of Microbiology and Biotechnology 28 3081–3095.

Pophaly S , Singh R, Pophaly S D, Kaushik J K and Tomar S K (2012). Current status and emerging role of

glutathione in food grade lactic acid bacteria. Microbial Cell Factories 11 114.

Sangwan V, Tomar S K, Singh R R B, Singh A K and Ali B (2011). Galactooligosaccharides: Novel Components of

Designer Foods. Journal of Food Science 76 R103-111.

Iyer R, Tomar S K, Maheswari U T and Singh R (2010). Streptococcus thermophilus strains: Multifunctional lactic

acid bacteria. International Dairy Journal 20 133–141.

Iyer R and Tomar S K (2009) Folate: A Functional Food Constituent. Journal of Food Science 74 R114-122.

Patra F, Tomar S K and Arora S (2009) Technological and Functional Applications of Low-Calorie Sweeteners from

Lactic Acid Bacteria. Journal of Food Science 74 R16-R23.

166

Newer Probiotic Organisms for Different Physiological Conditions

Rashmi H. M


1. Introduction

Probiotic bacteria, which are the part and parcel of the natural human gut microbiota, benefits the host

by improving gut homeostasis. As gut microbiota continues to fascinate the scientists in many realms,

it is considered as a "super organ" with diverse roles in health and disease. In addition, with the

establishment of profound correlations between gut microbial changes and their activity with common

disorders such as cancer, hypertension, hypercholesterolemia, inflammatory bowel diseases, obesity,

oral health etc., attempts have been made to use probiotics as dietary interventions towards the

prevention and management of various diseases and disorders. Now, the challenge is to mine and

design newer probiotic strains and improved delivery systems with a specific focus on their utility as

therapeutics for a wide range of enteric (from chronic to acute infections), respiratory, metabolic and

neurological diseases and disorders.

2. Probiotics in improving Nutritional status of host

The role of probiotics in improving the nutritional status among people of different age groups

(neonates and adults/elder) and health status (diseased/normal) have been extensively studied by

various investigators in the last few decades. The mechanisms involved in improvement of nutritional

status of host are: 1) improved gut microbial balance for effective metabolism of nutrients, their

absorption and bio-availability 2) improved synthesis of bioactive components like organic acids,

bioactive peptides, short chain fatty acids, vitamins which contribute significantly towards the

betterment of nutritional status of the host. Recently, the application of ‘omic sciences’ in nutritional

research has unraveled various signaling pathways which are modulated by probiotics and their

metabolites that elicit specific responses in the host and enabled the proof of nutrient optimization

rather than waiting for disease symptoms to appear and/or progress (Bron et al., 2012; Ferguson,

2015).

3. Probiotics against allergic and inflammatory disorders

The ability of probiotic bacteria to modulate immune responses is widely used as strategic approach to

treat various allergic and inflammatory disorders (Jan et al., 2012). The main mechanisms of probiotic

action on immune status and inflammatory processes of host have been well documented backed up

with cytokine production profiles in various in vitro cellular and in vivo animal models and their

efficacy has been established with intervention studies in human clinical trials. The mechanism of

immunomodulation involves the secretion of IgA that provide the host with a first line of immune

defense, modulation of DC/NK interaction, maintenance of Th1/Th2 immunity, modulation of anti-

inflammatory response (Aparna et al., 2013), enhancement of Treg activity to modulate cytokine

secretion and expression of antioxidative enzyme cascade (Chauhan et al., 2014).

4. Probiotics and improved intestinal barrier function

The intestinal barrier plays an important role in the maintenance of human health by protecting the

host from penetration of dangerous macromolecules like LPS besides nutrient absorption. The

intestinal barrier is a complex multilayer system made up of an external "anatomic" barrier and an

inner "functional" immunological barrier. The interaction of these two barriers enables equilibrated

permeability that helps in maintenance of human health. However, the factors like gut microflora

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modifications, altered mucus secretion and epithelial damage were found responsible for decreased

barrier function of intestine and increased translocation of luminal contents to the inner

immunological barrier. The increased translocation of luminal contents through leaky epithelium

causes overstimulation of inner mucosal immune system that is responsible for development of

various pathophysiological consequences resulting into diseases or disorders like IRD, IBS, colitis

and Metabolic Syndrome (Moeinian et al., 2014).

Probiotics are the attractive therapeutic option for improving the gut barrier function through the

modulation of gut microbial composition, increased mucus secretion, increased expression and

structural rearrangement of tight junction proteins in the small bowel and colon that results into

improved health of humans suffering from inflammatory and metabolic diseases and disorders

(Rokana, 2015).

5. Probiotics and antimicrobial effects against potential pathogens

The antimicrobial activity of probiotics against potential enteric pathogens like Salmonella enterica

serovar Typhi, Salmonella enterica serovar Typhimurium, Escherichia coli, Enterococcus faecalis,

Staphylococcus aureus and Clostridium difficile, Klebsiella pneumonia and vaginal pathogens like

Gardnerella vaginalis and Candida albicans has been extensively studied (Tejero et al., 2012;

Rokana, 2015). The main antimicrobial mechanisms include the production of organic acids such as

lactic and acetic acids that consequently lowers the culture pH, secretion of bacteriocins and

antimicrobial peptides, production of H2O2 and nitric oxide besides metabolities like short chain fatty

acids that are antagonistic to potential pathogens.

6. Mechanisms of probiotics action in the prevention and treatment of intestinal diseases

Changes in gut microbial composition with increased numbers of potentially pathogenic species is the

main cause in the development of gastrointestinal disorders and infections like antibiotic-associated

diarrhoea and Clostridium difficile associated diarrhoea, functional bowel problems (constipation and

irritable bowel syndrome), inflammatory bowel diseases [Crohn's disease (CD) and ulcerative colitis

(UC)] among people of various age groups (Malaguarnera et al., 2012). This dysbiosis in gut

microbial composition alters the gut barrier function, intestinal immune system and inflammation that

are responsible for development of above mentioned intestinal diseases. However, probiotics have

been found effective in the treatment of these diseases with improved gut microbial balance, enhanced

intestinal barrier function, improved exclusion of pathogens from intestinal epithelium and

modulation of immunity and improved bowel dysmotility (Chauhan et al., 2014; Rokana, 2015)

7. Mechanisms of probiotics in the management of metabolic disorders

Probiotics have recently emerged as the prospective biotherapeutics in the management of metabolic

syndrome with proven efficacy demonstrated in various in vitro and in vivo animal models adequately

supported with their established multifunctional roles and mechanism of action for the prevention and

disease treatment. The major mechanisms under this involves improved gut microbial balance with

conjugated linoleic acid (CLA) production, decreased food intake, decreased abdominal adiposity and

total cholesterol, decreased inflammatory tone with improved mucosal integrity in the management of

metabolic syndrome (MetS) like obesity and type 2 diabetes (Mallappa et al., 2012; Panwar et al.,

2013).

8. Conclusion:

From general gut health, to immune, respiratory, metabolic and mental health, the probiotic research

thus continues to build. Over the past decade, there has been extensive work in animal models on how

probiotics modulate host metabolism and physiology. However, there are very few good, doubleblind,

placebo-controlled clinical trials that can prove the potential of probiotics on modulating human

http://www.ncbi.nlm.nih.gov/pubmed?term=Malaguarnera%20G%5BAuthor%5D&cauthor=true&cauthor_uid=22499466

168

metabolism. Therefore, elucidation of mechanisms and substantiation of animal studies in humans

with a specific focus on their utility as therapeutics will open new horizon in the area of probiotic

research.


Aparna S V, Panwar H, Chauhan R, Duary R, Rathore R, et al. (2013) Modulation of anti-inflammatory

response in lipopolysaccharide stimulated human THP-1 cell line and mouse model at gene expression level

with indigenous putative probiotic lactobacilli. Genes & nutrition 8 637-648

Bron P A, van Baarlen P and Kleerebezem M (2012) Emerging molecular insights into the interaction between

probiotics and the host intestinal mucosa. Nat Rev Micro. 10 66-78.

Chauhan R, Sudhakaran Vasanthakumari A, Panwar H, Mallapa RH, Duary RK, et al. (2014) Amelioration of

Colitis in Mouse Model by Exploring Antioxidative Potentials of an Indigenous Probiotic Strain of

Lactobacillus fermentum Lf1. BioMed Research International 2014 12.

Ferguson L R (2015) Nutritional modulation of gene expression: might this be of benefit to individuals with

Crohn’s disease? Frontiers in Immunology 6.

Jan R L, Yeh K C, Hsieh M H, Lin Y L, Kao H F, Li P H, Chang Y S and Wang J Y (2012) Lactobacillus

gasseri suppresses Th17 pro-inflammatory response and attenuates allergen-induced airway inflammation in

a mouse model of allergic asthma. British Journal of Nutrition. 108 130-139.

Malaguarnera G, Leggio F, Vacante M, Motta M, Giordano M, Bondi A, Basile F, Mastrojeni S, Mistretta

A, Malaguarnera M, Toscano M A, Salmeri M (2012) Probiotics in the gastrointestinal diseases of the

elderly. The Journal of Nutrition Health and Aging 16 402-410.

Mallappa H R, Rokana N, Duar R K, Panwar H, Batish V K and Grover S (2012) Management of metabolic

syndrome through probiotic and prebiotic interventions. Indian Journal of Endocrinology and Metabolism.

16 20-27.

Moeinian M (2014) Beneficial effect of butyrate,Lactobacillus caseiand L-carnitine combination in preference

to each in experimental colitis. World Journal of Gastroenterology 20: 10876.

Panwar H, Rashmi H M, Batish V K and Grover S (2013). Probiotics as potential biotherapeutics in the

management of type 2 diabetes - Prospects and Perspectives. Diabetes Metabolism Research and Reviews

29:1 03-11 2.D OI: 10.10021dmrc.2376

Rokana N (2015) Mechanistic study of a potential indigenous probiotic Lactobacillus strain and its fermented

milk formulations in enhancing intestinal barrier function in mouse model. (Doctoral dissertation).National

Dairy Research Institute, Karnal

Tejero-Sarinena S, Barlow J, Costabile A, Gibson G R and Rowland I (2012) In vitro evaluation of the

antimicrobial activity of a range of probiotics against pathogens: Evidence for the effects of organic acids.

Anaerobe. doi:10.1016/ 08.004

http://www.ncbi.nlm.nih.gov/pubmed?term=Malaguarnera%20G%5BAuthor%5D&cauthor=true&cauthor_uid=22499466

http://www.ncbi.nlm.nih.gov/pubmed?term=Leggio%20F%5BAuthor%5D&cauthor=true&cauthor_uid=22499466

http://www.ncbi.nlm.nih.gov/pubmed?term=Vacante%20M%5BAuthor%5D&cauthor=true&cauthor_uid=22499466

http://www.ncbi.nlm.nih.gov/pubmed?term=Motta%20M%5BAuthor%5D&cauthor=true&cauthor_uid=22499466

http://www.ncbi.nlm.nih.gov/pubmed?term=Giordano%20M%5BAuthor%5D&cauthor=true&cauthor_uid=22499466

http://www.ncbi.nlm.nih.gov/pubmed?term=Bondi%20A%5BAuthor%5D&cauthor=true&cauthor_uid=22499466

http://www.ncbi.nlm.nih.gov/pubmed?term=Basile%20F%5BAuthor%5D&cauthor=true&cauthor_uid=22499466

http://www.ncbi.nlm.nih.gov/pubmed?term=Mastrojeni%20S%5BAuthor%5D&cauthor=true&cauthor_uid=22499466

http://www.ncbi.nlm.nih.gov/pubmed?term=Mistretta%20A%5BAuthor%5D&cauthor=true&cauthor_uid=22499466

http://www.ncbi.nlm.nih.gov/pubmed?term=Mistretta%20A%5BAuthor%5D&cauthor=true&cauthor_uid=22499466

http://www.ncbi.nlm.nih.gov/pubmed?term=Malaguarnera%20M%5BAuthor%5D&cauthor=true&cauthor_uid=22499466

http://www.ncbi.nlm.nih.gov/pubmed?term=Toscano%20MA%5BAuthor%5D&cauthor=true&cauthor_uid=22499466

http://www.ncbi.nlm.nih.gov/pubmed?term=Salmeri%20M%5BAuthor%5D&cauthor=true&cauthor_uid=22499466

http://www.ijem.in/

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Isolation of Potential New Probiotic Bacteria

Diwas Pradhan


1. Introduction

According to FAO/WHO (2002), ‘Probiotics are live microorganisms, which when administered in

adequate amounts confers a health benefit on the host’. A number of health benefits has been

attributed to probiotic bacteria such as protection against pathogenic microorganisms, decrease of

incidence and duration of antibiotic and rotavirus associated travelers’ diarrhea, alleviation of

symptoms of lactose intolerance, reduction of allergic reactions, anticolon cancer and antimutagenic

activities, antihypertensive or anticholesterol effects etc. Amongst different microbial types, members

of Lactobacilli and Bifidobacteria genera have been explored most extensively as potential probiotics

for human health. These bacteria have a long history of safe use in food fermentation and also form a

significant part of microbial population in human GI tract.

The selection of a strain to be used as an effective probiotic is a long and complex process. It starts

with the isolation process, followed by identification of the isolate and finally validation of essential

probiotic properties through appropriate tests. FAO/WHO (2002), at the international level and

ICMR/DBT (Ganguly et al., 2011) at the national level has laid down the detailed guidelines for the

evaluation of microorganisms to be used as potential probiotics. The isolation process is perhaps the

first step in the selection of effective probiotic strains. Various niches such as human gut, breast milk,

fermented dairy products, animal gut etc. which are normally inhabited by lactic acid bacteria are

most commonly used for probiotic isolation, although it is generally considered that the specificity of

the action is more important rather then the source of the microorganism. Nonetheless a human origin

isolate is more preferred over other niches since human isolates are better adapted to the human

environment (host specificity). A human origin probiotic isolate may be obtained from faecal matter

of healthy adult or infants, breast milk, vaginal flora, oral cavity etc.

2. Isolation Protocol

1. Samples (Fecal matter/dairy products) are collected in sterile containers.

Label the container with relevant information like name, age, date etc.

It is advised to process the sample immediately or refrigerated stored.

Anaerobic transport media may be used to maintain the viability of anaerobic

organisms.

2. A measured amount of sample is placed into a stomacher bag and sample homogenized in a

stomacher.

3. The homogenate is then serially diluted to make adequate dilutions with buffers (saline and

PBS) or pre-reduced buffers or broths (anaerobic dilution buffer, 0.25 x ringer solution

supplemented with cysteine, Wilkins-Chargren broth, saline with cysteine etc.) (Endo and

Gueimonde, 2015).

4. The diluted samples are then plated on respective selective/non-selective media (Table 1).

5. Plates are then incubated at 37°C under aerobic (Lactobacilli) or anaerobic conditions

(Bifidobacteria/Lactobacilli) for 24-48 h.

6. Single colonies are then transferred into selective broth tubes and incubated accordingly.

170

7. Further tests on the isolate maybe then carried out or glycerol stocks of the same may be

prepared and stored at -80°C for future use.

Note: Strict anaerobic conditions at each step needs to be followed for Bifidobacterium sp.

Anaerobic workstations should be used for working with Bifidobacterium sp.

Table 1: Isolation media for Lactobacilli and Bifidobacteria (Coeuret et al., 2003; Ashraf and

Shah, 2011; Endo and Gueimonde, 2015)

Probiotic bacteria Medium of Isolation

Lactobacilli MRS (de Mann Rogosa)

LBS Agar (Lactobacillus Selection Agar)/ Rogosa Agar

Lactobacillus anaerobic MRS agar with Vancomycin and Bromocresol

(LAMVAB) medium

Lb. acidophilus MRS + Maltose agar

MRS + Clindamycin agar

MRS + Salicin agar or MRS + Sorbitol agar

Lb. casei MRS-LP (Lithium chloride & Sodium Propionate) agar

MRS + Maltose agar + 4% Salt

Bifidobacteria BL medium + NPNL (neomycin, paromomycine sulphate, nalidixic acid

and lithium chloride)

MRS + NPNL; MRS-raffinose agar

RMS-PPNL agar (Modified Rogosa agar with sodium propionate,

paromomycin sulphate, neomycin sulphate and lithium chloride)

BLOG agar (blood-glucose-liver agar with oxgall and gentamycin)

MCAP medium (Columbia agar base media with propionic acid)

Wilkins-Chalgren (W-C) agar with muporicin

3. Identification of probiotic organisms

Correct identification of a probiotic strain is of utmost importance. This is important not only

for reliably identifying a bacterial strain but is also required for proper labelling. Conventionally,

bacterial identification methods mainly relied on a set of phenotypic tests. However in the recent

times phenotypic tests as a standalone approach is not used for identification of probiotic cultures. A

polyphasic approach consisting of a combination of phenotypic and genotypic tools is generally

followed for reliably establishing the strain identity.

4. Phenotypic identification

Phenotypic test mainly relies on an organisms phenotypic characteristics such as cell morphology,

growth at different temperatures, pH, salt concentration, analysis of fermentation products, ability to

utilize various carbohydrate etc. In the same line already known phenotypic characters of either

lactobacilli or bifidobacteria can be utilized to have a preliminary identification of the isolate. Ideally,

Bifidobacteria are Gram-positive, non-sporing, non-motile, strictly anaerobic intestinal rods of

variable morphology (bifurcated), which possess fructose-6-phosphate phosphoketolase (F-6-PPK),

the key enzyme of the bifid-shunt. Similarly, Lactobacilli are a wide group of Gram-positive rods that

are microaerophilic to anaerobic in oxygen requirement. Additionally these are catalase negative and

may ferment lactose to lactic acid alone or other products. Strains of lactobacilli and bifidobacteria

can also be grouped according to their ability to ferment a range of carbohydrates.

These test are relatively easy to perform, needs no special equipment or expertise. However these

methods lacks reproducibility and has a poor discriminating power at the species/strain level. These

methods are also lengthy and labor intensive, which has now led to the emergence of polyphasic

approach for establishing the strains identity.

171

5. Genotypic methods for bacterial identification and strain typing

PCR based methods are widely used to reliably identify bacterial isolates. 16S ribosomal RNA

(rRNA) gene sequence analysis is usually regarded as the best tool for the taxonomic positioning of

probiotic cultures. Housekeeping genes or 16S-23S rRNA genes intergenic spacer region is often used

in case of certain closely related species where high sequence similarities in 16S rRNA genes are

present. Housekeeping genes such as hsp60, dnaJ1, pheS, rpoA, atpD, recA, dna etc. have been used

for probiotic bacteria identification.

Probiotic isolates must also be identified at the strain level since probiotic properties are highly strain

specific. A good strain typing method should give reproducible results. Although many other strain

typing methods such as RAPD, MLST, Rep-PCR, RFLP etc. are available, DNA macro restriction

followed by pulse field gel electrophoresis (PFGE) is considered the gold standard for strain

identification. Whole genome sequencing by far constitutes the best method to genetically fingerprint

a given strain. However due to its time consuming nature, cost, equipment availability, method

sophistication etc. the method is still not widely used.

6. Selected Reading

Ashraf R and Shah N P (2011) Selective and differential enumerations of Lactobacillus delbrueckii subsp.

bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium

spp. in yoghurt—A review. International journal of food microbiology 149 194-208.

Coeuret V, Dubernet S, Bernardeau M, Gueguen M and Vernoux J P (2003) Isolation, characterisation and

identification of lactobacilli focusing mainly on cheeses and other dairy products. Le Lait 83 269-306.

Endo A and Gueimonde M (2015) Isolation, identification and characterisation of potential new

probiotics. Advances in Probiotic Technology, 1.

FAO/WHO. (2002) Guidelines for the evaluation of probiotics in food, Joint FAO/WHO working group report

on drafting guidelines for the evaluation of probiotics in food. London, ON, Canada, April 30th May 1st.

Ganguly N K, Bhattacharya S K, Sesikeran B, Nair G B, Ramakrishna B S, Sachdev H P S, Batish V K,

Kanagasabapathy A S, Muthuswamy V, Kathuria S C, Katoch V, Satyanarayana K, Toteja G S, Rahi M, Rao

S, Bhan M K, Kapur R and Hemalatha R (2011) ICMR-DBT guidelines for evaluation of probiotics in

food. The Indian journal of medical research 134 22.

172

Preservation Potential of Plant Essential Oils in Dairy Foods

Chand Ram Grover and Rohit PanwarDairy Microbiology Division

1. IntroductionDairy foods are prone to contamination by variety of microbes including pathogens and spoilageduring production, processing, post processing, transportation and handling. India is highest milkproducer but lag behind to meet international standards w.r.t. microbial quality that result about 5%loss due to souring of milk. The contamination of dairy foods with spoilage and pathogenic microbesmake it unfit for consumption causing huge economic loss to manufactures and consumers as well asnation’s health. Further, huge export potential of Indian dairy foods has remained untapped due tonon-conformation of food safety and quality norms of developed countries. Thus, qualityimprovement of milk and milk products is the need of hour to remain competitive in domestic andinternational market. Efforts have been made to enhance food safety and shelf life of dairy productsby use of alternate technologies such as mild heat processing, modified atmosphere and vacuumpackaging as well refrigeration. All these technologies suffer setback as they cannot completelyeliminate pathogens and spoilage microflora. Indiscriminate use of traditional preservatives &antimicrobials has also enhanced resistance in food borne microbes.

The “green consumerism” is a new concept of food safety that necessitates minimum use of syntheticpreservative due to their adverse health effects. Thus, use of “natural antimicrobials” including plantbioactive components would be better strategy to reduce or eliminate risk of pathogens and spoilagemicroorganisms to improve overall food safety and shelf life of dairy products.

2. Food antimicrobials and their requirements for use in foodsFood antimicrobials are chemical compounds which delay microbial growth or cause death in foodmatrix. These are usually classified into traditional/natural and synthetic. Essentials oils (EOs) aresecondary metabolites obtained from various plant parts and have been used for centuries incomplementary or alternative medicine for treatment of various ailments, perfumery and cosmetics.More than 3000 different EOs are known of which ~300 commercially used in flavour and fragrancesmarket and have been accorded GRAS status. Although, food industry primarily uses EOs asflavourings, however recently, these have been explored for food preservation and food safety due totheir broad-range antimicrobial activities. Dela Croix in1881 was the first to evaluate antimicrobialactivity of plant EOs. The antimicrobial nature of phyto-chemical is determined by its chemicalproperties, such as pKa value, hydrophobicity/ lipophilicity ratios, solubility, and volatility. The pHand polarity are the most prominent factors that influence the effectiveness of a food antimicrobial.Therefore, it is very important to know the specific characteristics of the food system that needs to bepreserved since a high proportion of lipids could limit the effectiveness of some antimicrobial agents(Owen and Palombo, 2007). The concentration thresholds required for inactivation of microbesdepends upon specific targets of antimicrobial substance including cell wall, cell membrane,metabolic enzymes, protein synthesis, and genetic systems.

3. Factors affecting antimicrobial efficacyThe application of bioactive components as food preservatives requires detailed knowledge abouttheir properties, i.e., minimum inhibitory concentration (MIC), range of target organisms, mode ofaction and effect of food matrix components on their antimicrobial activity. Generally, higherconcentrations of natural antimicrobials are required to achieve the same effect in food as compared to

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in-vitro assays. Various intrinsic (pH, salt, antioxidants and other additives) and extrinsic properties(temp, vacuum and modified atmosphere packaging, characteristic of microorganisms) of food play avital role in determining antimicrobial efficacy of bioactive components in food matrix. CinnamonEO was the most effective under refrigerated temp, inhibition of growth of B. cereus for at least 60days in a model refrigerated minimally processed food product made with carrots and fractionalheating. Lettuce leaf, beef and milk model media were used to study the efficacy of EOs againstListeria and spoilage bacteria, which was compared to the laboratory control media i.e. Tryptic SoyBroth (TSB). The efficacy of essential oils in the lettuce model media was 10 fold higher than in TSB,possibly due to the low fat content of vegetables. The EOs were less effective in beef extract than inTSB, probably high protein concentrations in beef extract promoted the growth of Listeria species.

4. Essential oils & extractsDifferent studies have demonstrated the effectiveness of EOs and their active compounds to control orinhibit the growth of pathogenic and spoilage microorganisms. The antibacterial activity is dependson pH, chemical structure and concentration of bioactive compound, besides the number and type ofmicroorganisms. The bacterial susceptibility to EOs increases with a reduction in pH of the food,since at low pH the hydrophobicity of the oil increases, enabling it to more easily soluble in the lipidsof cell membrane of the target bacteria (Burt, 2004). Partition coefficients of the EOs might also havean effect on activity by influencing its diffusion rate through the cell membrane, as higher partitioncoefficient of citral as compared to cinnamaldehyde and eugenol resulted in the faster reduction of E.coli O157:H7 (Raybaudi-Massilia et al., 2009). Storage temperature also influences the antimicrobialeffectiveness of EOs, as the bactericidal activity of different EOs or their active components againstE. coli O157:H7 and Salmonella in apple juice was higher at 37 °C than at 4 and 21°C (Friedman etal., 2004). Owen and Palombo (2007) investigated the ability of Eremophila duttonii and E.alternifolia to control the growth of L. monocytogenes in full cream milk, skim milk, dilutedhomogenates of salami, pate and brie cheese, and reported that both the extracts inhibited the growthof L. monocytogenes in salami at 37°C. In another study on yoghurt, addition of anise EOs andoleoresin at 1.0 g/L conc. was effective in controlling the growth of spoilage microbes without anyadverse effect on physicochemical attributes and viability of LAB. Further, anise is advantageousbecause of its antioxidant and antimicrobial activity as well as nontoxic that can reduce oxidativedegradation of fatty substances as well as inhibit growth of spoilage microflora (Singh et al., 2011).

Table 1 Preservation potential of bio-active components in dairy foods

Products and storageconditions

Natural compounds Main results References

West African softcheese

Treatment witheucalyptus oil andlemongrass oi

The treatment of eucalyptus+lemon grass oil (75+25%) exerteda positive impact on nutritional,sensory & microbial quality ofWest African soft cheese

Belewu etal., 2012

Ricotta cheese storedunder modifiedatmosphere a t4◦C

Coating with achitosan/whey proteinedible film

The chitosan /whey protein filmslowed detrimental phenome-nafor LAB, mesophilic &psychrotrophic microbes. Thiswas significantly lower inchitosan/whey protein coatedcheese as compared to control

Di Pierro etal., 2011

Traditional MinasSerro cheese

Nisin

Nisin was effective @ 100IU/mLreducing S. aureus counts of 1.2& 2.0 log cycles in Serro cheesefrom 7th day of ripening Pinto et al.,

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2011Fresh cheese Tosèla Antimicrobial comp-

ounds produced bynon-starter lactic acidbacteria.

Cheese showed higher conc oflactobacilli (7.90 log CFU/g) andstreptococci (6.10 log CFU/g),lower development of coliformsand staphylococci than controlcheese

Settanni etal., 2011

Caprese saladpackaged underMAP(65%N2, 30%CO2, and5%O2)

Dipping with thymol(400 ppm)

The combined use of thymol andMAP reduced coliform from 5.65to 4.23 log CFU/g as well asPseudomonadaceae counts andextended shelf-life from 3.77 to12 days.

Bevilacquaet al., 2007

Gorgonzola cheeseNatamycin wasincorporated inpackaging film &lused for processcheese packaging

Films with 2 and 4% natamycinpresented satisfactory results forP. roquefortii inhibition

de Oliveira etal., 2007

Lassi (sweetened) Vanillin incorporatedin conjunction withmild heat treatment

Significantly enhancedinactivation rate of E. coliO157:H7

Gaare etal.,2015

4.1 Essential oil constituent classes

EOs constituent is a diverse family of low mol. weight organic compounds with large differences inantimicrobial activity. Identification of bioactive component is cumbersome as these are complexmixtures of upto 45 different constituents as well as their composition may vary depending on theseason of harvest and methods of extraction (Espina et al., 2011; Paibon et al., 2011). The bioactivecompounds are broadly divided into four groups according to their chemical structure: terpenes,terpenoids, phenylpropenes, and “others.”

4.1.1 Terpenes

These are hydrocarbons produced from combination of several isoprene units (C5H8) and synthesizedin the cytoplasm of plant cells. The main terpenes are monoterpenes( C10H16) and sesquiterpene(C15H24), but longer chains such as diterpenes (C20H32), triterpenes (C30H40),. Examples include p-cymene, limonene, terpinene, sabinene, and pinene. In vitro tests indicate that terpenes are inefficientantimicrobials when applied as single compounds.

4.1.2 Terpenoids

Terpenoids are terpenes that undergoes biochemical modifications via enzymes that add oxygenmolecules and move or remove methyl groups. These can be subdivided into alcohols, esters,aldehydes, ketones, ethers, phenols, and epoxides (e.g. thymol, carvacrol, linalool, linalyl acetate,citronellal, piperitone, menthol, and geraniol). Antimicrobial activity of most terpenoids is linked totheir functional groups. The hydroxyl group of phenolic terpenoids and presence of delocalizedelectrons are important for antimicrobial activity. Exchange of hydroxyl group of carvacrol withmethylether affects its hydrophobicity and antimicrobial activity.

4.2 Mode of action

The mechanism of action of bioactive components is not fully understood. The Gram -ve aregenerally less susceptible than Gram +ve bacteria; as outer membrane of former contain hydrophiliclipo-polysaccharides (LPS), which create a barrier toward macromolecules and hydrophobiccompounds providing them higher tolerance toward hydrophobic antimicrobials present in EOs.

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Terpenoids and phenolics results membrane disruption, whereas phenols and flavonoids causes metalchelation. Coumarin and alkaloids affect genetic material that inhibits the growth of pathogens andspoilage microorganisms. Degradation of cell wall, damage to cytoplasmic membrane and membraneproteins, leakage of intracellular contents, coagulation of cytoplasm and depletion of proton motiveforce can cause cell death.

Thymol, eugenol, and carvacrol cause disruption of the cellular membrane, inhibition of ATPaseactivity, and release of intracellular ATP and other constituents of target microbes. Cinnamaldehydeproduces a decrease in the intracellular ATP by ATPase activity. It also increases membranepermeability, leakage of cytoplasm and interact with enzymes located on cell membrane. Thesechanges may cause its disruption to proton motive force by leakage of small ions or it can inhibit theenzymes necessary for amino acid biosynthesis. Carvacrol and thymol appear to make the cellmembrane permeable by dissolving into the phospholipid bilayer. This distortion causes expansionand destabilization of the membrane increasing its fluidity. Thymol binds to the membrane proteinshydrophobically and changes the permeability characteristics of membrane. Vanillin showedantimicrobial effect by affecting membrane functions and through inhibition of respiration in severalbacteria. Terpenes accumulate in the membrane and cause a loss of membrane integrity anddissipation of the proton motive force as well as disrupt the lipid structures.

4.3 Stability of bioactive during food processing

Health promotion through diet is gaining importance at a very fast pace, therefore, understanding ofprocessing effects on bioactive components is critical as they not only preserve the foods, but alsohave beneficial effect on human health. Heat processing i.e. sterilization, pasteurization, anddehydration may result loss while in some cases, induces the formation of the novel compounds,which either maintain or even increase the potential of various bioactive ingredients. Thermalprocessing caused marked losses in total anthocyanins in black raspberries and blueberries. Thethermal stability of phytochemicals added to food depends on the matrix in which they are found andadded. Significant changes in individual isoflavone levels were observed during storage of UHTprocessed chocolate flavoured high protein beverage containing soy proteins isolates depending onstorage temperatures (4, 23 & 38°C). Microcapsule curcumin was found to have similar antibacterialand antifungal activities as curcumin after microencapsulation. The amino acid conjugation alsoretained its antibacterial, antioxidant and antimutagenic activities, indicating its stability to chemicalmodifications (Parvathy et al., 2009).

4.4 Toxicity evaluation

Majority of bioactive components of plant origin have been consumed for thousands of years,however, typical toxicological information such as acceptable daily intake (ADI) or no observedadverse effect level (NOEL) are not available. Although International guidelines exist for the safetyevaluation of food additives, however, due to problems in standardization of these components owingto their batch wise compositional variability, it is difficult to assign ADI or NOEL. The markercompounds in EOs are affected by variety of plant, geographical origin, plant part used, age andgrowth condition of plants, method of extraction or drying, preparation, packaging and storage.According to Dietary Supplement Health and Education Act (DSHEA), 1994, botanicals areexempted from food additive category, and GRAS submission of safety evidence is not required aslong as that ingredient was in market before October 1994. The International Life Sciences Institute-Europe has developed a comprehensive document on the use of plant materials in food products(Schilter et al., 2003), which stresses that the ingredient for use in food products must be wellidentified and characterized. (Speijers et al., 2010; van den Berg et al., 2011).

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4.5 Interaction of bioactive with food components and its effects on antimicrobial activity

Most of investigations on antimicrobial properties of plant EOs have been focused at the efficacyagainst planktonic cultures of food related bacteria. However, evaluation for antimicrobial activityagainst native microflora of foods at different storage times, changes during processing and packagingis better criteria e.g. lightly preserved fish products where certain ingredients such as salt or sugar areadded and mildly processed using cold smoke. This type of processing lowers the water activitythereby inhibiting the growth of spoilage organisms and enhancing the growth of lactic acid bacteria(LAB). Vacuum packing of meat inhibits aerobic Pseudomonas sp. causing a change in microbiota.

4.6 Synergies within bioactive components

The main difficulty in utilization of plant EOs in dairy foods is their adverse effect on sensorialquality. Therefore, optimisation of their levels in foods is utmost priority. Bioactive components incombination exert better antimicrobial effect in foods as compared to individual major constituents,indicative of minor components synergy. Green tea extracts alone (20 or 40 mg/ml) or in combinationwith tartaric acid (37.5 mM) reduced Salmonella, Listeria and E. coli by 3.5 log CFU/ml in brothculture. The antimicrobial activity of grape seed extract when combined with bacteriocins like nisinhas demonstrated more effectiveness than when used alone against L. moncytogenes.

4.7 Delivery systems

Many methods are available to incorporate plant bioactive into foods. The simplest method is directaddition, however, in foods where surface sanitisation is targeted food product can be dipped inbioactive component(s) or applied as a spray. These simple delivery modes have become moresophisticated with the advancement in packaging, encapsulation and nanotechnologies. Some of themodes of application available for plant antimicrobials include:

4.8 Bioactive packaging

Incorporation of antimicrobial components in films rather than direct addition with food providesbetter functional effect at the food surface, where most of the microbial growth is localized.Antimicrobial packaging would include systems such as addition of sachet into package, dispersion orcoating of bioactive in or on the surface or as part of edible packaging material. Incorporation ofgarlic oil at 100 μl/g in chitosan and forming a film was found to exhibit antimicrobial activity againstS. aureus, L. monocytogenes and B. cereus. Kakadu plum (Terminalia ferdinandiana) is one of theAustralian native fruits indentified for its antimicrobial properties, having gallic acid as one of thecomponents with antimicrobial efficacy. Films made from 3% kakadu plum powder were found tohave antimicrobial activity against S. aureus, methicillin-resistant S. aureus (MRSA), L.monocytogenes, B. cereus, B. subtilis, E. coli, P. aeruginosa and Acinetobacter baumannii.

4.9 Encapsulation

Application of phyto-chemicals as preservative in food depends on maintaining the stability andbioactivity of the plant antimicrobial. The adverse effect of strong aroma and taste of bioactiveantimicrobials can be overcome by encapsulation instead of direct addition as free component to thefood. The encapsulation technologies include spray drying, coacervation, liposome entrapment,inclusion complexation, co-crystallization, nano-encapsulation, freeze drying, yeast encapsulation andemulsions etc. Nano-encapsulation of bio-actives represent an efficient approach to enhance physicalstability, protect them from interactions with food ingredients and, improve their bioactivity due tosub cellular size. A mixture of terpenes and D-limonene was encapsulated into nano-emulsions basedon food-grade ingredients, prepared by high pressure homogenization. The minimum inhibitoryconcentration (MIC) and the minimum bactericidal concentration (MBC) of the nano-encapsulatedterpenes against E. coli, L. delbrueckii and S. cerevisiae were lower or equal to the values of the un-

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encapsulated mixture. Microencapsulation of Mexican oregano EOs by spray drying preservedantimicrobial efficacy against P. aeruginosa, S. aureus and E.coli, improved its solubility in water.Linalool and methylchavicol from basil when incorporated in Low density polyethylene significantlyreduced E. coli and L. innocua in Cheddar cheese.

4.10 Challenges of using plant bioactive in foods

Plant bioactive components are essential to inhibit natural micro-flora of foods. The challenges ofusing plant antimicrobials are:

Some plant bio-actives have adverse effect on sensorial quality, therefore it is essential to matchthe food with bioactive flavour or understand the synergies to decide on the concentration to beused.

Type of microorganisms present in the food that can cause spoilage and disease is critical tounderstand the antimicrobial effect of plant components as it is not the same for allmicroorganisms.

Incorporation of plant antimicrobials in food can promote growth and virulence of certainpathogens due to changes in microbial ecology. It is critical to understand their effect on behaviourof these microorganisms in complex food systems.

The growing environment of the source plants influences the levels of antimicrobial compounds inthem. In addition, the period of harvest, storage and extraction procedures used have an effect onthe levels of active components responsible for antimicrobial activity and this would be achallenge in using it as a functional food ingredient.

5. ConclusionsMicrobial food safety is still a major concern to health conscious consumers, regulatory agencies andfood industries over the globe. Many food preservation strategies have been used traditionally for thecontrol of pathogens and spoilage microflora but contamination of food and spoilage microorganismsis a continuous problem yet to be controlled adequately. The literature demonstrates that differentplant antimicrobials effectively reduce or inhibit pathogenic and spoilage microbes, and thus have apotential to become a good alternative to synthetic preservatives. Further, the use of naturalantimicrobials in combination with another or with other technologies in a multi-hurdle preservationsystem can enhance the performance of natural bio-actives. Natural antimicrobials offer uniqueadvantages for food processing w.r.t. improvement in shelf life and safety of foods; they may allowdevelopments of novel food products with enhanced food safety, shelf life and nutritional security.The applications of natural antimicrobial agents are likely to grow steadily in the future because ofconsumer demand for minimal processing and food containing naturally derived preservatives is onrise. Further, it is expected that plant extracts showing target sites other than those used by antibioticswill be active against drug-resistant microbial pathogens. The impact of product formulation andstorage parameters on the efficacy of natural antimicrobials as well as safety and toxicologyevaluation of these natural antimicrobials require an in-depth study.

6. Selected ReadingBelewu M A, Ahmed El-Imam A M, Adeyemi K D, Oladunjoye S A (2012). Eucalyptus oil and lemon grass oil:

effect on chemical composition and shelf-life of soft cheese. Environment and Natural Resource Research 2114–118.

Bevilacqua A, Corbo M R, Sinigaglia M (2007) Combined effects of modified atmosphere packaging andthymol for prolonging the shelf life of caprese salad. Journal of Food Protection 70 722–728.

Burt S A (2004) Essential oils: Their antibacterial properties and potential applications in foods – a review.International Journal of Food Microbiology 94 223–253.

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de Oliveira T M, de Fátima Ferreira Soares N, Pereira R M, de Freitas Fraga K (2007) Development andevaluation of antimicrobial natamycin-incorporated film in Gorgonzola cheese conservation. PackagingTechnology and Science 20 147–153.

Di Pierro P, Sorrentino A, Mariniello L, Giosafatto C V L, Porta R (2011) Chitosan/whey protein film as activecoating to extend Ricotta cheese shelf-life. Food Science and Technology 44 2324–2327.

Gaare M, Ram C and Suman (2015) Antimicrobial efficacy of vanillin in conjuction with mild heat treatmentagainst Escherichia coli O157: H 7 in sweetened Lassi. Indian Journal of Dairy Science 68 223-228

Owen Roxanne J, Palombo Enzo A (2007) Anti-listerial activity of ethanolic extracts of medicinal plants,Eremophila alternifolia and Eremophila duttonii, in food homogenates and milk 18 387–390.

Parvathy KS, Negi PS, Srinivas P (2009) Antioxidant, antimutagenic and antibacterial of curcumin-β-diglucoside. Food Chemistry 115 265–271.

Pinto M S, Fernandes de Carvalho A, Pires A C S, Campos Souza A A, Fonseca da Silva P H, Sobral D, dePaula J C J, de lima Santos A (2011) The effects of nisin on Staphylococcus aureus count and thephysicochemical properties of Traditional Minas Serro cheese. International Dairy Journal 21 90–96.

Raybaudi-Massilia R M, Mosqueda-Melgar J, Sobrino-Lòpez A, Soliva-Fortuny R and Martín-Belloso O (2009)Use of malic acid and other quality stabilizing compounds to assure the safety of fresh-cut “Fuji” apples byinactivation of Listeria Monocytogenes, Salmonella Enteritidis and Escherichia coli O157, H7. Journal ofFood Safety. 29, 236–252.

Schilter B, Andersson C and Anton R, (2003) Guidance for the safety assessment of botanicals and botanicalpreparations for use in food and food supplements. Food Chemical Toxicology 41 1625- 49.

Settanni L, Franciosi E, Cavazza A, Cocconcelli P S and Poznanski E (2011) Extension of Tosèla cheese shelf-life using non-starter lactic acid bacteria. Food Microbiology 28 883–890.

Singh G, Kapoor I P S and Singh P (2011) Effect of volatile oil and oleoresin of anise on the shelf life of yogurt.Journal of Food Processing and Preservation 35 778–783.

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Feeding Strategies and Rumen Microbial Interventions for Enhancing the Nutritional

and Therapeutic Properties of Milk

Amrish Kumar Tyagi and Sachin Kumar

Dairy Cattle Nutrition Division

1. Introduction

Designer foods are the foods that are formulated to have higher contents of nutraceuticals,

antioxidants, and secondary metabolites than the traditional nutrients they contain, so as to promote

beneficial health properties (IFIC, 2011). Foods might inherently possess these supposedly beneficial

qualities, or they may be fortified/modified and/or genetically altered (FSSA, 2006). Designer milk

has vast scope and application by augmenting human health benefits, improving processing and

technological aspects. From human health view point it includes increase in proportion of unsaturated

fatty acids (USFA) in milk fat, reduce lactose content in milk in order to relish persons suffering from

lactose intolerance and remove b-lactoglobulin (b-lg) from milk. Designer milk may have altered

composition, concentration and structural variation in milk constituents. For example there may

change in primary structure of milk protein such as casein, lactalbumin; variation in lipid profile to

incorporate high level of beneficial fatty acid like CLA, and other essential fatty acids, altered

concentration of protein (higher or lower), reduced lactose and absence of beta-lactoglobulin (beta-

LG) etc.

Previously, manipulation in genetic makeup of dairy cattle has considerable implication in

augmenting the nutraceutical value of milk and milk products that impart human health benefits

(Karatzas and Turner 1997). But now-a-days, feeding strategies to the animals have highly influenced

the formation of designer foods primarily targeting the lipid profile. In this context, use of feed

additives is gaining momentum as they play a pivotal role in optimizing nutrient utilization by the

animals; thus affects the overall well beings of the animals. Vitamins, vitamin-like compounds,

minerals, essential fatty acids, probiotics, phytochemicals and others nutraceuticals are under

investigation by different groups in order to explore their effectiveness in the animal performance,

thus the quality of animal origin food.

2. Feeding strategies for designing milk with lipid profile: High conjugated linoleic acid (CLA)

Milk fatty acids (FA) are the group of compounds that have attracted the greatest interest under the

changing life style as typical cow milk fat is composed of 5% polyunsaturated fatty acids (PUFA),

70% saturated fatty acids (SFA) and 25% monounsaturated fatty acids (MUFA) (Tyagi et al., 2012).

Large amounts of SFA can increase the chronic and cardiovascular diseases in humans. Furthermore,

FA content and composition, as well as the fat soluble vitamins are some of the compounds that are

easiest to manipulate through feeding and through selection of animal breed. The main focus with

respect to FA has been to replace a proportion of saturated FA with mono- (MUFA) and

polyunsaturated fatty acids (PUFA) in accordance with the evidence that such replacement will

provide health benefit. However, the milk contains a specific fatty acid, conjugated linoleic acid

(CLA; cis-9, trans-11 C18:2) (Sieber et al., 2004) which has been proved to have anti-carcinogenic,

anti-allergic and anti-inflammatory effects (Kathirvalen et al., 2008; Chinnadurai et al., 2013). So,

the strategy is to make designer milk which should contain more amounts of CLA than is present

originally.

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3. Biosynthesis of CLA

The CLA refers to a mixture of positional and geometric isomers of linoleic acid (c-9, c-12, C18:2)

with two conjugated double bonds at various carbon positions in the fatty acid chain. The presence of

CLA in milk fat in dairy animals relates to the isomerization and biohydrogenation of unsaturated

fatty acids (FAs) by rumen bacteria as well as the ∆9-desaturase activity in the mammary gland (Fig.

1). The cis-9, trans-11 CLA comprises 75–90% of total CLA and is derived from linoleic acid and

linolenic acid (Bauman et al., 2003). Linoleic acid (cis-9, cis-12, 18:2) is first isomerized to the CLA

cis-9, trans-11 by cis-12, trans-11 isomerase and then hydrogenated by Butyrivibrio fibrisolvens to

vaccenic acid (VA, trans-11 18:1) in the rumen. These initial steps occur rapidly. There is a strong

positive correlation between the trans isomers of 18:1 (VA, trans-13–14, trans-15, and trans-16) in

milk fat and the level of linoleic acid in the diet. The hydrogenation of VA to stearic acid appears to

involve a different group of organisms and occurs at a slow rate. For this reason, VA typically

accumulates in the rumen. This main trans FA is responsible for the formation of the CLA isomer cis-

9, trans-11, which occurs by desaturation (D9-desaturase) of the ruminally derived VA in the

mammary gland. The pathway for the formation of the CLA is presented in flow diagram (Collomb et

al., 2006). Dietary supplementation of Butyrivibrio fibrisolvens alters fatty acids of milk and rumen

fluid in lactating goats (Shivani et al., 2015).

Dietary factors that affect CLA content have been grouped into four categories related to the potential

mechanisms through which they act:

1. The first category includes dietary factors that provide PUFA substrates for rumen production

of CLA and trans-11 18:1.

2. The second group consists of dietary factors that affect rumen bacteria involved in bio-

hydrogenation, either directly or via changes in rumen environment.

3. The third category includes dietary factors that involve a combination of lipid substrates and

modification of rumen bio-hydrogenation.

4. The fourth category is dietary supplements of CLA or trans-11 18:1 fatty acids.

4. Feeding of grains

Replacement of forages with grains in the diet reduces the rate of lipolysis and bio-hydrogenation.

Lipolysis and hydrogenation reactions were observed to occur more rapidly with feed particles

ranging from 1-2 mm size than from 0.1-0.4 mm size (Gerson et al., 1988).

5. Feeding on pasture and conserved forages

Pasture feeding can increase in the short term milk fat CLA concentrations in lactating dairy cows

when changed from indoor winter feeding and that milk fat CLA content increases with increasing

proportions of pasture in the diet (Dhiman et al., 1999). The CLA-enriching effect of pasture has

been attributed to higher α-linolenic acid and its biohydrogenation as a lipid substrate for the

formation of VA in the rumen and its subsequent desaturation to cis-9,trans-11 CLA in the mammary

gland (Bauman et al., 2003). With rising altitude, which is accompanied by a decrease in the

proportion of grasses and a corresponding increase in dicotyledonous species, there was an increase

in CLA levels from lowlands to mountains and highlands (Collomb et al., 2004). Animals fed with

fresh cut pasture than conserved fodder produce more CLA in milk. It because wilting of the grasses

cause oxidative loss of PUFA in grasses. Maturity of the pasture has a negative effect on CLA

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content of animal tissue (Loor et al., 2002). Deceased leaf stem ratio, maturation of leaves,

initiation of flowering and leaf senescence can cause a decreased total FA and LNA content in

grasses.

Fig. 1 (Adopted from Collomb et al., 2006)

The CLA content was increased by 300 percent in milk when the green fodder in diet increased from

33 to 100 percent (Tyagi et al., 2007). Green fodder increases the CLA content, vitamin A and E

content in goats milk (Tyagi et al., 2008). When ruminants are raised on pasture and green fodder,

their meat and milk contain much higher levels of n-3 PUFA and CLA as compared to products from

animals raised in stall feeding with concentrates (Aurousseau et al., 2004). Cows grazing natural

permanent pastures have 500% higher CLA content in milk compared with cows fed typical dairy

cow’s diet containing preserved forage and grain in a 50:50 ratio (Dhiman et al., 1999).

6. Feeding of diet with supplemented oils

The supplementation of different oils containing high levels of PUFA, including soybean, corn,

peanut, sunflower, linseed, mustard and fish oils to the cattle results in an increase in t-VA

production in the rumen, as well as CLA content in milk (Hossain, 2013). Plant oils from different

oilseeds have quite different FA compositions and accordingly would be expected to have different

effects on milk fat CLA concentrations. Comparisons between different types of plant oils suggest

that those rich in linoleic acid increase CLA concentration most effectively (Collomb et al., 2004).

Different dietary oil treatments (peanut oil, high in oleic acid; sunflower oil, high in linoleic acid;

linseed oil and flaxseed, high in α-linolenic acid) have been shown to exert different degrees of

enrichment of milk fat with CLA.

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The calcium salt of oils also increased the CLA content in milk fat. Feeding Ca salts of FAs from

soybean oil caused the highest level of CLA compared with canola and linseed oil (Chouinard et al.,

2001). Processing of seeds has a definite effect on CLA. Inclusion of Extruded seed in diet of

animals increases CLA content in milk (Lerch et al., 2012). Kathirvelan and Tyagi (2007) reported

that buffaloes fed with three different concentrate mixtures having different fatty acid composition

resulted in 185% increase in milk CLA in the diet having mustard oil and mustard cake. Mustard oil

based diet (2% of mustard oil) not only enhances the CLA content of milk but also increases the milk

fat and quantity (Tyagi and Kathirvelan, 2009).

Equivalent amount of dietary fish oils are more effective than plant oils for increasing milk CLA

content due to increased rumen output of Vaccenic Acid (VA). Supplementation of plant oils along

with fish oil can produce increased milk CLA than feeding fish oil alone (AbuGhazaleh et al.,

2002). Feeding mixtures of plant and marine oils have an effect on CLA content in milk fat and it is

observed that there was three- to four fold increase in CLA content after feeding mixture of plant and

marine oils.

Mierlata and Vicas, (2015) conducted an experiment to quantify the differences between maize-based

(MS) and grass-silage-based (GS) diets supplementing with camelina seed (Cs). Feeding the MS diets

increased net energy for lactation (NE) intake, raw milk yield and fat, protein and lactose yields.

Feeding +Cs increased energy corrected milk (ECM), milk fat content and fat yield. Maize silage

consumption is associated with an increased proportion of hypercholesterolemic fatty acids (HFA)

and a higher value of the atherogenicity index. However, an MS diet led to an increased share of t11-

C18:1 and c9,t11-conjugated linoleic acid (CLA) in milk. Milk FA profile in ewes fed GS diet was of

higher quality for human beings owing to higher concentrations of α-linolenic acid and a lower

content of HFA. Supplementing with camelina seed resulted in a higher concentration of t11-C18:1,

c9,t11-CLA and C18:3n-3 in milk fat. The trolox equivalent antioxidant capacity (TEAC) value of

milk was higher in milk from MS-fed ewes compared with that of their counterparts fed GS. Dietary

supplementation with camelina seed increased the oxidative stability of milk samples.

7. Conclusion

The paper describes the most effective feeding strategies applied to animals in order to enhance the

healthfulness of foods. Some research studies of designer milk with high CLA, Omega–3 fatty acids,

and high PUFA have been cited which better describes the role of feeding in enhancing their

production. Thus, nutritional and feeding strategies play an important role for making designer milk

having therapeutic potential to augment the health.

8. Selected Reading

Abu-Ghazaleh A A, Schingoethe D J, Hippen A R, Kalscheur K F and Whitlock L A (2002) Fatty acid profiles

of milk and rumen digesta from cows fed fish oil, extruded soybeans or their blend. Journal of Dairy Science

85 2266–2276.

Bauman D E, Corl B A and Peterson D G (2003) The biology of conjugated linoleic acids in ruminants.

Advances in conjugated linoleic acid research, 2 146-173.

Chinnadurai K, Kanwal H K, Tyagi A K, Stanton C and Ross P (2013) High conjugated linoleic acid enriched

ghee (clarified butter) increases the antioxidant and antiatherogenic potency in female Wistar rats. Lipids

Health Disease, 12 121. DOI:10.1186/1476-511X-12-121.

Chouinard P Y, Corneau L, Butler W R, Chilliard Y, Drackley J K and Bauman D E (2001) Effect of dietary

lipid source on conjugated linoleic acid concentrations in milk fat. Journal of Dairy Science 84 680–690.

183

Collomb M, Schmid A, Sieber R, Wechsler D and Ryhänen E L (2006) Conjugated linoleic acids in milk fat:

Variation and physiological effects. International Dairy Journal, 16 1347-1361.

Collomb M, Sieber R and Bu tikofer U (2004) CLA isomers in milk fat from cows fed diets with high levels of

unsaturated fatty acids. Lipids, 39 355–364.

Dhiman T R, Anand G R, Satter L D and Pariza M W (1999) Conjugated Linoleic Acid content of milk from

cows fed different diets. Journal of Dairy Science 82 2146-2156.

FSSA. 2006. Food Safety Standards Authority of India, New Delhi, India. http://www.fssai.gov.in.

Gerson T, King A S D, Kelly K E and Kelley W J (1988) Influence of particle size and surface area on in vitro

rates of gas production, lipolysis of triacylglycerol and hydrogenation of linoleic acid by sheep rumen

digests or Ruminococcus flavefaciens. Journal of Agriculture Science Cambridge, 110: 31.

Hossain, A.S. 2013. Diversity study of CLA producing indigenous Butyrivibrio spp. and its subsequent

application as a feed additive in ruminants. Ph.D thesis. National Dairy research institute, karnal, Haryana,

India.

IFIC, 2011. Background on functional foods. http://www.foodinsight.org.

Karatzas C N and Turner J D (1997) Toward altering milk composition by genetic manipulation: Current status

and challenges. Journal of Dairy Science 80, 2225–2232.

Kathirvelan C, Tyagi A K and Krishnamurthi P (2008) Influence of conjugated Linoleic acid ghee feeding on

cancer incidences and histopathological changes in 7, 12 dimethly benz (a) anthrazene induced mammary

gland carcenogenesis in rats. Veterinary Archive, 78 511-20.

Kathirvelan, C. and Tyagi A.K. 2007. Influence of mustard oil/cake feeding on CLA content of buffalo milk.

Indian Journal of Animal Nutrition, 24 237-240.

Lerch S, Ferlay A, Shingfield K J, Martin B, Pomiès D and Chilliard Y (2012) Rapeseed or linseed

supplements in grass-based diets: Effects on milk fatty acid composition of Holstein cows over two

consecutive lactations. Journal of Dairy Science 95 5221–5241.

Loor J J, Bandara A B P A and Herbein J H (2002) Characterization of C18:1 and C18:2 isomers produced

during microbial biohydrogenation of unsaturated fatty acids from canola and soya bean oil in the rumen of

lactating cows. Journal of Animal Physiology and Animal Nutrition, 86 422–432.

Mierlita D and Vica S (2015) Dietary effect of silage type and combination with camelina seed on milk fatty

acid profile and antioxidant capacity of sheep milk. South African Journal of Animal Science, 45 1-11.

Shivani S, Shandilya U, Srivastava A and A K Tyagi (2015) Dietary supplementation of Butyrivibrio

fibrisolvens alters fatty acids of milk and rumen fluid in lactating goats. Journal of the Science of Food and

Agriculture, (Accepted)

Sieber R, Collomba M, Aeschlimanna A and Jelenb P (2004) Impact of microbial cultures on conjugated

linoleic acid in dairy products-a review. International Dairy Journal, 14 1-15.

Tyagi A K and Kathirvelan C (2009) Conjugated linoleic acid content of milk from buffaloes fed a mustard oil-

based diet. International Journal of Dairy Technology 62 141-146.

Tyagi A K, Hossain S A and Tyagi A (2012) Feeding strategies for enhancing the nutritional and therapeutic

potential of milk. In: Regional Training on Quality Control of Milk during Production, Processing and

Marketing and Introduction to Novel Technologies for Dairy Products Diversification, India, pp. 145-149.

Tyagi A K, Kewalramani N, Dhiman T R, Kaur H, Singhal K K and Kanawjia S K (2007) Enhancement of CLA

content in buffalo milk through green berseem feeding. Animal Feed Science Technology 132 15-19.

Tyagi A K, Saluja M, Kathirvelan C and Singhal S S (2008) Enhancement of the conjugated linoleic acid and

vitamin A and E contents in goat milk through green fodder feeding International Journal of Dairy

Technology, 62 7-14.

184

Marketing Strategy of New Food Products

Smita Sirohi and Divya Pandey

Division of Dairy Economics, Statistics and Management

1. Introduction

The changes in demand pattern for agricultural and food products, fueled by growing population, rising

incomes, and changing lifestyles, is a vividly known trend, that will re-enforce itself in the times to come.

In the modern day market-led economies where ‘consumer is the king’, an assessment of the customer’s

expectations, preferences and aversion becomes a pre-requisite for success of a new product in the

market. The technology tailors the products to meet consumer needs, but it is the demand, not supply, that

drives product offerings and eventually sophisticated business models deliver them to the customer in a

secure manner. Product and process development are the lifeblood of smart business strategy. Failure to

develop new and improved products relegates firms to competing solely on price which favours the

players with access to the lowest cost inputs (land, labour etc).

Business entities are continuously on the lookout for new products as existing products are vulnerable to

changing consumer needs and tastes, new technologies, shortened product life cycles, and increased

domestic and foreign competition. A new product can have various connotations, such as,

- Innovative and new to the world

- Product lines that allow a company to enter an established market for the first time (the product

is new to the company not the market)

- Products that supplement a company’s established products lines (package sizes, flavors, and so

on)

- Products that provide improved performance or greater perceived value and replace existing

product (improvements in features and benefits of a product)

- Products that provide similar performance at lower cost to the company

- Products that are targeted to new markets or market segments (to be called a new product there

must be some changes in the existing product to suit the new segments targeted).

The ultimate test of product development occurs in the market and a new product can only be considered

successful if it is a market and financial success. This lecture note briefly describes the conceptual

framework of marketing strategy and presents some relevant case studies of new food products.

2. Developing a Marketing Plan

Developing a sound marketing plan is important for new food products to be accepted in the market

strategically. There are several factors that need to be considered when developing the market plana:

product position in the market;

stage of the lifecycle for the product class, and therefore the position of the new product on the

product class life cycle;

a Earle, M.D. and Earle, R.L. Creating New Foods. The Product Developer's Guide - Product Commercialisation,

http://www.nzifst.org.nz/creatingnewfoods/index.htm

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relationship of the new product marketing mix to the overall company marketing mix;

interaction of the parts of the marketing mix - market channel with price, promotion with price,

product with promotion;

quantitative relationship between sales volumes and the various parts of the marketing mix;

marketing profitability and efficiency of the marketing mix;

reactions to the marketing mix of the industrial, social, legal and political environments.

The overall objective of marketing plan is to have high volume of sales, to cover a large market area

and to ultimately earn profit. The plan should consider how socio economic, political and industrial

environment will affect the product and whether there is a need flexible market plan or not. The plan

should also consider the various marketing channels and intermediaries which will be involved in the

product launch and the market targeted for the same.

For planning a product, its characteristics, uses, its branding also need to be considered. The cost born

by the company and the price whether company list price, retail price should also be taken into account.

The plan should also include a proper sales and distribution plan such as the means of transportation, and

location of store or warehouse for storing the product. Targets of sales persons’, of area, sales budgets

also need to be planned. Advertising of the product through press, radio, television, cinema, posters,

internet and promoting the product through providing free samples, reduced price are also a part of an

effective marketing plan. Production and distribution schedules such as the quantity which needs to

produced and distributed, losses in production and distribution are also a necessary part of the plan.

Market research provides the key inputs required by the technical development group for the setting of

appropriate design specifications for the new product or service. Market research is a systematic process

that collects, analyzes and draws conclusions from data gathered from consumers, business owners, or

other groups of interest. The goal of market research is to identify and assess how changing elements of

the marketing mix impacts customer behavior. As it is based on analysis of qualitative and quantitative

data about issues relating to marketing products and services, the market research minimizes risk,

provides business intelligence to make informed decisions and therefore, improves the chances of success

in launch of a new product or service, fine tuning existing products and services, expanding into new

markets, developing an advertising campaign, setting prices, and/or selecting a business location. There

are many ways to perform market research, of which the commonly used five basic methods are surveys

(include in-person surveys, telephone surveys, mail surveys and online surveys), focus groups, personal

interview, observation and field trials.

3. Marketing of New Food Products: Case Studies

3.1 Milk for Lactose-Intolerant (Source: Tetra Pak, 2004)

The case study is about Valio, Finland’s biggest dairy company, which launched low lactose milk for

those with lactose intolerance. About 15 to 20 percent of the Finnish population is lactose intolerant. The

challenge to Valio was to produce milk which was acceptable in terms of taste and could be tolerated by

lactose intolerant people. Valio was able to perfect a unique process to produce lactose-free milk after a

long period of research and development that tasted just as milk should. At first, Valio was not allowed to

call the product ‘milk’, as one of its natural constituents had been removed. Finally, it was launched as

‘light milk drink’. Even though the price is twice as high as normal milk, consumers were not deterred.

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The case underlines the importance of taste preferences. Consumers were willing to pay a price premium

for a unique product that met their needs.

3.2 Market Mix Plan : Low-calorie chicken hotpot

Here is an example of the market mix plan of a medium-sized canning company that moved into the

nutritional diet market through its product a low-calorie chicken hotpot. The marketing objectives of the

company were to enter the diet food market segment emphasising the nutritional/low calorie/ convenience

aspects of the product, recover development costs within two years and maximise profits. The first step is

to understand the market environment. At the time of introducing the product, the country was in an

economic recession but there was prediction of a minor resurgence in the economy, that would justify

the product launch in current year. The consumer preferences and availability of substitute products is

gauged by the market manager.

The appeal and USP of the product were than were established from the market trial and from this a

product image developed of 'calorie reduced, highly nutritious, convenience meal'. Factors such as 'good

for you', 'balanced', 'quick' and 'healthy' could be emphasised as product benefits. The following section

describing the various other steps in development of market plan are reproduced from :

http://www.nzifst.org.nz/creatingnewfoods/product_commercialisation4.htm#EX61

4. Product name

As the sponsoring company is diversifying into the product area of diet foods, it could be preferable to

establish a new brand with a 'health' image. Brand suggestions include 'LITEWEIGHT', 'VITALITE' or

'NUTRILITE'. The latter brand name tends to be better suited to the product image of a calorie reduced,

nutritional product line.

The product name decided on is 'CHICKEN HOTPOT' as this describes a chicken and vegetable mix

suitable for a quick but special meal. It also implies the product is different from competing canned meat

and vegetable products. This distinction must be emphasised as the developed product is establishing a

different product image.

As the product is to be canned, it is important that the label be distinctive to attract consumer attention.

The label must meet the Food Regulations.

5. Consumer

The product has to appeal to two distinct consumers:

consumers on calorie reduced/health food diets;

general consumers of convenience foods who would buy the product for the reduction in calories,

nutritional attributes and possibly taste preferences to competing convenience products on the market.

6. Price

There are three alternative pricing strategies:

price high, to the upper end of the diet market;

price intermediate, to the low calorie/convenience market;

price low, to the convenience canned meals market.

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One of the marketing goals set was to maximise profits. To achieve this, the company's demand, cost and

profit functions were analysed. The production cost was estimated at $1.71 and to cover company costs

and profits the wholesale list price was set at $3.22. If there was a retailer mark-up of 30%, this would

give a retail price of $4.19. As the product would have to be promoted, it was decided that the price

should be first set to the upper end, i.e. the diet market, and then as production increases and development

costs are recovered the price should be dropped to the low calorie/convenience market.

The price set allows for 'specials', 'discounting' and other retail discounts that may be necessary in the

marketing of the product and establishing good relationships with retail outlets.

7. Market channels

The alternative market channels are:

market through supermarkets and convenience stores via a wholesaler;

market direct to major supermarket chains (eliminating wholesaler);

market to smaller health food shops and delicatessens via wholesaler;

market to all retail stores directly.

It was established from the market trial that the main retail outlets at which the consumer would expect to

buy the product were supermarkets and convenience stores. A smaller proportion of the respondents

indicated buying the product at health food shops and delicatessens. The company could use the latter if

they adopted the high price strategy (i.e. price to the upper end of the market).

The market channel for sale of the product through a wholesaler to supermarkets and convenience stores

is already established. Using this would minimise the cost and marketing effort required in moving the

product through the channel. Marketing to delicatessens and health food shops requires marketing through

a wholesaler or using a manufacturers' agent to a large number of retail outlet in small volumes. This may

suit the initial small volume produced. Alternatively, during the initial low throughput, it may be more

useful to market the product only to one or two supermarket chains in one of the major cities.

On considering the effectiveness, experience and cost of the alternatives, the first alternative

(supermarkets and convenience stores via a wholesaler) would appear to have the greatest potential.

8. Physical Distribution

The product is canned and has an estimated shelf life of two years at ambient temperatures. Due to the

nature of the product, damage is restricted to dented cans and torn labels, occurring only with excessive

handling. The existing company's physical distribution system is by road or rail, which can be adapted to

the Chicken Hotpot. The product is distributed to warehouses in main city centres. On analysis of

transport costing, it would appear rail is the cheaper method for this initial distribution. Distribution to the

smaller centres could be by road or rail as dictated by local costs and availability of the transport.

The company adopts a policy of minimising the level of capital invested in inventory.

During the initial product launch, it is estimated a three-month supply of product is required to fill the

market channel. As the market establishes, this level of inventory in the warehouses can decrease to

approximately a two-month supply.

This does not allow for any possible seasonal trends such as increased consumption during winter; these

can only be established during the initial years of marketing the product.

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9. Promotion

The promotional mix consists of a combination of four promotional methods:

advertising;

personal selling;

sales promotion;

publicity.

The mix must be coordinated and conform to the overall market plan. The theme for all promotional work

is:

CHICKEN HOTPOT a calorie reduced, highly nutritious, convenience meal

This theme emphasises that the product is a convenience product giving a balanced meal of essential

vitamins and nutrients for those people on calorie-reduced diets or interested in weight control. It is felt

that the main product benefit to emphasise is convenience: quick to prepare, calorie-reduced meal.

Advertising aims are to stimulate sales, and generate the new product image and the NUTRILITE brand

image.

The advertising media available for marketing the product, in order of increasing cost and increasing

penetration, are:

1. newspapers;

2. magazines;

3. mail pamphlets with discount offers;

4. radio;

5. television.

Due to limited finance, the possibility of television as a promotional medium is eliminated. Radio tends to

be specific for local regions and has intense competition and short attention span. Thus it was also

eliminated. The final media are within the company’s budget allocation. Newspapers have a wide

coverage but date quickly and have a short attention span. Magazines reach a specialist target audience

and have a longer life due to magazine circulation. The mailing of pamphlets provides a rapid means of

informing the public (important during the product launch) but is relatively expensive.

Two possible magazines for advertising the Chicken Hotpot throughout the market could be:

a high circulation women’s magazine;

a high circulation general magazine.

Both magazines have a high reach (i.e. a large number of people exposed one or more times to the

advertisement). The frequency of exposure will be determined by the number of times the product is

inserted.

Coordinating the advertising schedules is important to achieve a high reach at product launch. A possible

schedule is outlined below:

Delivery of pamphlets to householders with a discount on the product.

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At the same time, concentrated magazine advertising in both magazines.

Periodical burst advertising in magazines as the product is established on the market.

Personal selling in the company consists of a sales force of two area managers with eight sales persons.

The sales persons establish contact with potential customers. To ensure the presentation is effective, sales

staff must be informed of the product, sales method, any possible discounts, trade benefits, advertising

and promotion to be used.

Sales promotions are to gain retailer and consumer confidence in the product. To gain trade acceptance of

the product and achieve prime shelf space and in-store displays, cooperative advertising and buying

allowances could be offered.

In-store displays portray the calorie reduced, convenience, health aspect of the product, for example a

poster with a slim, healthy young couple eating the casserole and a caption underneath stating the ease of

preparation. The displays could show methods of serving the product and, if possible, in-store cooking

demonstrations will be used. Samples could also be given. In-store promotion is important to show the

attractive eating qualities and to emphasise the lower calories than the existing canned meals.

Publicity is to gain widespread awareness of the product in the trade and among the consumers. At the

product launch it may be possible to obtain media coverage of the revolution in food - a calorie reduced,

convenience meal balanced in nutrients and vitamins. This is justified by the fact that the type of product

is not presently available but corresponds to the new awareness in health and fitness. Promotion of this

type would require careful planning to be effective but in general publicity has a high level of truth

attached to it, i.e. consumers tend to believe it is more authentic than advertisements.

Overall, the promotional mix will be informative, building up an awareness of the product at the time of

launching. As the product establishes a market, the mix will become more persuasive.

10. Timing and test market

The most suitable time for launching of the product is prior to winter. This is because the hotpot may

show a seasonal trend with increased demand in the winter months for a hot meal. The extent of the trend

can only be determined by actual marketing, but it will probably not be very marked due to the ‘light’

sauce. Prior to national launching of the product, it may be advantageous to test market the product in a

small region. The cost of this is justified by the newness of the product. It is essential the product is

launched soon to obtain maximum benefit from the change in consumer awareness of health and fitness

combined with the increasing demand for convenient, quick-to-prepare foods.

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Cost Estimation of Value added Dairy Products

A.K. ChuahanDairy Economics Statistics and Management Division

1. IntroductionIndian Dairy industry is undergoing transformational change (Labernede, 2015). During the next fewyears, till 2030, the demand for dairy products is expected to grow at a rate of 9-12 per cent and industryat a rate of 4-5 per cent. Clearly, the Indian industry will struggle to maintain hundred per cent self-sufficiency due to huge local demand, between 160 to 170 million tonnes of milk that would be requiredby 2030 (Chand et.al, 2015). The NSSO data on Per Capita Monthly Consumption expenditure on milkand milk products revealed an increasing trend from 1971 to 2010. India ranks first in milk production,accounting for 17 per cent of world production. During 2013-14, milk production peaked at137.69 MT,thus becoming an important secondary source of income for 70 million rural households engaged indairying. The average year on-year growth rate of milk, at 4.18 per cent vis-à-vis the world average of 2.2per cent, shows sustained growth in availability of milk and milk products for the growing population(Economic Survey, 2014-15). Consequently, the demand for new and diversified dairy products likemozzarella cheese is rising and hence private investment in the sector has also gone up.

With the potential to accommodate imports with home produced dairy products, the Indian industry willpresent to be a very lucrative market. The dairy sector is a dynamic global industry, with steadily growingproduction trends which are forecast to continue in the long-term. These trends are driven by an increasein demand for animal proteins that goes along with the population growth and income growth in emergingeconomies. According to a report Economic Times 20 Oct 2013, Dairy sector will touch $140 billion by2020.The size of Indian dairy industry in both organized and unorganized sectors is expected to double to$140 billion by 2020, on the back of growing demand and rising disposable income. The increase in milkproduction, elevated demand for processed dairy products and low-cost advantage, have attracted themultinationals and other private entrepreneurs to establish milk plants in India, resulting in the number ofmilk plants registered under milk and milk product order (MMPO) increased from 789 to 1065 during2006 to 2011 (Basic Animal Husbandry Statistics, 2014). In this context, dairy production and dairyprocessing clearly appear as industries of utmost importance in contributing to the global challenge offood security today and for decades to come. Dairy products are a major source of cheap and nutritiousfood to millions of people in India and the only acceptable source of animal protein for large vegetariansegment of Indian population. Due to the stiff competition in the market of milk and milk products, theprices of these products play an important role. To provide the product at reasonable prices to theconsumers, the plants manufacturing value added products need to make all efforts to curtail their costs.Therefore, costing of value added products is very useful management tool to take right decisions.

2. Cost estimation of value added dairy productsSome of the latest cost estimates of different milk and milk products from a study conducted atcooperative sector milk plant in Haryana is given below:

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Table 1: Component-Wise Cost of Full Cream Milk (Fat-6%, SNF-9%) Manufacturing (2013-14)

Sl. No. Cost Components Unit Cost (Rs/ Lit.) Percentage1 Raw Material 40.44 95.872 Labour 0.1 0.24

3 Electricity 0.08 0.194 Water 0.01 0.025 Steam 0.14 0.336 Refrigerator 0.61 1.457 Quality Control 0.09 0.21

8 Miscellaneous 0.19 0.459 Packaging Material 0.4 0.9510 Administration and Supervision 0.1 0.2411 Interest And Depreciation 0.02 0.05

Total 42.18 100.00

Cost of Full Cream Milk production is displayed in Table 1. Component wise cost analysis revealed thatraw materials alone accounted for 95.87 per cent of total expenditure. Next important cost component wasRefrigeration that accounted for 1.45 per cent of total expenditure followed by cost on packaging material(0.95 per cent).

Table 2: Component-wise Cost of Standardized Milk (Fat-4.5% SNF- 8.6%) Manufacturing (2013-14)

Sl. No. Cost Components Unit Cost (Rs/ Lit.) Percentage

1 Raw Material 34.52 95.57

2 Labour 0.09 0.25

3 Electricity 0.07 0.19

4 Water 0.01 0.03

5 Steam 0.14 0.39

6 Refrigeration 0.61 1.69

7 Quality Control 0.04 0.11

8 Miscellaneous 0.16 0.449 Packaging Material 0.40 1.1110 Administration And Supervision 0.08 0.22

Total 36.12 100.00

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Table 2 shows the Component wise cost analysis of Standardized Milk production. The table revealed thatraw materials alone accounted for 95.57 per cent of total expenditure. Next important cost component wasRefrigeration that accounted for 1.69 per cent of total expenditure followed by cost on packaging material(1.11 per cent). Rests of all other costs were less than one percent individually.

Table 3: Component-wise Cost of Toned Milk (Fat – 3.1% SNF- 8.6%) Manufacturing (2013-14)

Sl. No. Cost Components Unit Cost (Rs/ Lit.) Percentage

1 Raw material 30.08 95.042 Labour 0.08 0.253 Electricity 0.06 0.194 Water 0.01 0.035 Steam 0.14 0.446 Refrigeration 0.61 1.937 Quality control 0.04 0.138 Miscellaneous 0.15 0.479 Packaging material 0.40 1.2610 Administration and supervision 0.07 0.2211 Interest and depreciation 0.01 0.03

Total 31.65 100.00

Cost of Toned Milk production is displayed in Table 3. Component wise cost analysis revealed that rawmaterials alone accounted for 95.04 per cent of total expenditure. Next important cost component wasRefrigeration that accounted for 1.93 per cent of total expenditure followed by cost on packaging material(1.26 per cent). Rest all other costs were less than one percent individually.

Table 4: Component-Wise Cost of Double Toned Milk (Fat – 1.6% SNF- 9.1%)Manufacturing (2013-14)

Sl. No. Cost components Unit Cost (Rs/ Lit.) Percentage

1 Raw material 26.38 94.55

2 Labour 0.07 0.25

3 Electricity 0.05 0.18

4 Water 0.01 0.04

5 Steam 0.14 0.50

6 Refrigeration 0.61 2.19

7 Quality control 0.03 0.11

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8 Miscellaneous 0.13 0.47

9 Packaging material 0.4 1.43

10 Administration and supervision 0.07 0.25

11 Interest and depreciation 0.01 0.04

Total 27.9 100.00

The components wise cost of double tonned milk is exhibited in Table 4. The component cost analysisrevealed that the raw material cost was observed 94.55% in the total cost followed by refrigeration(2.19%), packaging material (1.43%) and less than one percent for all other cost components.

Table 5: Component-wise Cost of Paneer Manufacturing (2013-14)

Sl. No. Cost components Unit Cost (Rs/ Lit.) Percentage

1 Raw material 227.78 96.802 Labour 3.25 1.383 Electricity 0.12 0.054 Water 0.21 0.09

5 Steam 0.45 0.19

6 Refrigeration 0.45 0.197 Quality control 0.26 0.118 Miscellaneous 1.05 0.459 Packaging material 1.2 0.5110 Administration and

supervision0.54 0.23

Total 235.31 100.00

Table 5 revealed that the cost of production of Paneer turned out to be Rs 235.31 per kg. In the total cost,processing cost was 3.29 per cent and the rest (96.80 per cent) was raw materials cost. In processing cost,the labour cost contributed 1.38 per cent, packaging material costs 0.51 per cent and miscellaneous 0.45per cent of the total cost. The shares of administration and supervision, steam and refrigeration were alsosignificant.

Table 6: Component-wise Cost of Ghee Manufacturing (2013-14)

Sl.No.

Cost components Unit Cost (Rs/ Lit.) Percentage

1 Raw material 323.99 97.112 Labour 1.72 0.523 Electricity 0.41 0.12

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4 Water 0.15 0.045 Steam 0.48 0.146 Refrigeration 0 0.00

7 Quality control 0.38 0.11

8 Miscellaneous 1.53 0.46

9 Packaging material 1.65 0.4910 Administration and

supervision0.78 0.23

11 Interest and Depreciation 2.53 0.76Total 333.62 100.00

Table 6, gave the details on the cost components for Ghee. It is revealed that total cost of ghee productionwas Rs.333.62 per litre. Raw materials accounted for 97.11 per cent and Interest and depreciation, 0.76per cent of the total cost., Next came labour (0.52 per cent), packaging material (0.49 per cent) andmiscellaneous (0.46 per cent).

Table 7: Component-wise Cost of Chhachh Manufacturing (2013-14)

Sl. No. Particular Cost1 Raw material 6.352 Processing charge 0.763 Packaging material 0.5

Total 7.61

Table 7, shows the component wise cost of manufacturing Chach in the plant. Raw materials accountedfor 6.35 percent followed by processing charge (0.76 percent) and packaging materials. 0.5 percent.

Table 8: Component-wise Cost of Dahi Manufacturing (2013-14)

Sl. No. Particular Cost

1 Raw material 12.032 Processing charge 0.78

3 Packaging material 3.22

Total 16.03

Table 8, shows the component wise cost of manufacturing Dahi in the plant. Raw materials accounted for12.03 percent followed by packaging materials (3.22 percent) and processing charge (0.78 percent) oftotal cost.

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Table 9: Profitability in Different Dairy Products (2013-14)

Products Price Received ByPlant(Rs.)

Cost of Manufacturing(Rs.) Profit Margin Profit %

Full Cream Milk (Lt.) 46 42.18 3.82 9.06

Standardised Milk (Lt.) 40 36.12 3.88 10.74

Toned Milk (Lt.) 37 31.65 5.35 16.90

Double Toned Milk (Lt.) 32 27.9 4.1 14.70

Paneer (kg) 237.5 235.31 2.19 0.93

Ghee (kg) 376 332.62 43.38 13.04

Dahi (400ml) 23.8 16.03 7.77 48.47

Chach (500ml) 8.8 7.61 1.19 15.64

Profitability of the dairy products is presented in Table 9. The information contained in the table revealsthat Dahi turned out to be the most profitable product (48.47 per cent) followed by Toned Milk (16.90 percent), Chach (15.64 per cent), Double Toned Milk (14.70 per cent) and Ghee (13.04 per cent),Standardised milk (10.74 per cent) and Full Cream Milk (9.06 per cent). Among different productsmanufactured, Paneer manufacturing turned out to be the lowest profitable proposition (0.93 per cent).There is need to explore the possibility to increase the profits in paneer manufacturing by increasing theprice of the product or to adopt some suitable steps to use the residual whey for by products such as wheybased beverages, whey powder and in pharmaceutical industry to bring down the cost.

Selected ReadingChand A, Swami V and Tipnis J (2015) Structural Changes in Dairy Farming For Better Margins and Local

Economy Development in Indian Context. Abhinav International Monthly Refereed Journal of Research inManagement and Technology. 4 1-10.

Government of India (2014). Basic Animal Husbandry and Fisheries Statistics, Ministry of Agriculture, Departmentof Animal Husbandry, Dairying & Fisheries, Ministry of Agriculture, Krishi Bhawan, New Delhi.

Government of India (2015). Economic Survey 2014-15.Ministry of Finance, New Delhi.Labernède C. (2015). Indian share in global milk production, Food and Beverages News. 03 January 2015. Print.

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Soft Computing in Dairy and Food Processing

A.K. Sharma

Dairy Economics, Statistics & Management Division

1. Introduction

Soft Computing (SC) is the use of inexact solutions to computationally hard tasks for which there is

no known algorithm that can compute an exact solution. For such complex tasks, conventional

methods like stochastic and statistical methods (called hard computing) have not been able to produce

cost-effective, analytical, or complete solutions. Unlike hard computing, SC deals with imprecision,

uncertainty, partial truth, and approximation to achieve practicability, robustness and low solution

cost. Hence, SC is a set of ‘inexact’ computing techniques, which are able to model and analyse very

complex problems. Actually, the role model for soft computing is the human mind. SC is extensively

studied and applied to scientific research and engineering computing. Scientists and engineers have

developed SC models based on Fuzzy Logic (FL), Artificial Neural Networks (ANN’s), Genetic

Algorithms (GA’s), Decision Trees (DT’s) and Support Vector Machines (SVM’s) to analyse various

aspects of dairy and food processing. SC became a formal area of study in Computer Science in the

early 1990’s. Conventionally, computational approaches could model and precisely analyse only

relatively simple systems. More complex systems arising in fields such as biology, medicine,

management sciences, etc., remained intractable to conventional mathematical and analytical

methods. However, the simplicity and complexity of such systems are relative; and many

conventional mathematical models have been both challenging and very productive.

It is evident from literature (as well as author’s experience) that no single SC technique generally can

be considered as the optimal technique for problem-solving. However, hybrid techniques have been

found to work better in solving various problems since each intelligent technique has a particular

computational property that suits them appropriately to a particular problem. Several real-time

applications including dairy and food processing applications have been efficiently solved using the

hybrid of ANN and FL models called neuro-fuzzy models. Similarly, hybrids of ANN’s and GA’s

have been employed for various problem-solving applications.

Recent trends tend to involve evolutionary and Swarm Intelligence (SI) based algorithms and

biologically-inspired computation. This lecture presentation describes the science of soft computing

mainly focusing commonly used Sc techniques such as ANN, FL, GA and their hybrids and their

application to dairy and food processing in which the fusion of soft computing and hard computing

has provided innovative solutions for challenging problems.

2. Artificial neural network model

ANN’s provide a way to emulate biological neurons to solve complex problems in the manner similar

to human brain. ANN’s are composed of a large number of highly interconnected processing elements

(artificial neurons) working in union to solve specific problems. An artificial neuron is a device with

many inputs and one output. An ANN model is configured for a specific application, such as pattern

recognition or data classification, through a learning process. Learning in biological systems involves

adjustments to the synaptic connections (weights) that exist between the neurons. In 1986, the advent

of ANN learning algorithm, viz., Error Back Propagation (EBP) algorithm, gave a strong impulse to

subsequent research and resulted in the largest body of research and applications in ANN’s although

many other ANN architectures and training algorithms have been developed and applied

simultaneously. ANN paradigm is founded on principle of learning by example rather than being a

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rigid number crunching procedure. ANN’s have remarkable ability to derive meaning from

complicated or imprecise data. Thus, ANN models are used for solving complex problems like pattern

recognition and fast information processing and adaptation.

3. Fuzzy logic

Fuzzy logic deals with reasoning that is approximate rather than precisely deduced from classical

predicate logic. It is based upon the theory of fuzzy sets. Fuzzy truth represents membership in

vaguely defined sets, like likelihood of some event or condition; and fuzzy sets are based on vague

definitions of sets, not randomness. Since fuzzy logic modelling is a probability based modelling, it

has many advantages over the conventional rule induction algorithms that deal with extraction of

formal rules from a set of observations. That is, fuzzy logic based rule induction can handle noise and

uncertainty in data values.

Fuzzy Systems (FS’s) use soft linguistic variables (e.g., hot, tall, slow, light, heavy, dry, small,

positive, etc.) and a range of their weigthages (or truth values), called membership functions, in the

interval [0, 1], enabling the computers to make human-like decisions. Thus, fuzzy logic is basically a

multi-valued logic that allows intermediate values to be defined between conventional evaluations

like yes/no, true/false, black/white, etc. Notions like or can be formulated

mathematically and processed by computers. In this way, an attempt is made to apply a more human-

like way of thinking in the programming of computers. In essence, FL deals with events and situations

with subjectively defined attributes.

4. Genetic algorithms

GA’s are based on Darwin’s theory of survival of the fittest. It is an optimisation and heuristic search

paradigm that uses techniques inspired by evolutionary biology such as inheritance, mutation,

selection, and crossover. GA works simultaneously on a set (population) of potential solutions

(individuals) to the problem. The algorithm starts with a set of solutions (representing chromosomes)

called a sub-population. The fitness to which solutions meet some performance criterion is evaluated

and used to select surviving individuals that will reproduce a new, better sub-population. Then, the

individuals will conduct alterations similar to the natural genetic mutation and crossover. The

selection scheme makes the process towards high performance solutions. A careful selection of GA

structure and parameters can ensure a good chance of reaching the globally optimal solution after a

reasonable number of iterations. GA’s have become an important method of soft computing.

5. Neuro-fuzzy computing models

The Adaptive Neuro-Fuzzy Inference System (ANFIS) is a combination of the ANN and FL as the

ANN’s are used to determine the parameter of the FS. That is, it combines the advantages of fuzzy

logic, which deal with explicit knowledge that can be explained and understood; and that of the neural

networks, which deal with implicit knowledge acquired through learning. The FL also enhances the

generalisation capability of an ANN by providing more reliable output when extrapolation is needed

beyond the limits of the training data. This approach provides means of training a family of

membership functions to emulate a nonlinear, multi-dimensional mapping function. The ANFIS

approach integrates the basic elements and functions of a conventional fuzzy system into the neural

network connective structure, which distributes the learning ability to obtain the membership

functions and fuzzy logic rules.

6. Neuro-genetic computing models

The idea of combining GA’s with ANN’s to solve difficult problems has been a field of intense

research since 1980’s. The procedure for optimising ANN with GA involves steps: i) initialisation of

ANN;

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ii) chromosome in GA is constructed by concatenating weights from the network; iii) minimisation of

Mean Square Error (MSE) is used as objective function for GA optimisation; and iv) weights evolved

by GA are fed to ANN. Flowchart for a typical neuro-genetic model is given in Fig.1.

7. Application of soft computing in dairy and food processing

Food including milk and milk products is complex term. Several investigators have attempted to apply

SC models especially ANN models for prediction of food properties, and changes during processing

and storage of foods. ANN models are useful tools for food safety and quality analyses, which include

modelling of microbial growth for predicting food safety, interpreting spectroscopic data, and

predicting physical, chemical, functional and sensory properties of various food products during

processing and distribution. ANN models hold a great deal of promise for modelling complex tasks in

process control and simulation, and in applications of machine perception including machine vision,

electronic longue (e-tongue) and electronic nose (e-nose) for food safety and quality control. For

example, ANN based food sensory evaluation system; identification of registered designation of

origin areas of Portuguese cheese defined by microbial phenotypes and ANN models; prediction of

thermal conductivity, specific heat, and density of milk with ANN models; prediction of shelf-life of

milk and milk product based on artificial predicting neural networks; ANN methods to model and

predict the pH of cheese curd at various stages during the cheese-making process; modelling and

control of food extrusion process using ANN model; an intelligent system for pasteurised milk quality

assessment and prediction to support quality decision makers to assess and predict the pasteurised

milk quality using expert system and ANN techniques; using ANN, ANFIS, Neuro-Genetic-

Algorithm (NGA) techniques for modelling moisture sorption characteristics in various Indian dairy

products such as dried dairy products, viz., Dried Acid Casein, Fortified Nutrimix (weaning food),

Skim Milk Powder, Whey Protein Concentrate, Gulabjamun Mix; and for high moisture dairy

products, i.e., Peda and Kheer,; modelling and simulating the cross-flow ultra-filtration of milk using

Fuzzy Inference System (FIS); FL based reasoning system for evaluating Bulk Tank Milk (BTM)

quality and herd’s milking practice using BTM microbiology test results; classifying raw milk based

on microbiological and physicochemical qualities using FL technology; milk powder spray-drying

based on composite fuzzy control technology; optimisation of the viability of probiotics in a new

fermented milk drink by the GA for response surface modelling; nuero-fuzzy hybrid model for

modelling and simulation of the fouling of milk heat exchangers; predicting antioxidant capacity of

whey protein hydrolysates using ANN and neuro-fuzzy models; precision model based on ANN and

GA methods to predict the protein content in milk powder; optimisation of whole milk powder

processing variables with ANN and GA methods; modelling and optimisation of viscosity in enzyme-

modified cheese using FL and GA techniques; etc.

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Figure 1 Flowchart depicting steps involved in a typical neuro-genetic-algorithm model.


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Initialisation of ANN

Generation of initial population

Selection

Crossover

New

Population

Mutation

Termination

criteria

reached?

Best fitness chromosome

representing optimised weights

No

Yes

Optimised weights fed to ANN for

test data

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production using neural networks. International Dairy Journal 15, 1156–1174.

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total protein in yogurt by infrared spectrometry. Microchemical Journal 91 47–52.

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neural network approach. International Journal of Food Properties 3 531–539.

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properties. International Journal of AgriScience 2 1168–1178.

Meng X, Zhang M and Adhikari B (2012) Prediction of storage quality of fresh-cut green peppers using

artificial neural network. International Journal of Food Science and Technology 47 1586–1592.

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fuzzy logic and genetic algorithm. Computers and Electronics in Agriculture 62 260-265.

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An Overview of SAS Enterprise Guide in Dairy and Food Sciences

Ravinder MalhotraDairy Economics Statistics & Management Division

1. IntroductionSAS Enterprise Guide (or SAS EG) is a windows application that provides a point-and-click interface tothe SAS System. SAS EG does not itself analyze data, instead it generates SAS program. Every time werun a task in SAS EG, it writes a SAS program.SAS EG can be used to connect to SAS server on remotesystem or on the local system also. SAS Enterprise Guide communicates with the SAS System to accessdata, perform analysis, and generate results. From SAS Enterprise Guide one can access and analyzemany types of data, such as SAS data sets, Excel spreadsheets, and third-party databases. One can eitheruse a set of task dialog boxes or write its own SAS code for performing the analysis.

SAS Enterprise Guide provides following features

a. access to much of the functionality of SASb. ready-to-use tasks for analysis and reportingc. easy ways to export data and results to other applicationsd. transparent access to datae. a code editing facility

2. Start SAS Enterprise GuideTo open SAS Enterprise Guide click the Start → SAS → Enterprise Guide 4.2 (or the version availableon your system) from menu bar, otherwise double click the shortcut icon Enterprise Guide 4.2 on thedesktop of your system. Every time you open SAS EG, it brings up SAS EG window in the background,with welcome screen (shown in Fig 2.1) in the foreground. It allows one to choose options like openprevious saved project, new project, new SAS program etc.

The first time you start SAS EG, the windows are arranged in the default application layout. This layoutconsists of the Project Tree, Resource Pane, and the Workspace window.

The Project Tree window displays tree structure of the project. Resource Pane window shows Server List, Task List, SAS folder etc. The Workspace window is the container for the process flow, results, data grids, SAS code etc.

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Fig 2.1At first, the process flow (shown in Fig 2.2) is the only window that is open in the workspace area. Whenyou generate reports or open data, other windows open in the workspace with a tabbed interface. You canalso use the recently viewed items menu in the upper-left corner of the workspace to navigate between thewindows.

Fig 2.2If one wants to customize layout by changing the position of any window or by closing the window, thenit gets automatically saved for the current session of SAS EG. If you close any of the application thenclick on Menu option View and select the window name you want to reopen. If one wants to restore thedefault layout then click on Tools → Options from menu bar then click on Restore Window Layoutbutton.

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2.1 Creating new SAS Data set or Entering Data

To get Data grid click File → New → Data from menu bar. A New Data wizard opens (as shown in Fig2.3). This is the first step for entering the data in which mention the name of Data Set or (SAS data table)in the first text box. Then select library where you want to save the data set. By default WORK library isselected but this is a temporary storage location so better to select SASUSER library or any User definedlibrary. Click Next to proceed to next step. In second window (Fig 2.4) one can assign column or variablename and there properties. By default there are six columns one can add more or delete as perrequirement. Set properties like Name, Label, Type of variable Numeric or Character etc.

Fig 2.3 Fig 2.4After making necessary entries click on Finish Button. A new data table appears in the data grid form inworkspace window (shown in Fig 2.5). A shortcut icon of Data Set name is also there in the Project treeunder process flow.

Fig 2.5After completing data entry you need to protect data before start working on it. To protect data click onEdit → Protect Data from Menu bar. If you forget to protect data and start working then SAS EGautomatically ask to protect data. One can insert or delete Row and Column from data set by selectingrow or column where you want to perform action and by clicking right mouse button and choosing insertrows or delete rows and insert column or delete.

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2.2 Importing Files other than SAS Data set

Click on File → Import Data from menu bar. A window appears where you mention name of the fileyou need to open for eg. Lactation.xls. Then clicks on open button. An Import Data wizard will openhaving 4 steps in which one can found various option if you want no change in the data then simply clickon Finish button to create SAS Data Set of the file. A SAS Data Set will open in workspace area withshortcut icon in Project tree.

3. Creating New ProjectIn this step one create a new project to store the data and results. Select File→ New→ Project. If youalready had a project open in SAS Enterprise Guide, you might be prompted to save the project. Select theappropriate response. The new project opens with an empty Process Flow window. There is also a Newbutton on the toolbar to accomplish the same function.

4. Save the ProjectOne can save the project in a single file at any desired location on local computer as well as server also (ifyou are connected with server). Select File → Save Project As… from menu bar. A Save window opensin which you can select the location where you want to save the project file (as shown in Fig 4.1). Enterfilename in the textbox; file will be saved with .egp extension. Click on save button.

Fig 4.1To open saved projects select File → Open → Project. An open window appears, one can select locationeither local or server and then select the project you need to open.

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5. The Project TreeProject tree is hierarchal representation of active project (Fig 5.1). It shows related data, results file, tasks,and programs. One can manage items in the project from project tree. You can rename, rearrange anddelete objects from the project.

Fig 5.1

6. The Workspace and Process flowWhen one creates a new project, an empty Process Flow window opens (as shown in Fig 6.1). As you adddata, run tasks, and generate output, an icon for each object is added to the process flow. The process flowdisplays the objects in a project, any relationships that exist between the objects, and the order in whichthe objects will run when one run the process flow.

Fig 6.1

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7. Resource PaneThis window consists of four sub options Task List, SAS Folder, Server List, and Prompt Manager.

7.1 Task List

One can use task to do manipulation in data, execute analytical procedures, create graphs and generatereports etc. One can select any task from task list or can have Tasks as an option in menu bar. You canview listing of Task list based on Category, Name or Task template (as shown in Fig 7.2).

7.2 SAS Folder

It displays list of all of your stored processes, information maps, and projects. You can select an itemfrom this list and open it.

7.3 Server List

It displays a list of all the available SAS servers.

7.4 Prompt Manager

It displays a list of all the available prompts (as shown in Fig 7.2).

Fig 7.1 Fig 7.2

8. SAS EG HelpSAS EG provides us a comprehensive help for our ease of access. Select Help → SAS Enterprise GuideHelp. In Help window you can browse through the table of content and index or you can use searchfeature (Fig 8.1).

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Fig 8.1

9. Menu BarSAS EG has following list of Menu. While clicking on any menu option sub-menu items appears in dropdown format.

Menu Functions

File Open and save project, data, code, report, and process flow.

Import and export data. Print process flow.

Edit Modify or copy text, search and replace data. Expand or collapse data.

View Customize the look of the SAS Enterprise Guide window by selecting toview the tool bars for Project Flow, Task List, and Task Status.

Tasks Perform statistical procedures to manage data, create graphs, and producedescriptive and inferential statistics.

Program Open new or existing program (where one type SAS code to performanalyses), run or stop current program.

Tools Combine multiple reports into one. Set style of report. Set options such aswindow layout and enabling particular features.

Help Get help on SAS Enterprise Guide tasks. Getting Started.

10. Working with TasksIn SAS EG, one can use Tasks to do statistical analysis procedures as well as for creating reports. Oneway to select tasks is from Menu Bar (as shown in Fig 10.1) and other way by using the Task List (asshown in Fig 7.1). As you scroll down the Task List you see tasks in the Statistical Analysis, Graph etc.categories. In each task, there are certain steps that you must complete before running the task. Forexample, you must specify which variables you want to analyze, how to analyze them, format in whichone can save its results, mentioning analysis title etc. Once you have specified the necessary informationto run the task, the Run button becomes available and one can run the task and get the results.

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Fig 10.110.1 Exploring Task Window

Every task has two lists of variables, (as shown in Fig 10.2) Variables to assign and Task roles. TheVariables to Assign list displays all the variables from the data that you have selected. In Task Roles listyou assign variables to roles in the task. This is how you tell SAS EG how you want to analyze your data.The Task Roles list displays all the ways that variables can be used in a task.

Fig 10.2

To assign a variable to a task role, one can select the variable and drag it to the role. You can also selectthe variable by clicking the right arrow, and select the role from the menu that appears. On the left hand

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side of window there is a Selection Pane having options like Results, Titles, and Options etc. You can setthese values according to your need. You can see the code and modify it also, if required by clicking thePreview code button in the lower-left of each task window.

10.2 How to Analyze Data

During this analysis we will be using Body_weight SAS Data set which consist of following variablesWT_FC (Weight at First Calving), AFC (Age at First Calving), FLY (Milk Yield at First Lactation), FLL(First Lactation Length), FCI (First Calving Interval) and FSP (First Service Period).

Fig 10.3

10.3 Descriptive Statistics

Select Tasks→ Describe→ Summary Statistics. On the Summary Statistics Wizard select theAFC, FLL, FLY variable from Variable to assign list and drag it to Analysis Variable under TaskRole window.

One can select desired Basic Statistics like Mean, Standard deviation, Standard Error etc. fromSelection Pane

After selecting desired variables you can click on Run. Result will be displayed in the result window (Fig 10.4)

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Fig 10.4

t Testt Test (one Sample) Select Tasks → ANOVA → t Test. In the wizard select One Sample option. Click on Data option and select the AFC, FLL, FLY variable from Variable to assign list and drag

it to Analysis Variable under Task Role window. Click on Run and result will be displayed in the result window (Fig 10.5)

Fig 10.5

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10.4 Two SampleDuring this analysis we will be using following SAS Data set which consist of following variables Trt(Treatments), Score (Acceptance Score).

Select Tasks → ANOVA → t Test. In the wizard select Two Sample option. Click on Data option and select the Score variable from Variable to assign list and drag it to

Analysis Variable under Task Role window. Select the Trt variable from Variable to assign list and drag it to Classification Variable under

Task Role window. Click on Run and result will be displayed in the result window (Fig 10.6)

Fig 10.610.5 Correlation and Regression

The task that generates results for correlation is Correlations.

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In the Process Flow window, select the data set on which you want to perform analysis. Then selectTask → Multivariate → Correlations.

Select the FLY variable on which you want to perform analysis and drag it to Analysis Variablesunder Task Role window.

Select the variable AFC to which you want to view correlation and drag it to Correlation withunder Task Role window.

Select Option from Selection Pane, by default type of correlation is selected as Pearson. One canselect any correlation type by clicking on the check box. By checking Fisher Options, one canselect level of significance, one can also select type of alternative hypothesis as lower, upper or twosided from the drop down list.

Select Results in Selection Pane, here one can select show statistics for each variable check box orshow significance probabilities associated with correlations

After selecting desired variables you can click on Run.

Result window will be opened in the process flow area showing results of your analysis. From thereyou can get options to create report, export report in any defined format, and modify the task etc.(as shown in Fig 10.7).

Fig 10.710.6 Partial Correlation

In the Process Flow window, select the data set on which you want to perform analysis. Then selectTask → Multivariate → Correlations.

Select the FLY variable on which you want to perform analysis and drag it to Analysis Variablesunder Task Role window.

214

Select the variable FLL to which you want to view correlation and drag it to Correlation withunder Task Role window.

You need to select a variable under Partial Variables option to perform partial correlation. So,select the FCI variable and drag it to Partial Variables under Task Role window.

Select Option from Selection Pane, by default type of correlation is selected as Pearson. One canselect any correlation type by clicking on the check box. By checking Fisher Options, one canselect level of significance, one can also select type of alternative hypothesis as lower, upper or twosided from the drop down list.

Select Results in Selection Pane, here one can select show statistics for each variable check box orshow significance probabilities associated with correlations


Result will be displayed in the result window (Fig 10.8)

Fig 10.810.7 Multiple Regression

In the Process Flow window, select the data set on which you want to perform analysis. Then selectTask → Regression → Linear Regression.

Select the FLY variable and drag it to Dependent Variable under Task Role window.

Select the variable AFC & FLL and drag it to Explanatory Variable under Task Role window.

You can also select any variable and drag it to Group Analysis by under Task Role window.

215


Result will be displayed in the result window (Fig 10.9)

Fig 10.910.8 One-Way ANOVA

During this analysis we will be using following SAS Data set which consist of following variables Trt(Treatments), Rep (Replication), Score (Acceptance Score).

In the Process Flow window, select the data set on which you want to perform analysis. Then selectTask → ANOVA → One-Way ANOVA.

Select the Trt variable from Variable to assign list and drag it to Dependent Variables under TaskRole window.

216

Select the Score variable from Variable to assign list and drag it to Independent Variables underTask Role window.

Under Means in the selection pane, select Comparison. If you reject the null hypothesis of equality of treatment effects, then use multiple comparison

procedure for all possible pair wise treatment comparisons to determine which of the mean aredifferent. A desired Multiple Comparison Procedure from the available options, say Tukey’sstudentized range test (HSD).

For this example one can select Tukey’s studentized range test (HSD). Tukey’s method examinesthe difference between all possible combinations of two treatment means.

Click Run to run the One-Way ANOVA. Result will be displayed in the result window (Fig 10.10)

Fig 10.1010.9 Two-Way ANOVA

During this analysis we will be using following SAS Data set which consist of following variables Trt(Treatments), Mth (Methods), Mois Cont (Moisture Content).

217

In the Process Flow window, select the data set on which you want to perform analysis. Then selectTask → ANOVA → Linear Models.

Select the Mois Cont variable from Variable to assign list and drag it to Dependent Variablesunder Task Role window.

Select the Trt and Mth variable from Variable to assign list and drag it to Classification Variablesunder Task Role window.

In the Selection Pane, select Model option. In the Class and Quantitative variables list, select Trt and Mth and click on main button. For desired type of Sum of Squares select from Model Options. For multiple comparison procedure, select from Post Hoc Tests, Least squares and then select the

trt under the options for means tests. Click Run to run the Two-Way ANOVA. Result will be displayed in the result window (Fig 10.11)

Fig 10.1110.10 Factorial Randomized Complete Block Design

During this analysis we will be using following SAS Data set which consist of following variables Stain(First Factor), Time (Second Factor) , Rep (Replication), Fat Content.

218

In the Process Flow window, select the data set on which you want to perform analysis. Then selectTask → ANOVA → Linear Models.

Select the Fat Cont variable from Variable to assign list and drag it to Dependent Variables underTask Role window.

Select the Stain, Time and Rep variable from Variable to assign list and drag it to ClassificationVariables under Task Role window.

In the Selection Pane, select Model option. In the Class and Quantitative variables list, select Stain, Time and Rep and click on main button,

then press ctrl and select Stain and Time and click on Cross button. For desired type of Sum of Squares select from Model Options. Click Run to run the Two-Way ANOVA. Result will be displayed in the result window (Fig 10.12)

Fig 10.12

11. Export a SAS ReportSuppose you would like to export Linear Regression Report as a step in a project, and you would like it tobe an HTML file. For this purpose you need to follow following steps.

219

Click on Export → Export SAS Report → Linear Regression1 As A Step In Project. The first page of the Export wizard enables you to select the file that you want to export. In this

case, select SAS Report → Linear Regression1. Click Next.

Fig 11.1

The second page of the Export wizard enables you to select the file type of the exported file. Tosave the report as an HTML file, select HTML documents.

Fig 11.2 The third page of the Export wizard allows you to specify a location for the exported file. If you

would like to change the name of the file, simply click on Browse button and mention newfilename and destination path. No need to change file extension (.html). Click Next.

220

Fig 11.3 The fourth page of the Export wizard enables you to review the selections that you have made.

Click Finish.

Fig 11.4Selected ReadingsSusan J. Slaughter & Lora D. Delwiche. The Little SAS Book for Enterprise Guide 4.2.http://support.sas.com/http://web.iasri.res.in/sscnars/sas_manual

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Is it difficult to file Patent in India?

Y.S. Rajput, Rajan Sharma and Dheraj Nanda

1. Introduction

Intellectual Property means the legal rights granted to a person which result from intellectual activity

in the industrial, scientific, literary and artistic fields. These include (i) Patents, (ii) Copyrights, (iii)

Trademark, (iv) Industrial designs (v) Layout designs of integrated circuits, (vi) Geographical

indications, (vii) Registration of plant varieties and (viii) Trade secrets.

Patent is an exclusive right given by Government to an inventor for a limited period of time, for

protecting against illegal use of invention. The duration of patent in 20 years and is counted from

the Priority Date i.e. the date of filing patent application.

For an invention to be patentable, three basic criteria must be satisfied:

1. Novelty - The invention should not have any prior art. Any material published, presented

or in public use globally will constitute prior art. Any invention which is published before

filing patent application becomes part of prior art and thus cannot be novel. Annual

reports, thesis, journals, periodicals etc are published materials. If student wants to obtain

patent from the work carried out in his/ her dissertation, he/she must file patent first before

submitting thesis. Alternately, thesis is written in a way that inventiveness is not disclosed.

2. Non-obviousness - The invention should not be obvious to the person skill in art. A

skilled person has reasonable knowledge in the subject of invention.

3. Commercial value - The invention must have industrial application.

A patent application is filed only when applicant is satisfied from all the three criteria. There are two

broad types of patent applications. First one is Provisional Patent Application while the other one is

Complete Specification Application.

2. Contents of Patent Application:

A patent application should contain:

1. Application for grant of patent in Form-1.

2. Provisional / complete specification in Form-2.

3. Statement and undertaking in Form-3, which is filed either along with the application or within 6

months from the date of application.

4. Declaration as to inventorship in Form 5 for Applications accompanying a Complete Specification

or a Convention Application or a PCT Application designating India.

5. Power of authority in Form-26, if filed through a Patent Agent.

6. Priority document is required in case of Convention Application (under Paris Convention) or PCT

National Phase Application. The priority document may be filed along with the application or

before the expiry of eighteen months from the date of priority, so as to enable publication of the

application.

7. Every application shall bear the Signature of the applicant or authorized person / Patent Agent

along with name and date. The drawing sheets should bear the signature of an applicant or his

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agent in the right hand bottom corner. On every sheet, name of applicant is written on top-left hand

corner while total number of drawing sheets and sheet number are written on top-right corner.

8. In case of a biological material obtained from India, the applicant is required to take the permission

from the National Biodiversity Authority before the grant of the patent. The Application form shall

also indicate clearly the source of geographical origin of any biological material used in the

Specification, wherever applicable.

Form 1: This form contains information about the applicant, inventor and the title of invention, the

particulars of same patent filed in convention country or through PCT. It also has columns for patent

of addition in case an inventor makes an improvement in invention specified in original patent

application and divisional patent, in case a patent application contains more than one invention and

the applicant wish to file a separate application for second invention.

Form 2: Form 2 contains Provisional/ Complete Specification. The Specification is a techno-legal

document containing full scientific details of the invention and claims to the patent rights. The first

page of the Form 2 shall contain:

a) Title of the invention;

b) Name, address and nationality of each of the applicants for the Patent; and

c) Preamble to the description.

3. Provisional Specification:

When the applicant finds that his invention has reached a stage wherein it can be disclosed on paper,

but has not attained the final stage, a written description may be submitted to Patent Office as a

Provisional Specification. A Provisional Specification secures a priority date. Immediately on

receiving the Provisional Specification the Patent office accords a filing date and application number

to the Application. A complete specification is to be filed within twelve months from the date of filing

of the provisional specification.

A Provisional Specification shall contain the title and description of the invention and shall start with

a preamble ‘The following Specification describes the invention.’ Claims may not be included in

the Provisional Specification as the purpose of filing a Provisional Specification is to claim a

priority date. The description starts from the second page starting with the field of invention and

containing the background of the invention, object of the invention and statement of the invention.

4. Complete Specification

Every complete specification shall fully and particularly describe the invention and discloses the best

method of performing the invention which is known to the applicant for which he is entitled to claim

protection and end with a claim or set of claims defining the scope of the invention for which the

protection is claimed and is to be accompanied by an abstract. A complete specification shall contain

the title, description, drawings, abstract and claims.

4.1 Title

The subject matter of the invention and shall disclose the specific features of the invention.

4.2 Description

The description should preferably begin with a general statement of the invention e.g. “This invention

relates to …………………”. Description of an invention is required to be furnished in sufficient detail

so as to give a complete picture of the invention and follows the Summary of invention. The nature of

improvements or modifications effected with respect to the prior art should be clearly and sufficiently

described. The details of invention described should enable a person skilled in the art to reproduce the

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invention into practice without further experimentation. Any biological material described in the

specification must be submitted with the International Depository Authority under the Budapest

Treaty or the International Depository Authority in India, Microbial Type Culture Collection and

Gene Bank (MTCC) – Chandigarh. Sequence listing should also be given in electronic form and the

fees with respect to the corresponding number of pages should be paid.

4.3 Drawings

Drawings shall be on separate sheet(s), shall be prepared neatly and clearly on durable paper sheet, of

standard A4 size with a clear margin of at least 4 cm on the top and left hand and 3 cm at the bottom

and right hand of every sheet, shall be sequentially or systematically numbered and shall bear in the

left hand top corner, the name of the applicant, in the right hand top corner, the number of the sheets

of drawings, and the consecutive number of each sheet and in the right hand bottom corner, the

signature of the applicant or his agent.

4.4 Abstract

The abstract shall contain a concise summary of the matter contained in the specification. The

summary shall indicate clearly the technical field to which the invention belongs, technical problem to

which the invention relates and the solution to the problem through the invention and principal use or

uses of the invention. The abstract may not contain more than one hundred and fifty words.

4.4 Claims

A claim is a statement of technical facts expressed in legal terms defining the scope of the invention

sought to be protected. In a complete specification the description is followed by claims. No

exclusivity is obtained for any matter described in the Complete Specification unless it is claimed in

the claims. What is not claimed in the ‘claims’ stands disclaimed, and is open to public use, even if

the matter is disclosed in the description.

4.5 Scope of claims

Claims must not be too broad to embrace more than what the applicant has in fact invented. A Claim

which is too broad may encroach upon the subject matter which is in public domain or belongs to

others. However, a claim may not be too narrow also because such a Claim would not be sufficiently

effective against potential infringement. A good drafting may begin with broad claims and develops

towards claims that are narrower in scope. The “statement of Claims” may be preceded by the

prescribed preamble, “I / We Claim” as the case may be. Each claim should be in a single sentence

and should be clearly worded.

The first claim is always an independent claim also known as ‘Principal Claim’ and should clearly

define the essential novel features of the most preferred embodiment of the process/product that

constitutes the invention. It is advisable to limit the number of claims, as well as the number of

independent claims in a single application so that the claims are all of cognate character and are linked

so as to form a single inventive concept. A dependent claim derives antecedence from an independent

claim and reads into it the features of the independent claim and may contain additional non-essential

features and even the minute aspects and optional features. Examples: “A wrapper as claimed in

Claim 1, wherein a narrow area of the tear tape, spaced from each edge of the tear-tape, is united to a

narrow area of the wrapper defined on each side by a line of perforations which are covered by the

outer portions of the tear-tape, the perforations facilitating tearing of the wrapper to remove the

portion bounded to the tear-tape.”

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4.6 Fee:

Fee payable under the Act may either be paid in cash or through electronic means or may be sent by

bank draft or cheque payable to the Controller of Patents and drawn on a scheduled bank at the place

where the appropriate office is situated. If the draft or cheque is sent by post, the fee shall be deemed

to have been paid on the date on which the draft or cheque would have reached the Controller.

4.7 Publication and Examination: Application is published after 18 months of submission of patent

application in journal of patent office and can be requested for early publication (form 9) by paying

fee (Rs 2750/- for individual, Rs 6,875/- for small entity and Rs. 13750 for others except small entity)

and early publication can occur within 3-4 months. The patent application is not examined by

examiners of patents (in patent office) unless it is published. After publication, request for

examination along with fee is submitted (form 18). Examiners may send examination report to

applicant and report has to be appropriately responded back within 12 months.

4.8 Maintenance of patent: After grant, a patent is maintained by paying renewal fee every year. If

renewal fee is not paid, the patent will cease to remain in force and invention becomes open to public.

4.9 Fee structure for physical filing:

Ten percent additional fees shall be paid in case of physical filing of patent application as compared to

e-filing. As given in Patent Amendment, 2014, small entity refers to:

An enterprise where the investment in plant and machinery does not exceed Ten crore Indian

Rupees (in case of an enterprise engaged in the manufacture or production of goods).

An enterprise where the investment in equipment does not exceed Five Crore Indian Rupees (in

case of an enterprise engaged in providing or rendering of services).

Following is the revised fee structure for physical filing:

Particular Natural

person

(INR)

Other than Natural

person (INR)

For small entity For others except

small entity

For filing patent application 1760 4400 8800

For each sheet of specification in

addition to 30

176 440 880

For each claim in addition to 10

claims

352 880 1760

Request for Early Publication 2750 6875 13750

Renewal fee (per year) 3rd

to 6th

year

880 2200 4400

Renewal fee 7th to 10

th year 2640 6600 13200


th year 5280 13200 26400


th year 8800 22000 44000

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Sensory Evaluation of Milk and Milk Products

Kaushik Khamrui and Writdhama PrasadDairy Technology Division

1. IntroductionSensory evaluation is the measurement of a food product’s quality based on information received fromthe five senses i.e., sight, smell, taste, touch, and hearing. The signals generated at the nerve endings ofthe senses are transmitted via the central nervous system to the brain where they are integrated with pastexperiences, expectations, and other conceptual factors before the option of the response in summarized.Sensory evaluation of milk is of utmost importance. It has been estimated that forty six percent i.e.,approximately 64.4 MMT of India’s total milk production (140 MMT) is consumed at liquid milk.Finished milk products can not be better than the ingredients from which they are made. Milk being themajor raw material for producing all the dairy products proper sensory evaluation of both raw as well asprocessed milk is very important and needs special care. There are two broad categories of milk viz.,market milk and manufacturing milk. Market milk are meant for direct consumption whereas,manufacturing milks are used for the production of other dairy products. The classes of liquid milksaccording to the Food Safety and Standard Regulation (FSSR, 2011) of India are presented in Table 1.

2. Desirable Sensorial Attributes of MilkTypically flavour of milk should be pleasantly sweet and possess neither a foretaste nor an aftertaste. Thenatural richness in milk is due to presence of milk fat and the sweetness due to milk sugar (lactose). Thecolour of cow milk is yellowish and of buffalo milk is white. The colour may vary depending on theextent of mixing the two types milks. There shall be no fat globules/particles on the surface.

3. Score Card for MilkA 25 – point score card has been recommended by American Dairy Science Association (ADSA),whereas a 100 - point score card has been recommended by Bureau of Indian Standards (BIS) for thesensory evaluation of milk (Table 2). Full or perfect score is normally awarded when there is no defect inmilk, and zero score for an unsalable product.

4. Order of Examination and Scoring Technique of Milk4.1 Containers and closure

The package and its closure in which milk is supplied should be carefully observed. Now-a-days milkinvariably packaged in polyethylene sachets. Hence, the evaluator must see that the packaging is noleakage/pilferage from the pouches/containers. The containers should be examined for the extent offullness (specified amount), cleanliness and printing.

4.2 Sediment

Milk samples should be observed for the presence of sediments. The kind, amount and size of sedimentparticles should be carefully observed by visual observation and scored against a chart of mental image. A3-point scale may be employed occasionally. The presence of any sediment in the processed milk isserious and should receive a zero score. One possible scoring system using a sediment disc could be:

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Amount of sediment ScoreNo sedimant 0

< 0.02 mg/disc 30.02 – 0.025 mg/disc 2

>0.025 mg/disc 1

4.3 Flavour

For the flavour evaluation, milk should be properly tempered between 13 and 18°C, preferably at 15.5°C.Samples should be served in clean, odourless glass or plastic bottles. For each evaluator, about 50 mlsample should be provided. Immediately after removing the lid the milk sample should be smelled andsimultaneously observe for the presence of cream or partially churned fat globules at the top. Then mixthe sample properly and take a generous sip, not less than 10 ml of milk, roll it around the mouth and notethe flavour and tactual sensation, then expectorate the sample, sometimes, the aftertaste may be enhancedby drawing a breath of fresh air very slowly through the nose. Slow agitation of milk leaves a thin film ofmilk on the inner surface of the bottle, which tends to evaporate thus readily giving off the odour present.Bureau of Indian standard has allotted a maximum score of 30 to body, which means consistency(watery/curdy).

4.4 Temperature

Raw milk as well as pasteurized milk should be stored at 7.2°C but lower then 4.4°C is preferred. Forpasteurized milk, if the temperature is above 7.2°C, the sample may be scored zero. One point may begiven is the temperature is between 4.4-7.2°C. Full two points may be given for a sample at or below4.4°C.

4.5 Bacterial count

The maximum permissible bacterial count in pasteurized milk in India is 30000/ml and coliform must beabsent in 0.1g. A sample containing a higher count than this limit should get zero score out of five. Thepractical count, however, cannot be done on every sample of raw milk for judging purpose. Hence, it isrecommended to perform the bacterial count test after a certain interval or in case of suspicion. Onepossible scoring for bacterial count in milk is presented in Table 3.

5. Defects in milkMilk is generally considered to have a flavour defect if it manifests an odour, a foretaste, an aftertaste, ordoes not leave the mouth a clean, sweet, pleasant condition after testing. Off-flavours in milk may becategorized into four major alphabetical groupings (A-B-C-D):

Absorbed (barny, cowy, feed, garlic/onion)

Bacterial (acid, bitter, fruity/fermented, malty, rancid, unclean)

Chemical (astringent, cooked, lacks freshness, light oxidized, metal oxidized, rancid)

Delinquency (flat, foreign, salty, unclean)

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5.1 Absorbed off-flavours

• Barny: Transmitted off-flavour due to poor maintenance, ventilation, cleaning of barn. It leaves apersistent, unclean after taste in the mouth.

• Cowy: Distinct cow’s breathe like odour & a persistent unpleasant, medicinal, or chemicalaftertaste imparted by acetone bodies & alkyl phenols in milk.

• Feed: Roughages impart aromatic taints if fed within three hours before milking.

• Garlic/onion (weedy): Recognized by characteristic pungent odour and somewhat persistentaftertaste.

5.2 Bacterial off-flavours

• Acid: Sour detected by smell & taste due to microbial conversation of lactose into lactic acid.

• Bitter: Due to rancidity, specific weeds consumed by cows and certain organisms especiallypsychrotrophs.

• Fruity: Certain microorganisms (e.g., Pseudomonas fragi) produce aromatic end products thatseriously taint milk. Quickly detected by odour which may resemble of vinegar, pineapple, appleand other fruits.

• Malty: Pronounced off-flavour suggestive of malt caused by the growth of Streptococcus lacticsubsp. maltigens bacteria in milk as the result of temp abuse (~18.2°C).

• Rancid: Extremely unpleasant flavour due to volatile fatty acids (butyric, caproic, caprylic, lauricand capric acids) formed through enzymatic hydrolysis of fat. Characterized by soapy, bitter,unclean & nauseating after taste.

• Unclean: Higher concentration of alkyl phenols leads to unclean flavour.

5.3 Chemical off-flavours

• Astringent: Tongue and lining of the mouth tend to feel shriveled, almost puckered. Indicatesextreme rancidity.

• Cooked: Appears when milk is heated 76°C or more, Four types of heat induced flavours i)cooked or sulfurus ii) heated iii) caramelized and iv) scorched. “Moderate heated” flavour is notobjectionable but “pronounced” is highly undesirable.

• Lacks freshness/Stale: Not pleasantly sweet and refreshing as is typically desired in milk. It canbe a fore runner of other oxidized or rancid off-flavours.

• Metal induced oxidized: Due to lipid oxidation induced by catalytic action of certain metals.Metallic, oily, cappy, cardboardy, stale, tallowy, paint-like, and fishy are terms used to describethis off-flavour.

• Light induced oxidized: Described as burnt protein, burnt feathers, cabbage-like, light-activated,sunlight or sunshine flavour. Light catalyzed lipid oxidation and protein degradation are involvedin development of this flavour. The aroma is similar wet card board or wet paper.

5.4 Delinquency off-flavours

• Flat: Flatness appears soon after the sample reaches the tongue, partly as a result in markedchanges in the mouthfeel. Could not be detected by smell.

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• Foreign (atypical): caused by improper use of various chemicals e.g., detergents, disinfectants &sanitizers; exposure to fumes from the combustion of diesel, petrol or kerosene; contamination ofinsecticides, drenching of cows with treatment chemicals; treatment of the udder withmedications.

• Salty: This-off taste is commonly associated with milk from individual cows that are in the mostadvanced stages of lactation or with milk from cows that have clinical stages of mastitis. Result inincrease of NaCl in the milk & decrease in other mineral samples.

• Unclean: This off-flavour may develop by the action of certain psychorophilic bacteria, whenstorage temperature is too high (>7.2°C)

6. General Guide for Scoring and Grading of Milk and Milk ProductsWhile judging a dairy product, the identification of a defect, if any, is important but equally important isto award correct scores for different attributes so that the difference among the sensory evaluators isminimum. Some of the defects are very serious, for example sour/high acid, rancid, oxidized and cowyflavour in fluid milk whereas others like flat, weedy and cooked flavour are not very objectionable. Thescores are thus based on the nature of defect and its intensity. Finally grading of samples is done on thebasis of total score. A general scoring guide is given below (Table 4) to help evaluators for consistentjudging of dairy product.

Table 1. Classes of liquid milks according to the Food Safety and Standard Regulation (FSSR,2011) of India.

Classes Designations Locality Milkfat (Min.) MSNF (Min.)

1 Buffalo milk Raw, Pasteurized,flavoured andsterilized

Haryana/Punjab 6.0 9.0

2 Cow milk Raw, Pasteurized,flavoured andsterilized

Haryana/Punjab 3.5 9.0

3 Mixed milk Raw, Pasteurized,flavoured andsterilized

All India 4.5 8.5

4 Standardizedmilk

Pasteurized,flavoured andsterilized

All India 4.5 8.5

5 Recombinedmilk


All India 3.0 8.5

6 Toned milk Pasteurized,flavoured andsterilized

All India 3.0 8.5

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7 Double tonedmilk


All India 1.5 9.0

8 Skimmed milk Raw, Pasteurized,flavoured andsterilized

All India Not more than0.5

8.7

9 Full creammilk

Pasteurized &Sterilized

All India 6.0 9.0

Table 2. Score card for sensory evaluation of milk

ADSA Score Card BIS Score Card

Attribute Perfect Score Attribute Perfect Score

Flavour 10 Odour 20

Sediment 3 Flavour 40

Package 5 Body(Consistency)

30

Bacteria 5 Colour &Appearance

10

Temperature 2 - -

Total 25 - 100

Table 3. Score card for bacterial count of milk

SPC (cfu/ml) Score

>30000 0

18000 - 30000 1

12000 – 18000 2

6000 – 12000 3

3000 – 6000 4

< 3000 5

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Table 4. General Scoring Guide for Milk and Milk Products

Selected ReadingBourne M (2002) Sensory Methods of texture and viscosity measurement. In: Food Texture and Viscosity.

Academic Press, London, UK. 257 – 291.

Clark, S., Costello, M., Deake, M, and Bodyfelt F (2009) Fluid milk and cream products. In: The sensory evaluationof Dairy products. Springer Science + Business Media, New York, USA. 73-133.

FSSR, (2011) Food Safety and Standard Regulation [Internet document]. www.fssai.gov.in. Accessed 26 ⁄ 08 ⁄ 2015.

Quality according tothe Grade of Dairy

Products

Grade Defect & Intensity Approximate Score(% of the Perfect

Score)

Excellent A No defect More than 90%

Good B Flavour: Flat, slight cooked/stale/barny/neutralized/ saltyConsistency/Texture: defects of onlyslight intensity

More then 80%But less then 90%

Fair C Flavour: Definite cooked/neutralized/feed/ flat. Slight rancid/oxidized/metallic/fishy/ yeasty/mouldy/ acidicConsistency/Texture: Any texture defectof definite intensity

More then 60%But less then 80%

Poor D Flavour: Any flavour defect of the higherintensity as given above for grade CConsistency/Texture: Pronounced defect

Less then 59%. Theproducts are generallyunacceptable at thisscore

231

High Pressure Processing of Milk and Milk Products

Ashish Kumar Singh, P. N. Raju, Sanket Borad


1. Introduction

Milk was among few commodities on which the first experimentation related to high pressure processing

(HPP) was carried out almost a century ago bit Hite & co-worker at Virginia Experimental Station.

Despite holding the promise to inactivate the microorganisms associated with milk spoilage HHP could

not be further utilized in milk and other food products owing to various issue related to equipment design

and their operation on commercial scale. However, research carried out recently indicate that HHP seems

a very promising processing intervention, as it offers numerous opportunities for developing shelf-stable

foods with better nutritional, excellent organoleptic characteristics and in delivering safe foods to

consumers (Fonberg-Broczek et al., 1999). The technology has potential to address certain critical issues

associated with conventional thermal processing in milk and milk products such as browning, loss of

nutrients and other bioactive components. Since the temperature employed in majority of the HHP

processes is in ambient range hence, it may provide a potential to reduce energy consumption and costs of

operation, as well as to improve sustainability of production (Toepfl et al, 2006). The most attractive

feature which has made the process worldwide acceptable is uniform processing ability as the pressure is

applied uniformly throughout the food material, independent of its mass and time. In the case of dairy

products, high pressure (up to 400 MPa) has been used to process milk successfully, which has resulted in

desriable characteristics of milk products like yogurt, cheese, cream, among others; in fact, there have

been more studies on these products than on fluid milk alone. The advantage of HHP is that it not only

homogenizes milk but also inactivates the microorganisms and extends the shelf life of the product

(O’Reilly et al., 2001), which is similar to milk processed under ultra high temperature conditions.

For low-acid foods, such as milk, mild temperatures in combination with high pressure are necessary to

ensure extended shelf life products with higher quality characteristics (Balasubramaniam, 2003). Pressure

in combination with heat can be used for sterilization of milk. Researchers have shown that the

application of ultra high pressure combined with mild temperatures can extend the shelf life of milk up to

45 d.

2. Effect of HHP on Milk Microorganisms

The resistance of microorganisms to pressure in food is very variable depending on HP processing

conditions (pressure, time, temperature, cycles,), food constituents and the properties and the

physiological state of the microorganism (Smelt, 1998). Bacteria are expected to be injured or killed by

high hydrostatic pressure (HHP). This depends on pressure levels, species and strain of the

microorganism and subsequent storage. Injured bacteria may be repaired which could affect the

microbiological quality of foodstuffs with an important safety consideration especially in low acid food

products. The pressure resistance of microorganisms can be affected by many intrinsic and environmental

parameters. Important among these is the nature of the suspending medium. Milk and cream protect

microorganisms against pressure. Pressurization of samples was carried out using a high pressure at 41 °C

and 448.0 MPa for11 min. Treatment of L. monocytogenes in milk under HPP was not as effective as in

fruit juices, because the high nutrient content in whole milk increased the resistance of the strain to HPP,

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and that HPP was suitable for inactivation of microorganisms in high and low acid food systems. Studies

conducted by Vachon et al. (2002) revealed that dynamic high pressure treatment inactivates the 3 major

food pathogens (Listeria monocytogenes LSD 105-1, Escherichia Coli O157:H7 ATCC 35150 and

Salmonella Enteritidis ATCC 13047) present in milk. Work carried out on combined application of HHP

and casein on microbiological quality of milk and milk products indicate that this intervention could be

more lethal as compared to HP treatment alone.

3. High Pressure Induced Effects on Milk Components

HP influences the physico-chemical and technological properties of milk. When milk is subjected to HP,

the Casein Micelles are disintegrated into smaller particles which in turn are accompanied by an increase

of caseins and calcium phosphate levels in the serum phase of milk and by a decrease in the both non-

casein nitrogen and serum nitrogen fractions (Law et al., 1998). Pressure above than 3000 atmosphere

tends towards irreversible denaturation as compared to reversible denaturation within range of 1000-3000

atmospheres (Jaenicke, 1981). Felipe et al. (1997) reported that a pressure treatment of 500 MPa at 25 °C,

denatures β-lactoglobulin and denaturation of the immunoglobulins and α-Lactalbumin only occurs at the

highest pressures and particularly at temp above 50° C which gives an idea of preservation of colostrums..

With the application of HP treatment as said above the size and number of casein micelles increases as

spherical particles changes to form chains or clusters of sub-micelles. Thus, reduces RCT (Rennet

Coagulation Time). In general, rennet coagulation properties of milk subjected to pressures of 100–500

MPa for 30 min are enhanced.

Liu et al. (2005) studied the effect of (HHP) treatments on hydrophobicity of whey protein concentrate

(WPC) and observed that treatment of WPC yields increase in the number of binding sites and led to

certain modifications of proteins and showed promising results for improving functional properties of

foods. Similar observations for improved hardness, surface hydrophobicity, solubility, gelation and

emulsifying properties were recorded by Lee et al. (2006) in whey proteins functionality.

Studies carried out by Gervilla et al. (2001) on Free fatty acids (FFA) content in ewe's milk have showed

that HP treatments between 100–500 MPa at 4, 25 and 50° C did not increase FFA content. Thus it is of

great interest to avoid production of off flavours which otherwise gets develop because of lipolytic

rancidity in milk. Hydrostatic pressure up to 500 MPa produces some modifications on size and

distribution of milk fat globules of ewe's milk. HP treatments at 25 and 50° C showed a tendency to

increase the number of small globules in the range 1–2 μm, whereas at 4°C the tendency was the reverse

(Gervilla et al., 2001). However, no damage on the milk fat globule membrane occurred, being proved by

the lack of increase in lipolysis as it has been mentioned above. This provides some advantages for HP-

treated milk, because HP treatment increases the stability of milk treated at 25 and 50° C, whereas at 4 °C

(low temperatures) increases the creaming-off. This aspect could improve cream separation in the

elaboration of butter. Lactose in milk and milk products may isomerise to lactulose by heating and then

degrade to form acids and other sugars. No changes in these compounds are observed after pressurization

(100–400 MPa for 10–60 min at 25 °C), suggesting that no Maillard reaction or lactose isomerization

occur in milk after pressure treatment (López Fandiño et al., 1996).

Contrary to thermal treatments, where covalent as well as non-covalent bonds are affected, HP treatment

at room and mild temperatures only disrupts relatively weak chemical bonds (hydrogen bonds,

hydrophobic bonds, ionic bonds). Thus, small molecules such as vitamins, amino acids, simple sugars and

flavour compounds remain unaffected by the HP treatment. HP treatment of milk at 400 MPa (@ 2.5

MPa/s for 30 min at 25 °C) results in no significant loss of vitamins B1 and B6 (Sierra et al., 2000).

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4. Effect on Milk Enzymes

The effects of HPP on inactivation of enzymes depend on the structure of the enzyme and the processing

conditions applied. Alkaline phosphatase used as indicator for determining the efficacy of pasteurization

process appears quite pressure-resistant, with no inactivation in raw milk after treatment at 400MPa for 60

min at 20°C. The enzyme is completely inactivated at 800 MPa for 8 min. Indigenous milk

lactoperoxidase, phosphohexoseisomerase and g-glutamyltransferase, are also resistant to pressures up to

400MPa at 20–25°C. Researchers have indicated that lipoprotein lipase (LPL) is pressure stable. In fact,

the activity of lipases in milk is enhanced by high pressure processing (Buffa et al., 2001). Combinations

of higher pressure and temperature are necessary to inactive lipases in milk.

5. High Pressure related Changes on Functional Characteristics of milk

Micelle disintegration induced by HP treatment also affects milk Colour. An study was carried out by

Harte et al. (2003) to observe the series of changes during combine treatment of thermal and HHP for

yogurt manufacture and it was observed that milk subjected to HHP or thermal treatment and then HHP,

lost its white colour and turns into yellowish colour and may be due to reduction in size of casein micelles

(Needs et al., 2000), whereas milk when first subjected to HP and then to thermal treatment regained its

whitish colour and is attributed to reversible nature of casein micelles (or reaggregation of disrupted

micelles) towards HHP treatment when applied in the range of 300-676 MPa followed by thermal

treatment. HPP has been observed to affect the heat coagulation time (HCT) of milk and pressure

treatment above 200 MPa was found to decrease the HCT. The effect on HCT could be attributed to HP

induced changes in casein and milk minerals. Likewise rennet coagulation time (RCT) of milk has also

been observed to be influenced by HPP, but the change in RCT is quite variable. Garcia Risco et al.

(2000) found that HP treatments at 400 MPa for 15 min at 40–60°C reduces the proteolytic activity, and

at 25–60° C improves the organoleptical properties of milk, suggesting that these combined treatments

could be used to produce milk of good sensory properties with an increased shelf life.

6. Prospective Applications of High Pressure Processing in Dairy Industry

In dairy products, high pressure (up to 400 MPa) has been used to process milk successfully, which has

resulted in significant effect on milk molecules that could also influence the characteristics of milk

products like yogurt, cheese, cream, etc. Some of the prospective applications have been discussed

hereunder.

6.1 HHP Induced Effect on Cheese Making Characteristics

Milk pasteurization destroys pathogenic and almost all spoilage microorganisms and it is the most

important heat treatment applied to cheese milk to provide acceptable safety and quality. However, milk

pasteurization is known for its adverse affects with respect to many sensory characteristics of cheese,

leading to alterations in texture and often delayed maturation (Grappin & Beuvier, 1997). HP technology

can be used to increase the microbiological safety and quality of milk to produce high quality cheeses. As

it has been mentioned above, HP processing of milk at room temperature causes several protein

modifications, such as whey protein denaturation and micelle fragmentation, and alters mineral

equilibrium. It has been observed that denaturation of whey proteins due to applied pressure results in

interaction between denatured whey protein and casein, which in turn increases the retention of former

within casein matrix in cheese. Thus, these changes results in modifying the technological aptitude of

milk to make cheese, improving the rennet coagulation and yield properties of cheese milk (Gonzalez et

al., 2004). Microbiological quality of cheeses from HP-treated milk (500 MPa for 15 min at 20 °C) was

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comparable to pasteurized milk (72 °C for 15 s) cheeses (Buffa et al., 2001). However, the application of

HP technology to cheese milk causes differences in cheese composition and ripening in comparison to

pasteurized milk cheese. The HP-treated milk cheeses have higher moisture, salt and total free amino

acids contents than raw or pasteurized milk cheeses. On the other hand, cheeses made from HP-treated

milk showed a similar level of lipolysis to cheeses made from raw milk, whereas the level of lipolysis in

cheese made from pasteurized milk was lower and this behaviour was explained by heat-sensitive but

partial pressure-resistant characteristics of the indigenous milk lipase. Also pressure treated cheese

showed more viscoelastic texture and had less resistance to flow (Messens et al, 1999).

Being cheese ripening a quite expensive process, acceleration of ripening is highly desirable. Most of the

work in this field has been done by elevation of ripening temperature, addition of cheese slurries or

exogenous enzymes or by the use of adjunct starters, either as such or in modified form. Experimental

Cheddar cheese samples were exposed to pressure from 0.1 to 300 MPa for 3 days at 25°C after cheese

making. Best results were obtained at 50 MPa, where cheese with free amino acid and taste comparable to

that of a 6 month old commercial cheese was obtained. In particular, the kind of starter bacteria added to

the cheese milk was highly proteolytic and at least 10-fold higher than conventional inoculation rates. In

certain cheese varieties such as Mozzarella and Gouda, pressurization increases rate of proteolysis on

exposure to pressure treatment of 400-600 MPa for 5-15 min. There is substantial scope for improving the

quality of cheeses made from Buffalo milk.

6.2 Yoghurt and Ice- Cream

Yoghurt, a very popular dairy product suffers from common defect of syneresis and low viscosity or

rigidity. Yoghurt quality can be improved in terms of its preservation and improved rheological properties

by pressurization treatment. Skim milk treated with combined treatments of high hydrostatic pressure

(400-500 MPa) and thermal treatment (85° C for 30 min) showed increased yield stress, resistance to

normal penetration, elastic modulus and reduced syneresis (Harte et al., 2003). Reps et al. (1999)

investigated the effect of pressurization on inactivation of microflora present in yogurt and found that HP

treatment of 400 MPa completely inactivates Lactobacillus bulgaricus but Streptococcus thermophilus

has been found to be more resistance towards pressure and its resistance varies from strain to strain & is

in the range of 35.3 to 99.9 % which will be beneficial in checking post-acidification in yoghurt a major

detriment for longer shelf life of yogurt.

HPP treatment Induces fat crystallization, Shortens the time required to achieve a desirable solid fat

content, & thereby thus reduces the ageing time of Ice Cream, and also enhances the physical ripening of

cream for making butter (Buchheim and Frede, 1996).

6.3 Prospective Applications in Functional Dairy Foods

Colostrum is the first mammary secretion produced during the first 72 hours after parturition, provides

nourishment for the newborn. It contains numerous immune system and growth factors as well as

essential nutrients, trypsin and protease inhibitors that protect it from destruction in the Gastrointestinal

tract. The most important components of colostrum can basically be divided into two major categories:

immune factors and growth factors. Colostrum is particularly rich in immunoglobulins, lactoferrin,

lactoperoxidase and other bioactive molecules, including growth factors. Colostrum has antioxidant and

anti-inflammatory properties and is a good source of many vitamins, minerals, enzymes and amino acids.

Heat treatment and freeze drying are two most common processing technologies which have been applied

so far for preserving the colostrum, however these cause substantial lossess of immunoglobulins, growth

factors and therapeutic minor whey protiens. There is increasing demand of colostrum powder because of

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its health promoting properties for infants, women, persons suffering with immune disorders including

HIV, Cancer. HHP technology have been reported to have minor effect on bioactivity of colostrum

preparations, however there is need to systematic carry out detailed investigations.

Probiotic foods are one of the fastest growing sectors of functional foods. One of the major obstacles in

efficacy of probiotic dairy products is the loss of viability of microorganisms during processing, storage

and consumption. Irrespective of the preservation method applied, probiotic cultures are exposed to

unfavourable environmental conditions, due to increased solute concentration, intracellular ice formation

in case of freezing and freeze drying, exposure to high temperatures during spray drying and in general,

dehydration. HP and PEF, particularly, might be highly useful techniques employed in the preservation of

ingredients used in processing liquid probiotic foods (Pereira & Vicente, 2009). In high pressure

processing (HP), the food (solid or liquid) is subjected to pressures above 100MPa upto 900MPa, with

pressures used in commercial systems between 400 and 700M. The probiotic micro organism should be

technologically suitable for incorporation in the food product retaining their viability and efficacy in the

food product. There is great scope for utilizing HP processing for the development of probiotic dairy

foods with higher viable counts.

7. Conclusion

HPP products are becoming choice of a new consumer in terms of health and safety aspects. Being one of

the emerging technologies in developing countries, High pressure technology offers the technologists an

opportunity to develop novel products with enhanced shelf life and higher safety with better sensory and

nutritional aspects of food intact within and being applicable to a wide range of products, this technology

offers food processors to manufacture minimally processed shelf stable products. However, looking at the

potential of High pressure treatment for shelf-life extension without affecting the nutritional value and

modification of functionalities of macromolecules for exploitation of improving the quality characteristics

of resultant products needs to be investigated. HPP offer considerable scope for delivering high-value

dairy products such as colostrum, probiotic dairy products; infant foods and even shelf-life enhancement

of human milk without affecting its nutritional and therapeutic virtues.

8. Selected Reading

Balasubramanian S and Balasubramanian V M (2003) Compression heating influence of pressure transmitting fluids

on bacteria inactivation during high pressure processing. Food Research International 36 661-668.

Buchheim W and Frede E (1996) Use of high-pressure treatment to influence the crystallisation of emulsified fats.

DMZ Lebensmittel industrie and Milchwirtschaft 117 228–237.

Buffa M, Guamis B, Pavia M and Trujillo A J (2001) Lipolysis in cheese made from raw, pasteurized or high-

pressure treated goat's milk. International Dairy Journal 11 175-179.

Chawla R, Patil G R and Singh A K (2011) High Hydrostatic technology in Dairy Processing: A Review. Journal of

Food Science & Technology. 48 260-268.

Felipe X, Capellas M and Law A R (1997) Comparison of the effects of high-pressure treatments and heat

pasteurisation on the whey proteins in goat's milk. Journal of Agricultural and Food Chemistry. 45 627–631.

Fonberg-Broczek M, Arabas J, Kostrzewa E, Reps A, Szczawiński J, Szczepek J, Windyga B, Porowski S. (1999)

High pressure treatment of fruit, meat, and cheese products: equipment, methods and results. Processing

Foods. Quality Optimisation and Process Assessment, eds CRC Press LLC. Pg. 281–300.

Garcia-Amezquita E L, Primo-Mora R A, Barbosa-Cánovas V G, Sepulveda R D (2009). Effect of non-thermal

technologies on the native size distribution of fat globules in bovine cheese-making milk. Innovative Food

Science and Emerging Technologies. 10 491–494.

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Gervilla R, Ferragut V and Guamis B (2001) High hydrostatic pressure effects on colour and milk-fat globule of

ewe’s milk. Journal of Food Science, 66 880 –885.

Gonzalez-Martin M F San, Chanes- Welti J S and Barbosa- Canovas G V (2004) Cheese manufacturing assisted by

ultra-high pressure. IFT Meeting, July 12-16, Las Vegas, NV.

Grappin R and Beuvier E (1997) Possible implications of milk pasteurisation on the manufacture and sensory

quality of ripened cheese: A review. International Dairy Journal 7 751–761.

Harte F, Luedecke L, Swanson B, Barbosa-Cánovas G V (2003). Low-Fat Set Yogurt Made from Milk Subjected to

Combinations of High Hydrostatic Pressure and Thermal Processing. Journal of Dairy Science 86 1074-1082.

Jaenicke R (1981) Enzymes under extreme conditions. Annual Review Biophysics Bioengineering. 10:1

Law A J R, Leaver J, Felipe X, Ferragut V, Pla R and Guamis B (1998) Comparison of the effects of high pressure

and thermal treatments on the casein micelles in goat’s milk. Journal of Agricultural and Food Chemistry, 46

2523 –2530.

Lee L and Kaletunc G (2010) Inactivation of Salmonella Enteritidis strains by combination of high hydrostatic

pressure and nisin. International Journal of Food Microbiology 140 49-55.

Lopez Fandino R, Carrascosa A V and Olano A (1996). The effects of high pressure on whey protein denaturation

and cheese-making properties of raw milk. Journal of Dairy Science 79 929 – 1126.

Messens W, Foubert I, Dewettinck K, Huyghebaert A (2000) Proteolysis of a high-pressure-treated smear-ripened

cheese. Milchwissenschaft 55 328–332.

Needs E C, Stenning R A, Gill A L, Ferragut V, Rich G T (2000) High pressure treatment of milk: effects on casein

micelle structure and on enzymic coagulation. Journal of Dairy Research 67 31–42.

O' Reilly C E, Kelly A L, Murphy P M, Beresford T P (2001) High pressure treatment: applications in cheese

manufacture and ripening. Trends in Food Science and Technology 12 51−59.

Reps A, Warminska Radyko I, Dajnowiec F (1999). Effect of high pressure on yoghurt. Advances in High

PressureBioscience and Biotechnology Heidelberg, Germany:Springer 453 –456.

Sierra I, Vidal Valverde C and López Fandiño R (2000) Effect of high pressure on the Vitamin B1 and B6 content in

milk. Milchwissenschaft 55 365–367.

Smelt J M (1998) Recent advances in the microbiology of high pressure processing. Trends in Food Science and

Technology 9 152 –158.

Toepfl S; Mathys A, Heinz V, Knorr D (2006) Potential of high hydrostatic pressure and pulsed electric fields for

energy efficient and enviornmentally friendly food processing. Food Reviews International. 22 405-423.

Vachon J F, Kheadr E E, Giasson J, Paquin P, Fliss I (2002) Inactivation of foodborne pathogens in milk using

dynamic high pressure. Journal of Food Protection. 65 345-352.


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Advances in Infant Food Formulations

D.K.Thompkinson

1. Introduction

Human infants should ideally be nursed on mothers milk which constitutes natures best food. However, in

the event of lactation failure, insufficient milk secretion and where mothers suffering from transmittable

diseases, human milk substitutes serve as savers of precious life during vulnerable stages of infancy. Most

of the breast milk substitutes utilize bovine milk due to easy availability. The pattern of bottle feeding is a

sequal to industrialization and urbanization. There has been an ever increasing reliance of formula

feeding practices both in developed and developing countries. Bovine milk based dried formulations have

become a prominent feature of infantile dietetics. The technological advances has contributed to the

fascinating evolution from simple condensed mix mixtures to sophisticated formulations of today. There

has been an tremendous growth in the dried milk industry for manufacture of infant formula. Today

nearly 185,000 tonnes of infant formula is manufacture d in India representing approximately 3.8% of

total milk production.

Emphesis has been laid on manufacture of formulations having compositional and biochemical

characteristics similar to human milk. This has changed the scenario of infant feeding where conventional

formulas have been constantly replaced with nutritionally improved formulations. Industrially prepared

formulae significantly lack in bioprotective factors, firstly because bovine milk is innately deficit in bio-

immune factors, and, secondly due to thermal processing treatments that tend to further reduce their

levels. Very little work has been done as yet for incorporating bio-immune factors that assume special

significance in the bottle-feeding practice for providing protection against enteropathogenic bacteria

among infants. Never the less, the most appropriate alternatives to breast milk will continue to be infant

formulae based on bovine milk.

2. Physiological response of infant to conventional formulae

The infant’s underdeveloped intermediary metabolism in the critical period of first three months of ‘extra-

uterine’ growth is expected to have certain physiological repercussions when fed on formulas that are

compositionally different from human milk. Although bovine milk seems to be the most perfect food in

the later stages of life, the requirements for nutrients are different in infancy, which can only be met with

human milk at least in the initial stages. It is, therefore, pertinent to review the influence of biochemistry

of human and bovine milk on the nutritional and physiological system of an infant.

3. Significance of Protein Profile

The quality of human milk proteins is most suited for the immature functioning of intermediary

metabolism. Bovine milk creates a variety of problems, if fed to infants without modification. Several

parameters, such as the protein level, protein make-up and amino acid composition should be considered

so as to suit the underdeveloped intermediate enzymes and organ function amongst infants, particularly

during first three months of infancy.

Amount of protein ingested by formula fed infants is far higher than in breast-fed infants. The protein

breakdown products exert an excessive load on the immature kidneys of these infants. On the other hand,

in breast-fed infants, low molecular weight protein breakdown products are efficiently utilized. Human

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milk has lower casein content (40%) as compared bovine milk (80%). The smaller amounts of casein in

breast milk allow softer milk curdling, shorter stay of the soft and flocculent curd in the stomach and

faster gastrointestinal digestion. In contrast to breast milk, the curd of bovine milk is coarse-flaky, large

and firm. Accordingly, bovine milk curd is more difficult to digest, increases the loss of nitrogen and

develops gastrointestinal dyspepsia. Babies fed on unmodified bovine milk respond with higher

concentration of blood urea compared to those given human milk. Studies on the solute load imposed by

bovine milk indicate the urea concentrations of 1007.3 mg per 100 ml in urine of bovine milk fed infants

as against 211.4 mg in breast-fed ones.

Besides the eight essential amino acids designated for adults, a few others are known to be essential for

infants. As the interconversion mechanisms of histidine from ribose-5-phosphate, cystine from

methionine and tyrosine from phenylalanine is not fully developed in infants, owing to the diminished

activity or even absence of their specific convertases, they are also considered to be essential for infants.

Human milk, although low in its protein content, provides all the eleven essential amino acids required for

infants. In cow milk, although other amino acids are in excess, cystine is considerably low. The

differences in the ratios of phenylalanine/tyrosine in human and cow milk are recognized as being

important or even critical in feeding the newborn babies when ‘transient tyrosinaemia’ is noted

frequently. In this context, buffalo milk once again excels cow milk in which phenylalanine/tyrosine ratio

is comparatively close to that of human milk. As taurine cannot be synthesized and infants are totally

dependent on its dietary intake, higher taurine content of human milk has gained considerable

significance.

4. Modification of milk proteins

Hypersensitivity to cow milk has caused a significant health problem since the introduction of mass-

produced infant formulas at the beginning of the twentieth century. Allergic symptoms normally appear

within the first two months of life and have been noted in 1 to 8 percent of formula fed infants. Although

the -lactoglobulin fraction has been implicated most often in allergic reactions to cow milk, the caseins,

-lactalbumin, serum albumin, and immunoglobulins and digests of these proteins are also allergenic in

infants and children. The -lactoglobulin can be removed and the remainder of the proteins blended for

use in infant formulas.

The caseins of cow milk also have been hydrolyzed and subjected to gel filtration to prepare a non-

antigenic fraction. Kuchroo and Ganguli (1980) prepared a humanized infant formula from buffalo milk

containing low levels of the antigenic s casein. Electrodialyzed skim milk was subjected to protein

hydrolysis with trypsin, pasteurized and fortified with lactose and vegetable oils. After homogenization,

vitamin mix was added and the mixture spray dried. Commercial hypoallergenic infant formulas such as

Neutramagin and Pregestamil from Mead-Johnson contain casein hydrolyzates.

Joglekar (1984) prepared an infant formula wherein the protein was exclusively derived from buffalo milk

to the extent that an adequate amount of limiting essential amino acid could be supplied at comparable

levels to human milk.

5. Significance of lipid profile

Lipids supply between 40 to 50 percent of the energy consumed in infancy. Beside energy, they are also

important sources of essential fatty acids and carriers of fat-soluble vitamins. The estimation of fat

requirement in infancy is, however, based on the types of fat, triglyceride and fatty acid make-up and the

recommended levels of fats. Human milk is better absorbed in comparison to bovine milk fat, which may

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be attributed to the presence of palmitic acid in beta-position in the triglyceride configuration where it is

absorbed much better than that in terminal position or free acids. Animal studies using rat models have

also demonstrated this phenomenon. Monoglycerides with palmitic acids in beta-position are more

efficiently solubilized and absorbed by human infants. Human milk fat also contains adequate amount of

linoleic acid or linoleates and other essential fatty acids, which are required for maintaining the integrity

of skin and normal growth.

The ratio of unsaturated to saturated fatty acids in infant formulas has been adjusted to that of human milk

by incorporation of soybean, oleo and safflower oils which are rich in unsaturated fatty acids and coconut

oil and milk fat, which are rich in saturated fatty acids. Corn oil may also be added to formula to increase

the oleic acid content. Since cow and goat milk fat also contain highly absorbable triacylglycerols with

palmitic acid in the Sn-2 position, the digestibility of infant formulas could be improved by blending

vegetable fats and milk fats. Such formulations have ratios of saturated : polyunsaturated fatty acid, short

: medium : long chain fatty acids and Linoleic acid content similar to human milk. In contrast to cow

milk, fat absorption from the infant formulas with vegetable oil mixture is similar to that of human milk.

6. Significance of carbohydrate profile

Although lactose is present in both human and bovine milks, the variations in its concentration as well as

higher incidence of mono- and oligosaccharides, imparts a superior status to human milk in infant

nutrition. Higher carbohydrate level in human milk plays a significant role in infant nutrition. It is well

documented that lactose is assimilated from the digestive tract more slowly than other carbohydrates. The

change of lactose into glucose and galactose in feeding human and bovine milk proceeds so slowly that

part of lactose remains intact almost up to the last section of digestive tract. The production of lactic acid

in the large intestine under the influence of bifidus microflora, makes the intestinal environment acidic

which is conducive to the growth of lactose fermenting rather than putrefactive organisms, thus

decreasing the likelihood of infection. Moreover, the extra lactose present in breast milk is helpful in the

synthesis of certain vitamins. Lactose also increases the absorption of calcium and iron.

There exist differences in the composition of mono- and oligosaccharides (galactose, glucose, fucose, N-

acetyl glucosamine, N-acetyl lactosamine etc.) of the carbohydrates in human and bovine milks.

Galactose plays an important role in the synthesis of galactosides and cerebrosides for myelin formation

and for the synthesis of collagen. Amongst the oligosaccharides, N-acetylglucosamine and N-acetyl

lactosamine possess bifidus-stimulating activity. These substances are present in substantial amounts in

human milk, about 40 times more than the amount found in bovine milk. In contrast to those in cow milk,

the glucides of human milk are only partly broken down and absorbed in infant, the remainder reaching

the lower gut and utilized by the bifidus microflora. The beneficial effects of bifidus flora in the intestinal

tract of infants include increased absorption of certain nutritionally important minerals like calcium,

phosphorus and iron, inhibition of enteropathogenic organisms, synthesis of ‘B’ group of vitamins and

detoxification influence in chronic liver diseases as reported by several workers (Gurr, 1981; Pahwa,

1982).

7. Modification of carbohydrates profile

Having adjusted protein and fat requirements, caloric density of the formulation was adjusted through the

incorporation of carbohydrates. After accounting for the energy available from protein and fat, the

carbohydrates requirement was calculated to be 61.10%. The bovine milk used in the formulation (mainly

as a source of proteins) accounts for only 18.9% of lactose. Balance amount for the total carbohydrate is

derived from the exogenous sources, namely maltodextrin and sucrose. The relative ratios of

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maltodextrins and sucrose control the level of sweetness in IF. It appears that at 4.55% level, sucrose

masks adequately extraneous flavour in LIF that may originate from vegetable oils used for formulation.

Thus maltodextrin and sucrose in the ratio of 37.7:4.5 may be recommended. The total carbohydrates

content of 61.6 %(W/W) would contribute 51% of the total calories available from the feed. In normal

human milk, lactose contributes about 41% of the total calories. The liquid feeds for infants must have

neutral taste to permit self regulation of feed intake. Thus maltodextrin and sucrose would be suitable

source of energy in the dietary management of lactose intolerant infants.

8. Significance of mineral profile

Bovine milk possesses significantly higher amount of ash and different mineral concentrations compared

to human milk, evoking different physiological response amongst infants. The renal solute load of cow

milk is much greater than that of human milk, thus creating an excessive load on the kidneys, which are

not fully developed yet during first three months of infancy. The higher renal solute load created in

bovine milk is due to the metabolic breakdown of products of high protein content, notably urea. In

addition, the plasma osmolality of breast milk-fed infants is also lower and approximately equals the

physiological level of plasma. The quantitative interrelationships among the various minerals in the

infant’s diet play a more significant role nutritionally than absolute amounts of a particular mineral.

Imbalances due to the calcium:phosphorus and sodium:potassium ratios often lead to physiological

distress that are specially stressful during the first three months of infancy.

8.1 Calcium:phosphorus Ratio

The concentration of calcium and phosphorus and their ratio in human milk lead to a superior utilization

of calcium. In breast-fed infants, calcium absorption amounts to 75 per cent of intake as against mere 20

per cent in infants given bovine milk. Higher calcium content of cow milk is associated with a much

greater incidence of neonatal hypocalcaemia accompanied by convulsions. The level of phosphorus

influences the calcium mal-absorption. Higher concentration of phosphorus in bovine milk and hence,

lower Ca:P ratio leads to increased phosphorus absorption and concomitantly diminished uptake of

calcium. The serum electrolyte estimations in neonatal hypocalcaemia shows both low calcium and high

phosphorus levels. Retention of phosphorus is 90 per cent or higher from human milk and between 25 and

37 per cent from bovine milk. The adequacy of vitamin D is a critical factor in deciding whether or not

the ratio is critical and not the Ca:P ratio.

8.2 Sodium:potassium Ratio

The sodium level in bovine milk is 3 to 4 times higher than in human milk. Excess sodium content of

bovine milk was, therefore, considered to cause hypernatraemic dehydration of bottle-fed infants, which

sometimes leads to convulsion and subsequent mental retardation. Severe hypernatraemia has a toxic

effect of sodium poisoning which could lead to irreversible brain damage and perhaps even mortality. The

hypertension that develops as a result of higher sodium content of bovine may be lowered by altering the

sodium:potassium ratio with higher levels of potassium.

8.3 Iron Status

Although the iron contents of breast and bovine milks are comparable, iron deficiency anaemia is rare in

breast-fed infants but more common in those fed bovine milk. This difference is attributed to the higher

bioavailability of iron from human milk, an average of 49 per cent compared to mere 10 per rcent from

cow milk. This is possibly due to the presence of lactoferrin in human milk (Gurr, 1981). Iron is

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distributed between high and low molecular weight fractions in human milk. The greater bioavailability is

related to the preponderance of the low molecular weight forms in human milk.

9. Considerations for formulation of infant formule

Infant formulae tend to vary in composition, but within fairly narrow and quite precise limits. In general,

as a complete substitute for human milk, formula should provide protein (of appropriate biological

quality) at 7 to 16 per cent of calories, fat at 30 to 54 percent of calories, linoleic acid at 2 to 3 per cent of

calories, and the remaining calories from carbohydrate sources. For best fat absorption, it is desirable to

have available a portion of monoglycerides with palmitic acid in the 2 position. Both cow’s and goat’s

milk fat represent sources of naturally occurring triglycerides with this fatty acid configuration. Vegetable

fats have little or no such chemical entities. Nonetheless, a reasonable likeness of human milk fat is

simulated through the judicious blending of various vegetable and milk fats. Possibly corn oil is

somewhat more acceptable because of its relatively high level of oleic acid. A complete replacement of

milk fat is possible, or blends of milk fat and vegetable fat possibly serve even better. The state of

scientific knowledge does not permit evaluation of the significance of cholesterol. Because many

contaminants or pollutants of food are soluble in fat, specially refined fats and oils provide better control.

To prevent conversion of cis to trans fatty acids, and loss thereby of essential fatty acids, low (or ultra

high) temperature treatment must be used throughout processing. In addition, evidence exists to suggest a

beneficial effect in fat absorption from presence of the amino acid taurine and the betaine carnitine.

Human milk contains 26.6 µmoles/100 ml of the former and 59 nmol/ml of the latter. Cow (and beef)

formulations are assumed to be equivalent or higher in content of carnitine, but soy formulas, lacking

supplementation, could be considered deficient. In human milk, the ratio of fat to protein is 2 (or more):1.

This balance in formulas is likewise preferably kept within those limits.

Protein in milk-based formulas is best divided between whey proteins and casein in a 60:40 ratio. Of the

major whey proteins in cow’s milk, -lactalbumin more nearly mirrors the whey proteins of mother’s

milk. Absence of the other major whey protein of cow’s milk (i.e., -lactoglobulin) eliminates a possibly

significant allergen. Given 0.9 percent -lactalbumin and 0.6 per cent casein, a reasonable likeness of the

protein profile of mother’s milk is produced. The ideal protein composition of formula would be 1.0 to

1.2 g protein/100 ml, and made consistent with human milk in the levels of taurine and cystine. Moreover,

certain glucosamines (of nonprotein nitrogen) may serve to stimulate growth of B. bifidum. For this

purpose, N-substituted D-glucosamines have been suggested, and also the co-enzyme A precursor,

pantetheine phosphate. In addition, lactulose (4-0-d-galactopyranosyl-d-fructose), a ketose derivative of

lactose, has been shown to enhance growth of the organism. This is true even though the compound is not

found to occur in raw (unheated) milk of either humans or cows. The formula in which the bifidus-

stimulating phenomenon was originally noted consisted of 1.2 g lactulose/70 kcal, with lactose content of

the diet held at 2.5 times the protein level. Work by different investigators put the desired lactulose

content at 1.2 to 1.5 percent of the diet. A heat-sterilized liquid infant formula causes conversion of

between 1 and 5 percent of total lactose to lactulose.

Caloric density of infant formulas of 670 kcal/liter appears nearly optimal for normal full-term infants.

The formulation should provide a calcium-phosphorus ratio of neither less than 1.1:1.0 nor more than 2:1.

The optimal ratio is near 1.5:1, at least through most of the first year of life. By 1 year of age, the

appropriate ratio is more nearly 1:1. Calcium should be of a chemical form that is biologically available

and should be present to a minimum of 50 mg/100 kcal. Minimum phosphorus level is 25 mg/100 kcal.

Minimum and maximum amounts of sodium, potassium and chloride must also be observed. These levels

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are met within the ranges 6 to 17, 14 to 34 and 11 to 29 milliequivalents (mEq), respectively, in a formula

providing 670 kcal/liter. Major sources of protein for infant formulas include milk, whey, and soy. In the

latter case, highly refined soy protein (soy isolate) is used in most instances. Nonetheless, various

methods of purifying soy protein create significant differences in the composition of associated

ingredients, particularly mineral components. All soy protein supplies should be carefully monitored, and

the protein obtained from different suppliers should not be assumed to be identical. Chemical analysis

will generally serve most monitoring and/or quality control functions. New protein sources (nonmilk,

nonsoy) should undergo extensive metabolic and clinical studies prior to use.

Carbohydrate sources include lactose (or milk and whey products that contain lactose), sucrose, corn

syrup solids (a source of glucose), and starch. Actually, modified starch is used not so much as source of

carbohydrate as a stabilizer. Although there is no reason to believe that starch poses a digestive problem

to infants, neither have studies been carried out to assess its relative digestibility. However, generally

more formulas are prepared without than with it.

Codex standards for infant food has approved various kinds and amounts of food additives such as

thickening agents, emulsifiers, antioxidants, and compounds for adjusting pH. Most of these agents have

GRAS status in the United States. Similarly, these standards also recommend the vitamin additives for

use in infant formula. In some cases, several different chemical forms of the vitamin or provitamin have

approval for use. Processing requirements, availability, and/or stability in the specific food system will

dictate which form(s) will serve best.

A large number of mineral compounds are approved and made mandatory for incorporation in formulated

infant foods . Suitability of any given mineral additive depends on composition and moisture level of the

food product. Furthermore, each food imposes its own requirements for flavor and/or textural stability.

Oxidative rancidity is an ever-present problem in iron and / or copper-fortified foods containing

unsaturated fats. Gelation is a potential problem in concentrated liquid infant formulas. The mineral

composition of dairy-based infant formula involves certain unique technological challenges. The content

of calcium in cow’s milk varies somewhat on a seasonal and regional basis. The level of certain trace

minerals – among them zinc, iodine, copper, sodium, manganese, and cobalt – vary to more or less an

extent by the amount present in feed. The iron content of cow’s milk seems uninfluenced by the intake of

feed. Some amount of iron, copper, molybdenum, manganese, and magnesium is associated with the fat

globule membrane and will be lost upon separation and exclusion of milk fat as an ingredient in formula.

Perhaps a third of both calcium and phosphorus is associated with casein and remains with casein upon

rennet coagulation. Most magnesium (about 75%) and essentially all zinc are complexed with casein. As

they exist in milk, some amount of some mineral components will be lost to the permeate in milk or whey

treated by ultrafiltration, reverse osmosis, electrodialysis, and other methods of concentrating protein. Of

course, a certain amount of mineral depletion is necessary to provide a formula of appropriate osmolar

strength. In the process, however, some amount of some trace minerals will be lost, for better or worse.

When trace minerals are added to formula, sulphate salts are commonly used. Because of the potential to

cause methemoglobinemia, nitrate salts are usually not added to formula. Some small (safe) amount may

occur in formula made up of vegetable products. Nitrates also occur, and occasionally at high levels, in

some water supplies. Copper is another potentially toxic component of water. Soy products not

adequately processed may contain goitrogens, necessitating the presence in formula of added iodine as a

defense against goiter. Minerals commonly added to formulas include calcium, phosphorus, magnesium,

iron, copper, iodine, zinc, potassium, sodium, manganese, and chlorine (as chloride).

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10. Formulations with bioprotective attributes

Despite the impressive advancement in developing technologies to upgrade the nutritional characteristics

of infant formulae similar to human milk, susceptibility of bottle fed infants to enteropathogenic

microorganism still contributes the largest health risk. The protective effect of human milk for infants, in

spite of lack of transfer of antibodies from gut into the circulatory system, points to a direct protective

role of human milk in the ecological system of the intestinal tract (Table 7). During early infancy, risk for

mortality and morbidity from common pathogens like coliform, salmonella, and shigella are very high. A

number of innate factors in human milk influence, the intestinal microflora of the neonate that ultimately

inhibit these enteropathogenic bacteria. These humoral protective factors are either derived from blood

serum, such as immunoglobulins or synthesized in mother’s memory glands like lactoferrin, lysozyme,

lactoperoxidase etc. Little attention has been paid for incorporating these bioprotective factors that

assume special significance in bottle-feeding practice for providing humoral protection among infants.

The following text describes possible actions and usage of these humoral protective factors for infant

formulae.

10. 1 Lactoferrin

Under physiological conditions, lactoferrin is capable of chelating iron, thus depriving bacteria of the iron

required for metabolism, resulting in their inhibition. Digestion of lactoferrin with certain proteolytic

enzymes does not significantly alter its iron binding property, suggesting that protein was quite capable of

exerting antibacterial activity against the E. coli and other enteropathogens in the digestive tract of the

infant. Lactoferrin also plays a useful role in the absorption of iron from the feed. Anti-carcinogenic

properties of lactoferrins, especially as a possible cure for leukemia have also been demonstrated.

Lactoferroxin, an opioid peptide with six amino acid residues originating from hydrolysis of lactoferrin

has been shown to be physiologically significant, although its usefulness in human health needs to be

further elucidated.

10.2 Lactoperoxidase

The lactoperoxidase system (LP) consists of three components – lactoperoxidase enzyme, hydrogen

peroxide and thiocyanate, an oxidizable substance. In the newborn infant saliva is quite rich in

lactoperoxidase activity and the enzyme is known to be resistant to low pH and proteolysis. The saliva

that enters the stomach and rich in lactoperoxidase is expected to reach the intestinal tract in an active

form for activation of LP system. The lactic acid bacteria metabolically generate hydrogen peroxide.

Thiocyanate, the third factor of LP system occurs naturally in animal tissues and secretions. Thus, the

system oxidizes the chemically inert SCN into OSCN. Exposure of bacterial cell to this oxidative product

results in leakage of potassium and amino acids from the cell and inhibition of carbohydrate uptake and

synthesis of DNA and RNA leading to cell lysis specially of the gram negative organisms. Bovine milk

that contains 30 µg/ml lactoperoxidase and 2 to 8 ppm thiocyanate could be an important source for the

supply of SCN required for activation of LP system. Controlled application of the system in infant

formula may be very useful for protecting neonatal health.

10.3 Lysozyme

In view of the antibacterial effect and possibly general immune system of neonates, there has been a

considerable interest in developing food applications of lysozyme. The incorporation of lysozyme in

infant formula could be useful in view of its influence under the ecological system of the intestinal tract.

The concentration of lysozyme in human milk is 300 times greater than bovine milk. The biological

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significance of lysozyme lies with its possible protective effect against gram-negative potential

pathogens. Although lysozyme is isolated from egg bovine lysozyme has greater lytic activity. It is stable

at low pH and resists gastric juices under in vivo situations. Lysozyme containing formulations tend to

enhance bifidobacterium population in the intestinal tract.

10.4 Immunoglobulins

It is well established that feeding of colostrum and milk increases the resistance of both human and

neonates against enteric infections. The secretory Ig-A is a major antibody in human milk. Ig-G and Ig-

M are known to involve to a greater extent in providing both the bacteriostatic and bactericidal effects.

These immunoglobulins if incorporated in an active form the infant formula, will complement the

bacteriostatic components present in the formula. Membrane driven processes have been standardised in

our lab for the preparation of immunoglobulins on pilot scale suitable for incorporation in the infant

formulas. In this manner, it may be possible to augment the future infant formulations with the unique

properties of human milk with respect to containment of humoral protective factors apart from the

nutritional properties to fulfil the requirements of an human infant in the absence of availability of

sufficient quantities of mothers milk or in case where mothers are unable to feed their infants due to

socioeconomic reasons or fear of transmittable diseases.

11. Pro- and/or prebiotics in infant feeding

Many of the probiotic organisms are now being proposed as remedies for a number of gastrointestinal

conditions such as infectious and non-infectious diarrhoea, inflammatory bowel disease and food

allergies. There are also reports suggesting their hypocholesterolemic and anti-cancer properties besides

improving lactose tolerance. The predominance of several of the probiotic organisms as natural

inhabitants of the gastrointestinal tract of breast-fed infants has led to detailed studies on their role on the

health and well being of infants, their association with the components of breast milk and their probable

incorporation in infant feeds. The industry is investigating means to make the gut ecology of formula-fed

infants similar to those of breast-fed infants.

The bifidus activity of milk of Indian women were studied by Pahwa and Mathur (1982) who found that

the activity was approximately 30 and 50 times as much as those of buffalo and cow milk respectively.

Cell concentrate of B. bifidum was added to an infant formula mix (BCF) and fed to 24 infants born in a

hospital in New Delhi. The infants were all of the same range of body weight. They were divided into

three groups of eight each and fed on breast milk, BCF and a commercial formula (CCF). The

physiological response of the BCF-fed infants was very close to the breast-fed children. Both the groups

had bifidobacteria implanted in their intestines. Animal bioassays involving the two formulae revealed

that the calcium and iron absorption was greater in the BCF-fed rats than in the CC-fed rats.

Langhendries et al. (1995) reported that the addition of lactobacilli to infant formula resulted in a gut

ecology similar to those of breast-fed infants. It has been demonstrated in independent studies conducted

at the Mount Washington Paediatric Hospital, Maryland (USA) and the Pakkred Babies Home, Bangkok

(Thailand) that supplementation of infant formula with B. bifidum and S. thermophilus can reduce the

incidence of acute diarrhoea and rotavirus shedding in infants admitted to hospital (Saavedra et al., 1994;

Phuapradit et al., 1999). In the US-based experiments, out of the 55 subjects evaluated for a total of 4447

patient days for 17 months, only 7% of those receiving the probiotic formula, but 31% of the control

group developed diarrhoea. Ten per cent of the children on the probiotic formula recorded rotavirus

shedding as against 29% in the control group.

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Haschke et al. (1998) reported that feeding follow-up formulas and growing-up milks containing

Bifidobacterium strain Bb12 (107-10

8/g formula powder) resulted in rotavirus infection, fewer periods

with hard bowel movements and a lower incidence of diaper rash. They also found that survival of the

organism during intestinal transit was confirmed when Bb 12 was given with milk-based formulas and

was not satisfactory with soy-based formulas. The use of antibiotics, particularly those with broad-

spectrum action, destroys the intestinal flora, causing diarrhoea. The incidence of antibiotic-associated

diarrhoea in children receiving broad-spectrum antibiotics ranges from 20% to 40% (Haschke et al.,

2001). Several studies mention the possibilities of employing probiotics as alternatives to antibiotics to

help the gastrointestinal tract to resist such aggression. Vanderhoof et al. conducted a trial in children

receiving antibiotics, wherein they were given Lactobacillus casei subsp. rhamnosus (Lactobacillus GG)

or placebo. The percentages of children with loose stools and diarrhoea were significantly lower in the

group receiving the probiotic organisms. Incorporation of oligosaccharides and other prebiotics in

humanised infant formulae may boost the survival of probiotic organisms in the gut of formula-fed

infants. The proportions, as well as the absolute numbers of bifidobacteria in the intestinal contents of

infants increase when they are fed formula supplemented with prebiotics (Boehm et al., 2001; Knol et al.

2001). The Scientific Committees on Food (SCF) of the European Commission has confirmed the safety

of oligofructose for use as an ingredient in baby food (Franck, 2002) after examining experimental

evidences. This prebiotic along with galacto-oligofructose can be used in follow-up foods in a

concentration of up to 0.8 g/dl in the product ready for consumption and can be given to infants aged

between 0 and 6 months.

In a double blind, controlled, randomised study, 123 healthy children (4-24 months) attending a day-care

centre were given a regular cereal or a cereal supplemented with 0.55 g/15 g of fructo-oligosaccharides

(Saavedra et al., 1999; Tchernis et al., 1999). The average daily intake of the fructo-oligosaccharide was

1.2 g. There was a reduction in cold symptoms, runny nose, antibiotic use and day-care absenteeism.

Although there was no significance difference in the frequency of reported diarrhoea, the incidence of

diarrhoea-associated symptoms (fever, medical attention required, vomiting, discomfort and

regurgitation) was reduced. Another study conducted simultaneously in Brazil, Mexico, Spain and

Portugal among 626 mild to moderately malnourished children (1-6 years) evaluated the incidence and

duration of sickness when they were given a nutritional supplement with or without synbiotics (L.

acidophilus, B. infantis and an oligosaccharide (Raftilose P95). The catch-up growth increased and the

number of sick days decreased in both the groups, the decrease being more pronounced in younger

children (1-2 years). The number of sick days for older children (3-5 years) as well as the constipation

days was lower in the symbiotic-fed group (Fisburg et al., 2000).

12. Transgenic technology for humanisation of bovine milk

While until recently, emphasis in the field of dairy-based biotechnology has been on breeding large

animals to produce more milk, the attention is now tuned to adding more value to milk and studying its

health implications. Milk composition can be altered by nutritional management or through the

exploitation of naturally occurring genetic variation among cattle. By combining the two approaches of

nutritional and genetic interventions, researchers are now hoping to develop 'designer milk' tailored to

consumer preferences or rich in specific milk components that have implications in health as well as

processing. Although few would debate over the suitability of mother’s milk as the best food for the

sucking infants, due to varying reasons, a number of infants are fed formulas based on bovine milk. The

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composition of these formulas could be greatly improved to suit the needs of the infant by incorporation

of ingredients that resemble those of human milk in bovine milk, thereby 'humanising' the latter.

Lactoferrin, the iron-binding protein has antimicrobial properties and may also mediate some effects of

inflammation and have a role in regulating various components of the immune system. Its level in human

milk is about one g/l (in human colostrum about seven g/l). As the levels of lactoferrin in cow milk is

only about one tenth of that in human milk, this has caught the attention of those involved in designing

human milk replacement formulas. The human lactoferrin (hLF) gene has already been expressed at low

levels (0.1 to 36 mg/ml) in the milk of transgenic mice and a transgenic bull that carries the gene for hLF

has been produced. Human milk contains 0.4 g/l of lysozyme (LZ), an enzyme that provides antibacterial

activity in human milk. Active human lysozyme (hLZ) has been produced in the milk of transgenic mice

at the concentrations of 0.78 g/l (Maga and Anderson, 1995). On the processing front, the expression of

LZ in milk results in the reduction of rennet clotting time and greater gel strength in the clot. A double

transgenic cow that co-expresses both hLF and hLZ in milk may also reduce the incidence of intra-

mammary infection or mastitis.

Yet another application of transgenic technology could be to produce the human lipase, which is

stimulated by bile salt in the milk of bovines. The lipase thus produced could be used as a constituent of

formulas to increase the digestibility of lipids especially in premature infants who have low lactose

activity (Lonnerdal, 1996).

Several children are allergic to cow’s milk, owing to the presence of -lg, which is not found in human

milk. Elimination of this protein by knocking out -lg gene from cow’s milk is unlikely to have any

detrimental effects, on either cow or human formula, and might actually overcome many of the major

allergy problems associated with cow’s milk.

Industrial interest has focused on the production of high value, low volume, therapeutic proteins in the

milk of domestic animals and in this context, several human proteins have already been expressed with

success. The major advantage of transgenic technology is that proteins can be produced at a very low

cost. Economic comparison of production costs of human tissue plasminogen activator (htPA) through

bacterial fermentation, mammalian cell culture and cow transgenic technology estimates the cost/g of

htPA to be 20000, 10000 and 10 US dollars respectively (Karatzas and Turner, 1997). Two proteins,

human antitrypsin and human antithrombin III have been purified from milk of transgenic ruminants.

Human antithrombin III, a plasma protein that helps prevent harmful blood clotting is also being tested.

13. Conclusion

During the past few decades, there has been a resurgence of interest in infant feeding and in the role of

human milk in particular. However, there is still a place for artificial milks, although the emphasis of

evidence suggests that breast-feeding is the ideal to be sought wherever possible. There is no doubt,

nevertheless, that there are circumstances which demand bottle-feeding. The need of the hour appears to

develop advanced infant foods with antimicrobial and protective components of milk such as lysozymes,

lactoperoxidases, lactoferrin, vitamin binding proteins. Such a formula should have abilities to alter

gastrointestinal microflora of infants and implant beneficial microorganisms removing pathogens. It

should contain concentrations of antimicrobials and proteins, which may offset losses in these biologicals

caused by pasteurization during manufacture of infant foods. Infant formulae with added prebiotics and

with or without probiotics are also gaining recognition. Such formulae help to improve the gut microflora

as well as nutrient absorption. ‘Humanising’ the bovine milk by the most sophisticated transgenic

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technologies suggest the scope of obtaining straight from the udder of the cow, milk that resembles

human milk in compositional and physiological attributes.


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Newer Packaging Technologies (Nanomaterial-based and Edible Packaging) for Dairyand Food Products

Narender Raju Panjagari, Mukesh Kumar Bishnoi, Pavan Kanade


1. IntroductionFood packaging is the largest user of plastics (~40%). In India, as per the Central Pollution ControlBoard, approximately 15,300 tonnes of plastic waste is generated per day (The Hindu, 2011). Thevolume of plastics discarded annually creates a substantial waste, which is causing a great threat toenvironment. Consequently, the approach of making materials from biodegradable materials that canbe disposed of through composting or recycling got momentum. As a result a number ofbiodegradable materials such as naturally occurring polymeric materials, polymers made bypolymerization of organic molecules and biodegradable polymers from petrochemicals have beeninvestigated for use as alternative to plastics. Biopolymers from agricultural food stocks, foodprocessing waste and other resources has the ability upon blending and/or processing to result inbiopolymeric packaging material called as biodegradable polymers or bioplastics (Davis and Song,2006). Unfortunately, so far the use of biodegradable films for food packaging has been stronglylimited because of the poor barrier properties and weak mechanical properties shown by naturalpolymers. Also, biopolymers cannot meet the requirements of a cost-effective film with mechanicaland barrier properties matching those of plastics (Kumar et al., 2011). The most frequently usedstrategies to enhance barrier properties are blending of polymers, coating with high barrier materialsand the use of multilayered films containing a high barrier film. Recently, a new class of materialsrepresented by bio-nanocomposites has proven to be promising option in improving the mechanical,barrier and thermal properties of these biopolymer-based packaging materials. Polymers can also beadded with suitable fillers to form composites for enhanced barrier properties.

2. Polymer nanocompositesPolymer nanocomposites are created by dispersing an inert, nanoscale filler throughout a polymericmatrix in which the filler has at least one dimension smaller than 100 nm. Filler materials could beeither flakes, fibers, whiskers or nanoparticles. The mechanical, thermal and barrier of nano-composites are often remarkably different from those of non-reinforced biopolymer-based materials.Addition of relatively low levels of nanoparticles (less than 5%) have been shown to substantiallyimprove the properties of finished plastic, increasing the deformability and strength, and reducing theelectrical conductivity and gas permeability. Further, polymers when incorporated with certainnanoparticles have the ability to interact with the food/environment and package and conferantimicrobial properties and while some continuously monitor and detect changes in the packageenvironment. Such nanocomposites have applications in active and smart packaging systems. Fillermaterials which have been used widely used by researchers include clay and silicate nanoplatelets,silica (SiO2) nanoparticles, carbon nanotubes, grapheme, starch nanocrystals, cellulose-basednanofibers or nanowhiskers, chitin or chitosan nanoparticles, silver nanoparticles (AgNO3), titaniumnanoparticles (TiO2), magnesium nanoparticles (MgO), copper nanoparticles (CuO), zinc (ZnO), etc.

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3. Advantages of nanocomposites in food packaging3.1 Enhancement of Barrier Properties

Critical issues in food packaging are that of permeability and migration. None of the packagingmaterials used for food applications is completely impermeable to atmospheric gases and watervapour. In general, permeability of a polymer to oxygen or moisture is dependent on a large numberof interrelated factors including polarity and structural features of polymeric side chains, hydrogenbonding characteristics, molecular weight and polydispersity, degree of branching or cross-linking,processing methodology, method of synthesis and degree of crystallinity (Duncan, 2011). Variousinorganic nanoparticles have been recognized as possible additives to enhance the polymerperformance. Among all, as of now the layered inorganic solids like clay have attracted attention bypackaging industry. This is not only due to their availability and low cost but also due to theirsignificant enhancements and relatively simple processability. The characteristic feature of clayminerals is the stacked arrangement of negatively charged silicate layers with a thickness of about 1nm and lateral extensions of about 100 m (Fig.1). Montmorillonite (MMT), hectorite, saponite andkaolinite are the commonly used layered silicates (Ray and Okamoto, 2003; Duncan, 2011). MMTcomprises of highly anisotropic platelets separated by thin layers of water. Each platelet contains alayer of aluminum or magnesium hydroxide octahedral sandwiched between two layers of siliconoxide tetrahedral. The faces of each platelet have a net negative charge, which causes the interstitialwater layer (known as gallery) to attract cations (Ca2+, Mg2+, Na+, etc.) (Fig.1). The structuralcharacteristics contribute to MMT’s excellent utility as a filler material for polymer nanocomposites,typically giving rise to impressive increase in polymer strength and barrier properties. Three types ofcomposites are created when clay is dispersed in polymer (Fig.2). Clay nanoplatelets tend toagglomerate when dispersed in polymer leading to tactoid structure (microcomposites) with reducedaspect ratios and reduced barrier efficiencies. Hence, the key requirement for the generation ofnanoclay composites is separation of the ultrafine layers, a process known as exfoliation. As the claynanoparticles are essentially impermeable crystals, gas molecules must diffuse around them (tortuouspath) rather taking a straight-line path that lies perpendicular to the film surface (Fig.3). In general,higher the degree of exfoliation of the nanoclay the higher the improvement in barrier and mechanicalproperties of the film.

When biopolymers are combined with nanoparticles, the resulting bio-nanocomposites exhibitsignificant improvements in the mechanical properties, dimensional stability and solvent or gasresistance with respect to the pristine polymer due to high aspect ratio and high surface area ofnanoparticles. The application of nanocomposites promises to expand the use of edible andbiodegradable films produced from agro-processing products and byproducts. Starch-clay is the mostoften cited biodegradable nanocomposites investigated for various applications including foodpackaging (Park et al., 2002; Avella et al., 2005; Chen and Evans, 2005; Yoon and Deng, 2006;Cyras, et al., 2008; Tang et al., 2008). Significant improvements in mechanical properties werereported with the addition of montmorillonite (MMT) clay.

Nanocomposites of amorphous polylactic acid (PLA) and chemically modified kaolinite were studiedby Cabedo et al. (2006). The combination of PLA and montmorillonite layered silicate may result in ananocomposite with barrier properties suitable for food packaging applications (Sinclair, 1996;Thellen et al., 2005). Yu et al. (2006) reported that through the use of high-powered ultrasonics, soyprotein/clay nanocomposites could be produced, which exhibited improvement in modulus. Kumar etal. (2010) studied the effects of the pH of film forming solution, MMT content, and extrusionprocessing parameters on the structure and properties of soy protein isolate-MMT bio-nanocompositefilms. Tunc et al. (2007) investigated that wheat gluten/MMT nanocomposite films could be prepared

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by casting. Voon et. al. (2012) studied the effect of addition of halloysite nanoclay and SiO2

nanoparticles on barrier and mechanical properties of bovine gelatin films. Biodegradable titaniumdioxide (TiO2)/whey protein isolate (WPI) blend films were made by casting denatured WPI filmsolutions incorporated with TiO2 nanoparticles (Zhou et al., 2009). Similar studies regardinginteractions on ZnO-whey protein nanocomposites have been reported (Shi et al., 2008). The reportedimprovements in barrier properties of biopolymer-based nanocomposites are given in Table 1.

Fig. 1. Structure of Montmorillonite(Source: Duncan, 2011)

Fig. 2. Polymer-clay composite morphologies(a) Tactoid; (b) Intercalated; (c) Exfoliated

(Source: Duncan, 2011)

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Fig.3 Illustration of tortuous pathway created by incorporation of exfoliated clay nanoplateletsinto a polymer matrix film

Table 1. Barrier Properties of Some Bionanocomposite Films

Film typeWater vapourpermeability@

(g mm hr-1 m-2)

Oxygenpermeability*

(m3 mm-2 s Pa)Reference

Corn Starch + 0% MMT 1.61

Tang et al. (2008)Corn Starch + 3% MMT 1.42Corn Starch + 6% MMT 1.06Corn Starch + 9% MMT 0.77Potato Starch + 0% MMT 1.81

Tang et al. (2008)Potato Starch + 3% MMT 1.22Potato Starch + 6% MMT 0.98Potato Starch + 9% MMT 0.84Soy protein isolate (SPI) 3.1a

Rhim et al. (2005)SPI + MMT 2.9a

SPI+ Bentonite 1.5a

SPI + Talc 2.3a

SPI + Zeolite 2.0a

Poly lactic acid (PLA)

Lagaron et al. (2005)PLA 11 x 10-19

PLA + 4% MMT 10 x 10-19

PLA + 4% Kaolinite 6 x 10-19

* 21C, 40% RH; @ 25C, 75%; a: ng.m.m-2.s.Pa3.2 Enhancement of Antimicrobial Properties

In addition to proving as a passive barrier, packaging can contribute to the control of microbialgrowth in food products, which cause spoilage or in case of pathogens, diseases and illness.Antimicrobial (nisin, silver oxide, zinc oxide, magnesium oxide) nanoparticle and antioxidantcoatings in the matrix of the packaging material can reduce the development of bacteria on or near thefood product, inhibiting the microbial growth on non-sterilized foods, maintain sterility of pasteurizedfoods by preventing post-contamination and prevent oxidative changes in food thereby improving thequality. Most antimicrobial activities of nanocomposites have centered on nanoparticles of silver andzinc oxide. Silver has a long history of being used as an antimicrobial agent in food and beverage

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storage applications. Silver nanoparticles have been found to be potent agents against numerousspecies of bacteria including Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Vibriocholera, Pseudomonas aeruginosa, Shigella flexneri, Bacillus subtilis, Salmonella enterica,Micrococcus luteus, Listeria monocytogenes and Klebsiella pneumonia (Duncan, 2011). Cellulose is agood carrier of silver nanoparticles. Fernandez et al. (2010) developed cellulose-silver nanoparticlehybrid materials (cellulose-based silver loaded absorbent pads) and demonstrated that when fresh cut“Piel de Sapo” melon was placed over the pads, the pads released silver ions after the melon juiceimpregnated the pad and controlled the spoilage-related bacteria.

Titanium dioxide (TiO2) coated packaging film has shown to considerably reduce E. colicontamination on food surfaces. TiO2 is a photocatalytic disinfecting material for surface coatings(Chawengkijwanich and Hayata, 2008). Page et al. (2007) reported that combining titanium dioxidewith silver has been shown to improve the disinfection process through improving photocatalysis.Zinc oxide also exhibits antibacterial activity and its antimicrobial property increases with decreasingparticle size (Yammamoto, 2001). Recently, a starch/ZnO-carboxymethylcellulose sodiumnanocomposite was prepared using ZnO nanoparticles stabilized by carboxymethylcellulose sodium(CMC) as the filler in glycerol plasticized-pea starch (Yu et al., 2009). Busolo et al. (2010) developeda novel silver-based nanoclay as an antimicrobial in PLA food packaging coatings and reported thatsuch compounds when incorporated at either 1%, 5% or 10% exhibited strong biocidal activity(99.9% CFU reduction) against Salmonella spp. when the sample taken was 1.5g.

3.3 Smart Packaging

A package that continuously monitors the internal environment and responds or communicates thechanges to external environment and/ or consumer, beyond performing the basic functions, is calledas a smart package and the technique as smart packaging. The unique chemical and electro-opticalproperties of nanoscale particles enable them to be part of a smart package. Such nanoparticles can beused to detect the presence of gases, aromas, chemical contaminants and pathogens. Mills (2005)reported development of a promising photoactivated indicator ink for in-package oxygen detectionbased on nanosized TiO2 or SnO2 particles and a redox-active dye (methylene blue). This detectorgradually changes colour in response to even minute changes in oxygen. Luechinger et al. (2007)reported development of porous metal films for optical humidity sensing from copper nanoparticlesprotected by a 2-3 nm carbon coating. The film is reported to have exceptional sensitivity with opticalshifts in the visible light range of up to 50 nm for a change of 1% in relative humidity. A non-invasivemethod of measuring CO2 content in modified atmosphere packages has been devised by Von-Bultzingslowen et al. (2002). It was reported to be based upon lifetime analysis of luminescent dyesstandardized by fluorophore-encapsulated polymer nanobeads. Many such applications ofnanoparticles in smart packaging are being reported.

4. Edible packagingEdible packaging consists of edible films, sheets, coating and pouches. Edible films and sheets arestand-alone structures that are preformed separately from the food and then placed on or between foodcomponents or sealed into edible pouches, whereas edible coatings are then placed on or betweenfood components or sealed into edible pouches. Whereas edible coatings are thin layers of ediblematerials formed directly onto the surface of food (Janjarasskul and Krochta, 2010). The edible filmscomprise of thickness of <254 µm, whereas edible sheets include upto thickness of 254 µm. Theedible packaging materials offer multifunction, like offer a selective barrier barrier to retard themigration of moisture, gas transport, oil and fat migration and solute transport; improve themechanical handling properties of foods; improve the mechanical integrity or handling characteristicsof the food; retain volatile flavour compounds and carry food additives such as antioxidants and

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antimicrobials (Falguera et al., 2011). The edible coatings and films are not meant to, nor could theyever, replace non-edible, petrochemical based packaging materials for prolonged storage of foods.The utility of edible films lies in their capacity to act as an adjunct for improving overall food quality,extending shelf life and improving economic efficiency of packaging materials.

Fig.1. Components of Edible Films and Coatings4.1 Biopolymers used for Edible Film making

The majority of edible films and coatings contain at least one component that is high molecularweight polymer, particularly if a self-supporting film is desired. Long-chain polymeric structures arerequired to yield film matrices with appropriate cohesive strength when deposited from a suitablesolvent. Increased structural cohesion generally results in reduced film flexibility, porosity andpermeability to gases, vapours and solutes. As polymer chain length and polarity increase, cohesion isenhanced. A uniform distribution of polar groups, along the polymer chain increases cohesion byincreasing the likelihood of inter chain hydrogen bonding and ionic interactions. A variety ofpolysaccharides, proteins and lipids derived from plants and animals have been utilized, either aloneor in mixtures, to produce edible films and coatings (Fig.1) and the materials of edible films includepolysaccharides (starch, cellulose, hemicellulose, chitosan, gums, etc.), proteins (milk proteins, soyproteins, cereal proteins, millet proteins, gelatin), lipids (natural waxes such as carnauba wax,candelilla wax, rice bran wax, bees wax and synthetic waxes such as paraffin and petroleum wax, aswell as mineral and vegetable oils, and edible resins such as shellac, terpene resin and wood resin)

Essential Components Optional ComponentsPolysaccharides

LipidsProteins Compositematerials

1. Starch2. Cellulose3. Hemicellulose4. Chitosan5. Gums1. Waxes2. Vegetable and mineral oils3. Edible Resins4. Mono, di and triglycerides

1. Collagen2. Milk protein3. Plant protein

Multicomponents1. Plasticizers2. Emulsifiers3. Antimicrobials4. Antioxidants5. Pigments

Edible Films or Coatings

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4.2 Edible Packaging Additives

A number of materials can be incorporated into edible films to enhance structural, mechanical andhandling properties or to provide active functions to the films. They include plasticizers, emulsifiers,antimicrobial compounds, antioxidants, colorants, etc. Among all, plasticizers have an important roleas they are added to improve film flexibility and durability. These include mono, di, oroligosaccharides such as glucose, fructose-glucose syrups and sucrose; polyols such as glycerol,sorbitol, lipids and derivatives such as phospholipids, fatty acids etc.

5. Methods to enhance the properties of edible filmsMany approaches have applied to improve the barrier properties of edible films by modifyingproperties of protein by chemical, enzymatic or physical method. The methods primarily focus onimproving the mechanical strength and moisture barrier properties.

5.1 Chemical method

Chemical treatments with acid, alkali or cross-linking agents (glyoxal, glutaradehyde andformaldehyde etc.) have been extensively used to improve the properties of films. Hydrolyzed proteinresults in grater greater solubility at high pH and high temperature. Denatured protein resultsformation of less flexible and transparent but more moisture resistant films (Guilbert, 1986).

5.2 Enzymatic treatments

The enzymatic treatments improve protein film functionality by modifying polymer network throughthe cross-linking of polymer chains. The enzyme transglutaminase is used widely as cross-linkingagents. Addition of covalent bonds by the use of transglutaminase increased the film’s integrity andheavy duty capacity as well as its capacity to stretch.

5.3 Combination with hydrophobic materials

Protein and polysaccharides based films are having good oxygen barrier properties but are poormoisture barrier due to their hydrophilic character. Combination of hydrophobic lipid with these filmscan improve their moisture barrier properties. High melting point lipids, such as beeswax or carnaubawax are generally used. A composite film made of a protein and a lipid can be divided into laminatesand emulsion.

5.4 Application of irradiation

To improve the functional properties of protein films, ionizing radiation have been tried. Gamma-irradiation affects proteins by causing conformational changes, oxidation of amino acids and ruptureof covalent bond and formation of protein free radicals. Cross-linking induced through gammairradiation was found to improve both barrier and mechanical properties of edible films and coatingsbased on protein. Gamma irradiation was observed to improve water vapour permeability, chemicalstability and resistance to microbial and enzymatic biodegradation (Ouattara et al., 2002).

5.5 Advantages of Edible Packaging Materials

The advantages of edible film over traditional petrochemical-based polymeric packaging materialshave been listed as follows (Gennadios, 2002):

i. They can be consumed with packaged product, leaving no residual packaging to be disposed of.

ii. Even if the films are not consumed, they are biodegradable and contribute to the reduction ofenvironmental pollution.

iii. The edible packaging materials can enhance the organoleptic properties of packaged foodsprovided that various food additives (flavourings, colourings, sweetners etc.) are incorporated intothem.

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iv. They can be carrier of nutrients like vitamins, minerals etc.

v. They can be used for individual packaging of small portions of food, particularly products thatcurrently are not individually packaged for practical reasons such as peas, beans, nuts andstrawberries.

vi. They can be applied inside heterogenous foods at the interfaces between different layers ofcomponents and tailored to prevent deteriorative inter-component moisture and solute migrationin foods such as pizzas, pies and candies.

vii. They can be very conveniently used for microencapsulation of food flavouring and leaveningagents to efficiently control their addition and release into the interior of foods.

viii.They can function as carriers for antimicrobial and antioxidants agents and be used at the surfaceof foods to control the diffusion rate of preservative substances from surface to the interior of thefood.

They could be used in multilayer food packaging materials together with inedible films, in which casethe edible films would be the inner layers in direct contact with the food.

6. Application of edible filmsi. Edible coatings and films can act as moisture barrier to prevent moisture loss from product and

maintain freshness of fruits and vegetables, frozen products etc.

ii. Edible films and coatings can act as gas barrier to delay fruit O2 uptake from environment andreduce ripening rate. Gas barrier properties can reduce rancidity in peanuts by reducing O2

concentration.

iii. Edible films can act as flavour carrier in addition to provide a protection to aroma loss.

iv. Application of antioxidants in edible films and coatings can reduce lipid oxidation in frozen meat,salmon etc.

v. Edible films or coatings containing antimicrobial compounds can reduce surface microbial loadand can enhance shelf life of cheese, fish, chicken, meat etc.

vi.

7. ConclusionsWith the worldwide growing scientific evidence of the potential benefits of nanotechnology in foodpackaging, there exists huge scope for us to get benefitted in India as such applications are at nascentstage. Biopolymers from agricultural food stocks, food processing waste and other resources could beexploited for developing nanocomposite films for packaging applications. Also, development ofsensor and antimicrobial based smart packaging materials could be developed. The use of edible filmsor coatings on various food products continues to expand. The many potential benefits of edible filmsand coatings as carriers of antimicrobial agents, flavours, antioxidants, colouring agents, vitamins,probiotics and nutraceuticals justify continued research in this arena of active packaging. However,the application of edible films and coatings in India is still at infancy. Data available in the regard ofapplication of edible films and coatings on dairy products especially indigenous dairy products arescanty. Research is necessary to increase understanding of film composition-structure-functionrelations and thus establish food applications. In addition, new legislations and standards in favour ofedible materials seem to be very helpful to spur market growth.

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8. Suggested ReadingsAvella M, De-Vlieger J J, Errico M E, Fischer S, Vacca P and Volpe M G (2005) Biodegradable starch/clay

nanocomposite films for food packaging applications. Food Chemistry, 93 467-474.

Azeredo H M C, Mattoso L H C, Wood D, Williams T G, Avena-Bustillos R J and McHugh T H (2009)Nanocomposite edible films from mango puree reinforced with cellulose nanofibers. Journal of FoodScience, 74 N31-N35.

Busolo M A, Fernandez P, Ocio M J and Lagaron J M (2010) Novel silver-based nanoclay as an antimicrobial inpolylactic acid food packaging coatings. Food Additives and Contaminants. 27 1617-1626.

Cabedo L, Feijoo J.L., Villanueva, M.P., Lagaron, J.M. and Gimenez, E. (2006). Optimization of biodegradablenanocomposites based on a PLA/PCL blends for food packaging applications. Macromol. Symp. Vol.233(1): 191-197.

Chawengkilwanich C. and Hayata, Y. (2008). Development of TiO2 powder-coated food packaging film and itsability to inactivate Escherichia coli in vitro and in actual tests. International Journal of Food Microbiology,Vol.123(3): 288-292.

Chen, B.Q. and Evans, J.R.G. (2005). Thermoplastic starch-clay nanocomposites and their characteristics.Carbohydrate Polymers, Vol. 61: 455-463.

Cyras, V.P., Manfredi, L.B., Ton-That, M.T. and Vazquez, A. (2008). Physical and mechanical properties ofthermoplastic starch/montmorillonite nanocomposite films. Carbohydrate Polymers, Vol.73(1): 55-63.

Das, K., Ray, D., Bandyopadhyay, N.R., Sahoo, S., Mohanty, A.K. and Misra, M. (2011). Physico-mechanicalproperties of the jute micro/nanofibril reinforced starch/polyvinyl alcohol biocomposite films. CompositesPart B:Engineering, Vol. 42(3): 376-381.

Davis, G. and Song, J.H. (2006). Biodegradable packaging based on raw materials from crops and their impacton waste management. Industrial Crops and Products, Vol.23: 147-161.

Duncan, T.V. (2011). Applications of nanotechnology in food packaging and food safety: barrier materials,antimicrobials and sensors. Journal of Colloid and Interface Science, Vol. 363:1-24.

Falguera, V., Qunitero, J.P., Jimenez, A., Munoz, J.A., Ibarz, A. (2011). Edible films and coatings: Structures,active functions and trends in their use. Trends in Food Science and Technology. Vol.22: 292-303.

Fernandez, A., Picouet, P. and Lloret, E. (2010). Cellulose-silver nanoparticle hybrid materials to controlspoilage related microflora in absorbent pads located in trays of fresh-cut melons. International Journal ofMicrobiology, Vol.142: 222-228.

Janjarasskul, T. and Krochta, J.M. (2010). Edible packaging materials. Annual Review of Food Science andTechnology. Vol.1: 415-448.

Kumar, P., Sandeep, K.P., Alavi, S. and Trong, V.D. (2011). A review of experimental and modeling techniquest odetermine properties of biopolymer-based nanocomposites. Journal of Food Science, Vol.76(1):E2-E14.

Kumar, P., Sandeep, K.P., Alavi, S., Truong, V.D. and Gorga, R.E. (2010). Preparation and characterization ofbio-nanocomposite films based on soy protein isolate and montmorillonite using melt extrusion. Journal ofFood Engineering, Vol.100: 480-489.

Luechinger, N.A., Loher, S., Athanassiou, E.K., Grass, R.N. and Stark, W.J. (2007). High sensitive opticaldetection of humidity on polymer/ metal nanoparticle hybrid films. Langmuir, Vol.23(6):3473-3477.

Mills, A. (2005). Oxygen indicators and intelligent inks for packaging food. Chemical Society Reviews, Vol.34(12):1003-1011.

Page, K., Palgrave, R.G., Parkin, I.P., Wilson, M., Savin, S.L.P and Chadwick, A.V. (2007). Titania and silver-titania composite films on glass-potent antimicrobial coatings. Journal of Materials Chemistry.Vol.17(1):95-104.

Ray, S.S. and Okamoto, M. (2003). Polymer/layered silicate nanocomposites: a review from preparation toprocessing. Progress in Polymer Science, Vol. 28:1539-1641.

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Shi, L., Zhou, J. and Gunasekaran, S. (2008). Low temperature fabrication of ZnO-whey protein isolatenanocomposite. Materials Letters, Vol. 62(28): 4383-4385.

Sinclair, R.G. (1996). The case for polylactic acid as a commodity packaging plastic. Journal of MassSpectroscopy and Pure Applied Chemistry. Vol. 33(5):585-597.

Tamer, C. and Copur, O.U. (2010). Chitosan: An edible coating for fresh-cut fruits and vegetables. ActaHorticulturae. Vol.877: 619-626.

Tang, X., Alavi, S. and Herald, T.J. (2008). Barrier and mechanical properties of starch-clay nanocompositesfilms. Cereal Chemistry, Vol. 85(3): 433-439.

The Hindu (2011). Cutting plastic waste. The Hindu, editorial section published on 28th February.

Thellen, C., Orroth, C., Froio, D., Lucciarini, J., Farrell, R., D’Souza, N.A. and Ratto, J.A. (2005). Influence ofmontmorillonite layered silicate on plasticized poly(l-lactide) blown films. Polymer. Vol.46(25): 11716-11727.

Tunc, S., Angellier, H., Cahyana, Y., Chalier, P., Gontard, N. and Gastaldi, E. (2007). Functional properties ofwheat gluten/montmorillonite nanocomposite films processed by casting. Journal of Membrane Science,Vol. 289(1-2): 159-168.

Voon, H.C., Bhat, R., Easa, A.M., Liong, M.T. and Karim, A.A. (2012). Effect of addition of halloysite nanoclayand SiO2 nanoparticles on barrier and mechanical properties of bovine gelatine films. Food and BioprocessTechnology. Vol.5(5): 1766-1774.

Yammamoto, O. (2001). Influence of particle size on the antibacterial activity of zinc oxide. InternationalJournal of Inorganic Materials, Vol.3:643-646.

Yoon, S. and Deng, Y. (2006). Clay-starch composites and their application in packaging. Journal of AppliedPolymer Science, Vol. 100(2): 1032-1038.

Yu, L., Dean, K. and Li, L. (2006). Polymer blends and composites from renewable resources. Progress inPolymer Science, Vol. 31(6): 576-602.

Zhou, J.J., Wang, S.Y. and Gunasekaran, S. (2009). Preparation and characterization of whey protein filmincorporated with TiO2 nanoparticles. Journal of Food Science, Vol. 74(7): N50-N56.

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Technological Advances in Manufacture of Low Calorie Dairy Foods

Narender Raju Panjagari and Ashish Kumar Singh


1. Introduction

Worldwide non-communicable diseases such as obesity, diabetes, cardiovascular diseases and cancer

have become major health problems due to changing lifestyle and dietary patterns among people. The

World Health Organization indicated that worldwide approximately 1.6 billion adults (age 15+) and

20 million children under the age of 5 years were overweight and at least 400 million adults were

obese in 2005 and projected that approximately 2.3 billion adults will be overweight and more than

700 million will be obese by the year 2015 (WHO, 2006). Further, recent estimations revealed that

worldwide more than 220 million people have diabetes (WHO, 2009). In 2005, an estimated 1.1

million people died from diabetes, with the number likely to be doubled by the year 2030 (WHO,

2009). India has the largest diabetic population with one of the highest diabetes prevalence rates in the

world (King et al., 1998; Bjrok et al., 2003). It is predicted that the Indian diabetic population would

rise to more than 80.9 million by the year 2030 (King, et al., 1998). An Indian National Urban

Diabetes Survey reported the average diabetes prevalence rate as 12.1% (Ramachandran, et al., 2001).

However, there was a large regional variation and the prevalence rates varied from 9.3% in Mumbai

to 16.6% in Hyderabad. Type-2 diabetes is a chronic progressive disease that requires lifestyle

changes (Knowler et al., 2002), the key lifestyle interventions being physical activity and a nutritional

plan with reduced caloric intake (Franz, 1997). The dietary factors such as high intake of fats, sugars,

milk and its products and low intake of fruits and vegetables were ascribed for the role in the non-

communicable diseases (Gupta et al., 2006). Being aware of the impact of high fat and high sugar on

health, today’s health conscious consumer is looking for the low-fat, low-sugar or sugar-free dairy

products. With the continuous invention of low-calorie and high-intensity sweeteners it has been

possible to develop dietetic dairy products for the benefit of health conscious consumers in general

and calorie conscious consumers in particular. In the present manuscript, technological developments

in the manufacture of artificially sweetened dairy products for the management of diabetes and alike

have been presented.

2. Intense sweeteners

Sweeteners elicit pleasurable sensations with or without energy and contribute to bulk and

characteristic colour. These attributes are desired attributes and hence positively regarded qualities in

food products. But, calorie conscious people in general and diabetics in particular need to control their

diet by cutting down their sugar and calories intake. Sweeteners can be classified, based on their

contribution towards energy, as nutritive and non-nutritive sweeteners. Nutritive sweeteners are those

substances, which when consumed, not only provide sweet taste but also contribute 4 kcal per gram of

substance. It includes sugar, honey, D-glucose, invert sugar, caramel, maltodextrin, high-fructose corn

syrup and dextrose syrup. Low-calorie sweeteners are nutritive sweeteners that are relatively less

sweet than sucrose and provide energy between 1 to 3 kcal per gram. Polyols are low-calorie

sweeteners (about 2 kcal per gram) that occur naturally in a number of fruits, all vegetables, cereals,

algae, mushrooms, seaweeds, etc. e.g. sorbitol, maltitol, lactitol and mannitol. Non-nutritive

sweeteners are those sweeteners that offer no energy such as aspartame, acesulfame-K, sucralose etc.

The intensity of the sweetness of a given substance in relation to sucrose is made on a weight basis

(Table-1).

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Table-1. Relative Sweetness of Sweeteners

Sweetener Approximate

Sweetness

Sucrose 1.0

Crystalline fructose 1.2 - 1.7

HFCS, 55% 1.0

HFCS, 90% 1.0

Hydrogenated starch

hydrolysates 0.4-0.9

Lactitol 0.4

Trehalose 0.45

Isomalt 0.45-0.65

Sorbitol 0.6

Mannitol 0.7

Maltitol 0.9

Xylitol 1.0

Aspartame 180

Acesulfame

potassium 200

Saccharin 300

Sucralose 600

Stevioside 300

Alitame 2000

Neotame 8000

3. Alternatively sweetened dairy products

The dairy industry has responded to the growing needs of health conscious consumers for low-calorie

foods. Consequently, a large number of dairy products made with low-calorie and/or non-nutritive

sweeteners have been developed and some of them can be witnessed in the super market shelves.

Some of the R&D efforts in this area are discussed here.

3.1 Ice-cream and frozen desserts

Olsen (1989) suggested an ice cream formulation with low fat and low sugar content having 3% fat,

0% sugar, 4% glucose syrup, 3% bulking agent, 0.05% aspartame and 0.7% stabilizer/emulsifier.

Palumbo, et al. (1995) developed aspartame sweetened ice cream and ice milk bulked with lactitol

and/or polydextrose. Olinger and Pepper (1996) described a process for frozen dessert sweetened with

acesulfame-K in combination with lactitol and hydrogenated starch hydrolysate was used as the bulk

sweeteners. Taste, texture, hardness, melting and overrun properties of the frozen dessert were

reported to be comparable to those in conventional products sweetened with sucrose and corn syrup.

Verma (2002) had developed frozen dessert using artificial sweeteners and reported that amongst the

various sweeteners attempted, aspartame produced the most acceptable product. Further, it was

reported that such frozen dessert contained 5.5% fat, 12.5% MSNF, 9.9% maltodextrin, 9.3% sorbitol,

1.5% WPC, 0.38% stabilizer and emulsifier and 400 ppm aspartame. Basyigit, et al. (2006) developed

a human-derived probiotic ice cream using sucrose and aspartame and reported that the probiotic

cultures remained unchanged in ice cream stored for 6 months regardless of the sweeteners used.

3.2 Fermented dairy products

Pinheiro, et al. (2005) reviewed the effect of different sweeteners in low-calorie yogurts. Keller, et al.

(1991) had formulated an aspartame-sweetened frozen dairy dessert with increased MSNF but without

bulking agents by treating it with lactase. It was reported that there were no significant differences in

the scores of lactase-treated and artificially sweetened frozen desserts. Malone and Miles (1984) was

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granted a patent by the US patents organization for the development of a gelled, artificially sweetened

yogurt prepared by mixing a stabilizer solution containing high methoxyl pectin (2-7%), low

methoxyl pectin (3-8%) and an aspartic acid-based sweetener (0.1-0.75%). Farooq and Haque (1992)

developed a non-fat low-calorie yogurt using aspartame and sugar esters and reported that sugar esters

had improved the overall quality of non-fat low calorie yoghurt. It was reported that yoghurt with

sugar esters, mainly stearate-type yoghurt with an HLB range of 5 to 9, had firmer body, texture, and

mouth feel than yoghurts without sugar esters. Further it was reported that skim milk yoghurts

sweetened with aspartame had 50% fewer calories per serving than regular yoghurt containing 3.25%

fat and 4% sucrose. Keating and White (1990) had developed plain and fruit-flavoured yogurts using

9 different alternative sweeteners including aspartame, sodium and calcium saccharins, and

acesulfame-K. It was reported that among all the plain and fruit flavoured yoghurts, yoghurts

sweetened with sorbitol and aspartame received highest sensory flavour scores. Fellows, et al. (1991)

developed a sundae-style yogurt using aspartame and reported that during the manufacture, aspartame

has excellent stability in fruit preparation.

3.3 Traditional Indian dairy products

Burfi, the most popular khoa based confection contains high amounts of fat (20%) and sugar (30%).

Prabha and Pal (2006) developed a technology for the production of dietetic burfi for a target group of

obese and diabetics and reported that aspartame and neotame showed poor stability in dietetic burfi

while sucralose provided the most desirable sweetness profile and excellent stability to the product.

Recently, Arora et al. (2010) reported that aspartame sweetened (0.065%) burfi resembled control

burfi in sweetness with 94% recovery of aspartame when stored at 6-8°C for 7 days. Chetna et al

(2004) optimized conditions for making alternatively sweetened gulabjamun, another khoa based

sweet, and reported that soaking of fried gulabjamun balls in sorbitol syrup of 54B strength added

with aspartame @ 0.25% maintained at 65C for 3 h yielded a good quality product. Technology has

been developed for the manufacture of sugar free rasogolla using artificial sweeteners for such a large

group of people. The use of 40% sorbitol and 0.08% aspartame was found to be optimum for cooking

of rasogolla balls. The higher sorbitol level resulted in hard body and unacceptable flavour where as

lower level caused flattening of rasogolla balls with surface cracks.

Misti dahi contains high fat (1-12%) and cane sugar (6-25%) contents. High fat and sugar contents in

misti dahi may pose a hurdle for its successful marketing in other parts of the country in the present

health foods regime. With an aim to completely replace cane sugar in misti dahi, Raju and Pal (2011)

attempted a blend of artificial sweeteners viz. aspartame and acesulfame-K along with different

bulking agents and reported that maltodextrin was found to be the most suitable bulking agent.

Further attempts to characterize the artificially sweetened misti dahi revealed that hardness and

lightness were the most affected properties by the different binary blends of artificial sweeteners and

bulking agents (Raju and Pal, 2012). Shrikhand has very high content of sugar (40). The effect of

sugar replacers on sensory attributes and storage stability of shrikhand was studied by Singh and Jha

(2005). Among various combinations of sugar and raftilose tired, shrikhand prepared with raftilose

(4%) and sugar (12.5%) was rated as most acceptable by the sensory panelists. Sugar and raftilose

exhibited significant effect (p<0.01) on flavour, body and texture and overall acceptability no

significant effect was observed on color and appearance.

Kumar (2000) developed a low calorie lassi, a traditional fermented refreshing beverage, by using

aspartame and reported that aspartame at a level of 0.08% was required to replace 15% of cane sugar

in lassi. Recently, George et al. (2010) studied the stability of multiple sweeteners in lassi and

reported that binary blend of aspartame and acesulfame-K was found to be the best as it resembled

control sample in all the sensory attributes up to 5 days of storage. Beukema and Jelen (1990) studied

262

the suitability of developing whey-based drinks using high potency sweeteners and reported that both

aspartame and acesulfame-K may be suitable sweetening agents in cottage cheese whey based fruit

drinks. It was further reported that, in such drinks, the total calories were reduced to almost 50%.

4. Conclusion

With growing evidence of the role of diet and dietary components especially sugar in non-

communicable diseases such as obesity and diabetes, worldwide people are cautious of what they eat.

With the continuous invention of food additives such as low-calorie and high-intensity sweeteners it

has been possible to develop dietetic dairy products that suit the palate of local consumers. R&D

efforts in India contributed for the development of low-calorie dairy products such as dietetic

rasogolla, burfi, misti dahi, kulfi, etc. for the benefit of health conscious consumers in general and

diabetics in particular.


Arora S, Gawande H, Sharma V, Wadhwa B K, George V, Sharma G S and Singh A K (2010) The development

of burfi sweetened with aspartame. International Journal of Dairy Technology, 63 127-135.

Arora S, Singh V P, Yarrakula S, Gawande H, Narendra K, Sharma V, Wadhwa B K, Tomer S K and Sharma G

S (2007) Textural and microstructural properties of burfi made with various sweeteners. Journal of Texture

Studies, 38 684-697.

Beaglehole R and Yach D (2003) Globalization and the prevention and control of non-communicable disease:

the neglected chronic disease of adults. Lancet, 362 903-908.

Bjrok S, Kapur A, King H, Nair J and Ramachandran A (2003) Global policy: aspects of diabetes in India.

Health Policy. 66 61-72.

Chetana R, Manohar B and Reddy S R Y (2004) Process optimization of gulab jamun, an Indian traditional

sweet, using sugar substitutes. European Food Research and Technology, 219 386-392.

Franz M J (1997) Lifestyle modifications for diabetes management. Endocrinology and Metabolism Clinics of

North America, 26 499-510.

George V, Arora S, Sharma V, Wadhwa B K and Singh A K (2010) Stability, physico-chemical, microbial and

sensory properties of sweetener/sweetener blends in lassi during storage. Food and Bioprocess Technology,

DOI 10.1007/s11947-009-0315-7.

Gupta R, Misra A, Pais P, Rastogi P and Gupta V P (2006) Correlation of regional cardiovascular disease

mortality in India with lifestyle and nutritional factors. International Journal of Cardiology, 108 291-300.

Keating K R and White C H (1990). Effect of alternative sweeteners in plain and fruit-flavored yogurts. Journal

of Dairy Science, 73 54-62.

King H, Aubert R E and Herman W H (1998). Global burden of diabetes, 1995-2025: prevalence, numerical

estimates, and projections. Diabetes Care, 21 1414-1431.

Knowler W C, Barrett-Connor E, Fowler S E, Hamman R F, Lachin J M, Walker E A, Nathan D M (2002)

Reduction in the incidence of type-2 diabetes with lifestyle intervention or metformin. New England Journal

of Medicine, 346 393-403.

Pinheiro M V S, Oliveira M N, Penna A L B and Tamime A Y (2005) The effects of different sweeteners in

low-calorie yogurts – a review. International Journal of Dairy Technology, 58 193-199.

Raju P N and Pal D (2011). Effect of bulking agents on the quality of artificially sweetened misti dahi (caramel

colored sweetened yoghurt) prepared from reduced fat buffalo milk. LWT-Food Science and Technology. 44

1835-1843.

Raju P N and Pal D (2012) Characterization of artificially sweetened misti dahi based on physico-chemical,

sensory and textural properties. Indian Journal of Dairy Science, 65 34-46.

WHO (2009) Diabetes. Fact sheet no. 312. Geneva, Switzerland: World Health Organization, Geneva,

Switzerland (http://www.who.int).

263

Metabolites of Dairy Microbes and their Food Application

R K Malik, Chhaya Goyal, Taufeeq


1. Introduction

In the last few years, concerns over food safety have increased their importance due to its dramatic impact

on public health, which feature prominently leads to food safety crisis and rapid spread of foodborne

illness resulting from the consumption of contaminated food. Food safety has become more than ever

important issue from economic point of view as consumers may trigger a sudden lack of confidence in

any food product which can lead to a dramatic fall in demand of a particular product. Undoubtedly, the

major threat to food safety are pathogenic & spoilage causing organisms. Changes in the food chain will

continue to create opportunities for the emergence of new diseases and the re-emergence of old ones

(Elmi 2004; Church 2004). In addition, the presence in food products of chemical additives and residues

of agrochemical and veterinary drugs are also perceived by consumers as a health risk, the use of

antibiotics in intensive animal production poses the additional risk of bacterial resistance, which

constitutes a microbiological hazard. On the basis of these data, the need emerges for solutions to the

problem of food hygienic quality. Consumers are increasingly demanding pathogen-free foods with

minimal processing, fewer preservatives and additives, high nutritional value, and intact sensory quality.

Against this background and relying on improved understanding and knowledge of microbial interactions,

milder preservation approaches such as biopreservation have been developed (Holzapfel et al., 1995;

Hugas, 1998).

2. Brief overview of biopreservative metabolites

Lactic acid bacteria has a long history of use as bio-preservatives for food and feed storage; they are

known to produce different antimicrobial compounds that are able to control pathogenic, spoilage

bacteria, undesirable spoilage yeast and fungi (Dalie et al., 2009, Messens, 2002). Their preserving effect

mainly relates to production of organic acid such as lactic acid and acetic acid, hydrogen peroxide,

competition for nutrients, production of bacteriocins and protein, or proteinaceous compounds (Stiles,

1996; Ström et al., 2002, Dalie et al., 2009). Compounds such as fatty acids (Corsetti et al., 1998),

phenyllactic acid (Lavermicocca et al., 2003), peptide, 4-hydroxyphenyllactic acid, cyclo (Phe- Pro),

cyclo (Phe-OH-Pro) and reuterin (Magnusson, 2003), and organic acids, hydrogen peroxide and diacetyl

(Messens and De Vuyst, 2002) were reported to have antifungal activity.

3. Biopreservative metabolites

3.1 Organic acids

Organic acids produced by LAB are mainly lactic acid and acetic acid beside some other acids depending

on the strain of LAB. These acids diffuse through the membrane of the target organisms in their

hydrophobic un-dissociated form and then reduce the cytoplasmic pH and stop metabolic activities. Other

factors that contribute to the preservative action of the acids are the sole effect of pH, the extent of

dissociation of the acid and the specific effect of the molecule itself on the microorganisms (Axelsson,

1998). Some food borne pathogens can multiply in such conditions and posed risk to the safety of raw,

processed and stored foods. Many of these microorganisms are Gram negative bacteria, including

http://en.wikipedia.org/wiki/Food_spoilage

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Yersinia enterocolitica and Aeromonas hydrophila; and some of mesophilic species like Salmonella spp,

pathogenic Escherichia coli, Gram positive Listeria monocytogenes that are capable of becoming virulent

during low temperature storage. The inhibition activity of LAB to the growth of pathogenic bacteria is

most likely due to the production of organic acids and bacteriocin (De Vos, 1993). Corsetti et al., (1998)

observed that Fusarium, Penicillium, Aspergillus and Monilia were inhibited by mixture of acetic,

caproic, formic, propionic, butyric and n-valeric acids. These compounds were detected from obligate

heterofermentative Lactobacillus spp and L. sanfrancisco CB1 had the largest antifungal spectrum.

3.2 Bacteriocin

Bacteriocins stand out among the wide variety of antimicrobial ribosomal peptides synthesized by

bacteria. They have been found in all major lineages of bacteria (Riley et al., 2002). Bacteriocin has been

found in several species of bacteria, but most of bacteriocins studied are from LAB because of their

generally recognized as safe (GRAS) status. Bacteriocins produced by LAB are small, ribosomally

synthesized, antimicrobial peptides or proteins that possess activity towards closely related Gram-positive

bacteria, whereas producer cells are immune to their own bacteriocins (Cotter et al., 2005). The first

report about bacteriocin was made by Rogers (1928) who showed the antagonistic activity for

Lactococcus lactis against L. bulgaricus. The substance was determined to be a polypeptide and named

nisin. Its antibacterial spectrum includes inhibition of streptococci, staphylococci, Bacillus spp, clostridia

and lactobacilli. In general, bacteriocins are cationic peptides that display hydrophobic or amphiphilic

properties and the bacterial membrane is in most cases the target for their activity. However, nisin and

bovicin HC5, a bacteriocin produced by Streptococcus bovis HC5, has the putative N-terminal lipid II

binding motif and thus inhibits peptidoglycan synthesis and forms pores at sensitive membranes upon

interaction with lipid II (Paiva et al., 2011).

Corsetti et al., 2005 reported bacteriocin-like inhibitory substances that maintain the inhibition activity.

Lactobacillus pentosus TV35b produced a bacteriocin-like peptide (pentocin TV35b) that have inhibitory

activity against the growth of Clostridium sporogenes, Cl. tyrobutyricum, L. curvatus, L. fermentum, L.

sake, Listeria innocua, Propionibacterium acidipropionici, Propionibacterium sp. and Candida albicans

(Okkers et al., 1999).

The classification of bacteriocins, however, remains controversial. A scheme proposed by Heng and

Tagg (2006), which also takes into account the previous classification schemes has been mentioned in the

table 1 below.

Class General features Producing lactic acid bacteria

I-Lantibiotics

Ia-Linear

Ib-Globular

Ic-Multi-component

Modified, heat stable, <15 kDa

Pore forming, cationic

Enzyme inhibitors, no cationic

Two peptides

Nisin, Lacticin 481, Plantaricin C

None

Lct3147, Plantaricin W

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II-Unmodified

peptides

IIa-Pediocin-like

IIb-Miscellaneous

IIc- Multi-

component

Heat stable, <15 kDa

Anti-listeria, YGNGV

consensus

Non-pediocin-like

Two peptides

Pediocin PA1/AcH, Enterocin A,

Sakacin A

Enterocin B, L50, Carnobacteriocin

A

Lactococcin G, Plantaricin S,

Lactacin F

III-Large proteins

IIIa- Bacteriolytic

IIIb-Non-lytic

Heat labile, >30 kDa

Cell wall degradation

Cytosolic targets

Enterolysin A, Lcn972

Colicin E2-E9

IV-Circular peptides Heat stable, tail-head peptide

bond

AS-48, Gassericin A, Acidocin B

3.3 Carbon dioxide

Carbon dioxide (CO2) is one of products produced by hetero-fermenters LAB. The activity of CO2 is due

to two factors firstly, it creates anaerobic condition and replaces the existent molecular oxygen in the

products and secondly, CO2 has antimicrobial activity and this activity is important in the vegetable

fermentation to prevent the growth of spoilage fungi. Common fruit spoilage organisms such as Botrytis,

Rhizopus and Penicillum are not inhibited by 10% CO2 but concentrations between 20 - 50% have strong

antifungal activity (Bliekstad et al., 1981).

3.4 Hydrogen peroxide

Hydrogen peroxide (H2O2) is produced by most of the LAB when oxygen is available. Reports from

(Venturini et al. 2002) suggested that the application of hydrogen peroxide to apple skin might be an

alternative to fungicides to inhibit P. expansum. Ponts (2006) reported that the rate of the spore

germination of F. graminearum may be affected by hydrogen peroxide. It is related to the strong

oxidizing effect on the bacterial cell, and to the destruction of basic molecular structures of cellular

proteins (Magnusson, 2003). The inhibition of food borne pathogens and food spoilage bacteria by LAB

has been ascribed at least in part to the activity of H2O2.

3.5 Reuterin

Reuterin (3-HPA) is a product produced by some strains of lactic acid bacteria (LAB) from the

fermentation of the glycerol. The production of reuterin was reported from L. reuteri (Chung et al., 1989).

Reuterin is active against many kinds of microorganisms including Gram-positive and Gram-negative

bacteria, yeast and fungi. Antifungal activity was shown against species of Candida, Torulopsis,

Saccharomyces, Aspergillus and Fusarium (Chung et al., 1989). Other reports also show the production

of reuterin from different LAB isolates such as L. brevis, L. buchneri (Elferink et al., 2001), L. collinoides

(Claisse et al., 2000). The addition of glycerol to some of the reuterin producing LAB isolates has

increased their antifungal activity (Magnusson, 2003). Slininger et al. (1983) described the metabolic

266

pathway for the production and further reduction or oxidation of 3-HPA. The glycerol dehydratase of L.

reuteri has been purified and characterized (Talarico et al., 1990). The production of reuterin was

reported from L. brevis, L. buchneri, L. collinoides and L. coryniformis (Claisse et al., 2000; Nakanishi et

al., 2002).

3.6 Phenyllactic acid

Phenyllactic acid is special organic acid produced by certain LAB and shows antifungal activity (Gerez et

al., 2009). Magnusson et al. (2003) and Ström et al. (2002), reported that phenyllactic acid isolated from

LAB had spectrum range to inhibit pathogen bacteria, spoilage bacteria and spoilage fungi. Prema et al.,

(2008) observed Lactobacillus plantarum strain isolated from grass silage, produced a broad spectrum of

antifungal compound 3-phenyllactic acid L. plantarum 21B that produce phenyllactic acid have antifungal

action against Aspergillus niger.

3.7 Antifungal proteinaceous compounds and Cyclic dipeptides

Several lactobacilli produce compounds that have antifungal properties. L. rhamnosus VT1 exhibited

strong antifungal properties and capable of inhibiting the growth of many spoilage and toxigenic fungi

including species in the genera Aspergillus, Penicillium and Fusarium (Stiles et al., 1999). Okkers et al.,

(1999) purified and characterized a peptide TV35b from L. pentosus with antifungal effect against

Candida albicans. Other kinds of antifungal dipeptides which are cyclic namely, cyclo (Phe-Pro) and

cyclo (Phe- OH-Pro) were produced by L. coryniformis ssp. coryniformis Si3 and were inhibitory to

Aspergillus sp. (Magnusson, 2003; Ström et al., 2002). The peptides are highly heat stable with an

estimated molecular weight 3 KDa. Lavermicocca et al., 2003 reported that different LAB isolate

(Weissella confusa, W. cibaria, Leuconostoc citreum, L. mesenteroides, Lactococcus lactis, L. rossiae and

L. plantarum) inhibited the growth of fungi Aspergillus niger, Penicillium roqueforti and Endomyces

fibuliger in sourdough system and suggested to use them as natural preservatives. Rouse et al. 2008 and

Mandal et al., 2007, observed that L. reuteri 1100 inhibited the growth of a range spoilage causing fungi

in different food materials.

4. Applications of microbial metabolites in food industry

Biopreservation refers to the use of antagonistic microorganisms or their metabolic products to inhibit or

destroy undesired microorganisms in foods to enhance food safety and extend shelf life. Antimicrobial

metabolites can be used to biopreserve the highly perishable or ready –to-eat food in three following

ways.

1) A purified or semi-purified form of antimicrobial compound can be added to food to inhibit

specific undesirable microbes in specific products

2) Addition of food grade microbes (as starter or adjunct culture) during manufacture of food

products for in situ production of antimicrobial metabolites

3) As a part of hurdle technology or as an antimicrobial agent in Anti Microbial Packaging System

So far bacteriocins of LAB have been used to preserve various food items in its purified of semi-purified

form. Though nisin is currently the only bacteriocin approved for use in the United States, many

bacteriocins produced by members of the LAB have potential application in food products. Nisin has been

shown effective against L. monocytogenes in dairy products such as pasteurized milk (Fleming et al.,

1985) and cheese (Davies et al., 1997). Nisin is commonly added to pasteurized processed cheese spreads

to prevent the outgrowth of clostridia spores, such as Clostridium tyrobutyricum (Schillinger et al., 1996).

267

One application of lacticin 3147, a broad-spectrum, 2-component bacteriocin produced by L. lactis subsp.

lactis DPC 3147, is to control cheddar cheese quality by reducing non-starter LAB populations during

ripening (Ross et al., 1999). In cottage cheese the population of L. monocytogenes was reduced by 3-

log10 cycles over a 1-wk ripening period when it was manufactured with L. lactis DPC4275; however,

the number of Listeria in the control cheese, manufactured with a non-lacticin 3147-producing starter,

remained unchanged (104 CFU/g). The lacticin 3147-producing transconjugant has also been used as a

protective culture to inhibit Listeria on the surface of a mold-ripened cheese. Presence of the lacticin 3147

producer on the cheese surface reduced the number of L. monocytogenes by 3-log10 cycles (Ross et al.,

1999). A bacteriocin (Pediocin 34) Based biopreservatives has been found to be effective in extending the

shelf-life of Paneer (an Indian soft cheese) by more than 60 days and Khoa (a desiccated milk product) by

more than 30 days under refrigerated conditions (7C) (Malik, 2008).

The application of bioprotective cultures to ensure hygienic quality is a promising tool. Fermentation with

food grade microbes (starter or adjunct culture, now termed as “Bioprotective Cultures”) during

manufacture of food products for in situ production of antimicrobial metabolites, is the most effective,

easy and general approach for biopreservation. “Protective cultures are preparations consisting of live

microorganisms (pure cultures or culture concentrates) that are added to foods with the aim of reducing

risks by pathogenic or toxigenic microorganisms” (Vogel et al. 2011). A strain combination of lactic acid

bacteria (LAB) and propionic acid bacteria (PAB), used as protective culture, was found to be the most

active against yeasts, molds and Bacillus spp. in fermented milks and in bakery products (Suomalainen et

al., 1999). A novel application of protective cultures is their use in dairy and food products to inhibit the

growth of pathogenic microorganisms (Vibrio, Listeria monocytogenes and histaminogen bacteria) and

spoiling microbiota including yeast and molds to enhance the safety and shelf life of the food.

Industrial starters like SafePro® (CHR Hansen, DK) and HOLDBACTM

(Danisco, DK) find several

applications in efficient spoilage and pathogen prevention in fermented dairy foods. It is used for control

of yeasts and molds and some heterofermentative lactic acid bacteria in fresh fermented foods, for growth

control of Leuconostoc, heterofermentative Lactobacilli and Enterococci in hard and semi-hard cheese.

MicrogardTM

(Wesman Foods, Inc. Beaverton, USA) is the pasteurized product of the fermentation of

skim milk by Propionibacterium freudenreichii spp. shermanii and its protective action has been

associated with diacetyl, propionic, acetic and lactic acid and probably due to a heat stable peptide. It

inhibits Gram-negative bacteria such as Pseudomonas, Salmonella and Yersinia as well as yeasts and

moulds. It has been approved by the FDA for use especially in Cottage cheese and fruit flavoured

yoghurt. Another commercial product is BioprofitTM

(Valio, Helsinki, Finland) which contains

Lactobacillus rhamnosus LC705 and Propionibacterium freudenreichii JJ. The product is reported to

inhibit yeasts and moulds in dairy products and Bacillus spp. in sourdough bread. ALTATM

2341 (Quest

International, USA) is produced from Pediococcus acidilactici fermentation and has to rely on the

inhibitory effects of natural metabolites, including organic acids and the bacteriocin pediocin. It can serve

as an effective barrier to help control the development of Listeria in dairy products. ALC 01 (Niebüll,

Germany) is also a patented antilisteral culture developed especially for soft cheese production. Its

protective activity is due to pediocin generated by Lactobacillus plantarum. It inhibits the growth of

Listeria on the surface of artificially and/or naturally contaminated Munster cheese after spray treatment.

FARGOTM

23 (Quest International, USA) contains the same metabolites as for ALTATM

2341, but live

culture producing pediocin is present in greater quantity. In France it is added to raw milk intended for

raw milk cheese production (Kesenkas et al., 2006).

268

5. Microbial metabolites as part of Hurdle technology and AMP

In general the antimicrobial metabolites of dairy microbes have relatively narrow activity spectra and

moderate antibacterial effects. To overcome these limitations, more and more researchers use the concept

of hurdle technology to improve shelf life and enhance food safety. Hurdle technology is the use of

hurdles of differing levels of intensity to bring microbiological growth under control”. The synergistic

effect between bacteriocins and other processing technologies on the inactivation of microorganisms has

also been frequently reported in the literature. Nisin enhances thermal inactivation of bacteria, thus

reducing the treatment time and resulting in better food qualities. For example, Budu-Amoako et al.,

(1999) found that nisin reduced the heat resistance of L. monocytogenes in lobster meat and significantly

reduced the treatment time compared with thermal treatment alone. Generally, Bacteriocins are not active

against Gram negative Bacteria. However, a few members of Lactobacillus genera have reported to

produce bacteriocin active against gram negative bacteria. Gram-negative bacteria become sensitive to

bacteriocins if the permeability barrier properties of their outer membrane are impaired. For example,

chelating agents, such as EDTA, can bind magnesium ions from the lipopolysaccharide layer and disrupt

the outer membrane of Gram-negative bacteria, thus allowing nisin to gain access to the cytoplasmic

membrane (Abee et al., 1995). Schlyter et al., (1993) reported synergistic effects between sodium

diacetate and pediocin against L. monocytogenes in meat slurries. The use of combinations of various

bacteriocins has also been shown to enhance antibacterial activity (Hanlin et al., 1993; Mulet-Powell et

al., 1998). When used in combination with nisin, leucocin F10 provides greater activity against L.

monocytogenes (Parente et al., 1998). There has been continued interest in the food industry in using

nonthermal processing technologies, such as high hydrostatic pressure (HP) and pulsed electric field

(PEF) in food preservation. It is frequently observed that bacteriocins, in combination with these

processing techniques, enhance bacterial inactivation. In addition, Gram-negative bacteria that are usually

insensitive to LAB bacteriocins, such as E. coli O157:H7 and S. typhimurium, become sensitive following

HP/PEF treatments that induce sublethal injury to bacterial cells (Kalchayanand et al., 1994). Studies

demonstrate that nisin enhances the pressure inactivation of spores of Bacillus coagulans, Bacillus

subtilis, and C. sporogenes (Roberts et al., 1996; Stewart et al., 2000).

Antimicrobial packaging film prevents microbial growth on food surface by direct contact of the package

with the surface of foods, such as meats and cheese. For this reason, the antimicrobial packaging film

must contact the surface of the food so that bacteriocins can diffuse to the surface. Incorporation of

bacteriocins into packaging films to control food spoilage and pathogenic organisms has been an area of

active research for the last decade. The gradual release of bacteriocins from a packaging film to the food

surface may have an advantage over dipping and spraying foods with bacteriocins. In the latter processes,

antimicrobial activity may be lost or reduced due to inactivation of the bacteriocins by food components

or dilution below active concentration due to migration into the foods (Appendini et al., 2002). Two

methods have been commonly used to prepare packaging films with bacteriocins (Appendini et al., 2002).

One is to incorporate bacteriocins directly into polymers. Examples include incorporation of nisin into

biodegradable protein films Two packaging film-forming methods, heat-press and casting, were used to

incorporate nisin into films made from soy protein and corn zein in this study. Both cast and heat-press

films formed excellent films and inhibited the growth of L. plantarum. Compared to the heat-press films,

the cast films exhibited larger inhibitory zones when the same levels of nisin were incorporated.

Incorporation of EDTA into the films increased the inhibitory effect of nisin against E. coli. Coma et al.,

269

(2001) incorporated nisin into edible cellulosic films made with hydroxypropylmethylcellulose by adding

nisin to the film-forming solution. Inhibitory effect could be demonstrated against L. innocua and S.

aureus. Another method to incorporate bacteriocins into packaging films is to coat or adsorb bacteriocins

to polymer surfaces. Examples include nisin/methylcellulose coatings for polyethylene films and nisin

coatings for poultry, adsorption of nisin on polyethylene, ethylene vinyl acetate, polypropylene,

polyamide, polyester, acrylics, and polyvinyl chloride (Appendini et al., 2002).

6. Conclusion and future perspectives

There has been growing interest in the use of food grade bacteria and/or their natural preservative agents

for pathogen-free foods that are minimally processed, have lesser preservatives, additives and have high

nutritional value. Several food grade metabolites have been well characterized to have biopreservatives

effects. Further efforts should be made to scale up the use of these metabolites up to industrial level by

careful selection of dairy microbes and their metabolites for their general and specific use in

biopreservation of different food items. More research is needed on the usage possibilities of metabolites

of dairy microbes in food industry

7. Selected Reading

Appendini P, Hotchkiss J H (2002) Review of antimicrobial food packaging. Innovative Food Science and Emerging

Technologies 3 113-126.

Axelsson L (1998) Lactic acid bacteria: Classification and physiology. In Lactic Acid Bacteria: Microbiology and

functional aspects, 2nd Edition, Revised and Expanded. Edited by S. Salminen & A. von Wright. pp. 1-72.

Marcel Dekker, Inc. New York

Bliekstad E, Enfors S O and Molln G (1981) Effect of carbon dioxide pressure on the microbial flora of poA stored

at 4 or 14°C. Journal of Applied Bacteriology, 50 493-504.

Budu-Amoako E, Ablett R F, Harris J, Delves-Broughton J (1999) Combined effect of nisin and moderate heat on

destruction of Listeria monocytogenes in cold-pack lobster meat. Journal of Food Protection 62 46-50.

Chung T C, Axelsson L, Lindgren S E and Dobrogosz W J (1989) In vitro studies on reuterin synthesis by

Lactobacillus reuteri. Microbial Ecology in Health and Disease, 2 137-144.

Church (2004) Major factors affecting the emergence and re-emergence of infectious diseases. Clinics in Laboratory

Medicine 24 559–586.

Claisse O. and Lonvaud-Funel A (2000). Assimilation of glycerol by a strain of Lactobacillus collinoides isolated

from cider. Food Microbiology, 17 513-519.

Coma V, Sebti I, Pardon P, Deschamps A, Pichavant F H (2001) Antimicrobial edible packaging based on cellulosic

ethers, fatty acids, and nisin incorporation to inhibit Listeria innocua and Staphylococcus aureus. Journal of

Food Protection 64 470-5.

Corsetti A, Gobetti M, Rossi J and Damiani P (1998) Antimould activity of sourdough lactic acid bacteria:

identification of a mixture of organic acids produced by Lactobacillus sanfrancisco CB1. Applied Microbiology

Biotechnology, 50 253-256

Corsetti A, Settanni L, Sinderen D, Felis G E, Dellaglio F and Gobbetti M (2005) Lactobacillus rossii sp. nov.,

isolated from wheat sourdough. International Journal of Systematic and Evolutionary Microbiology, 55 35-40.

Cotter P D, Hill C, Ross R P (2005) Bacteriocins: developing innate immunity for food. Nature Review

Microbiology 3 777–788.

Dalié D K D, Deschamps A M and Richard- Forget F (2009) Lactic acid bacteria - Potential for control of mould

growth and mycotoxins. A review. Food Control, 21 370-380.

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Detection of foreign fats in ghee by triglyceride profiling using low- resolution gas -

liquid- chromatography

Vivek Sharma and Tanmay Hazra


1. Introduction

Milk fat is an important component in the dairy industry, which plays a significant role in economic,

nutrition, physical and chemical properties of milk and milk products. Milk fat is one of the valuable

fats that continue to be a target of unscrupulous traders for the maximization of profits. Present trend

is the use the concoction of vegetable oils and body fats. Vegetable oil is one of the common

adulterants in concoctions being used by adulterators; hence tracer component ß- sitosterol has been

used as the marker in the RP-TLC based method. In the present practical another approach, which is

based on the principle of low- resolution GLC of triglycerides will be used to check the adulteration

of milk fat with foreign fast including body fats.

2. Low- resolution gas chromatography of chromatographic method

Fit a capillary column of 2.5 mts in the GC oven. The details of the method are as:

2.1 Sample Preparation

Take molten ghee and filter with what man 1or 4 filter paper to remove any residue of ghee. Take 25mg of ghee

in a 5 ml volumetric flask and make the volume to 5.0 ml with hexane to get a solution of 5mg/ ml concentration

and dissolve the contents. Use this solution for GC- injection.

2.2 Conditions of Gas- Liquid- Chromatographic analysis:

a) Injector condition: Temp of injector-350°C; Split ratio-1:50

b) Carrier gas: Nitrogen; Total Flow 79mL/min; Column Flow 1.49mL/min; Purge flow3mL/min.

c) Injection volume:1µl

d) Column: Capillary type, 5% Phenyl / 95% Dimethyl Polysiloxane (BP5/RTx5 etc); Length- 2.3m, Inner

Dia-.25mm,Film Thickness-.25µm; Max temp-335°C

e) Temperature programme for TAG profiling of ghee: Injection at 80°C and holding for 50 sec then

increase the temperature to 190°C @ 50°C and hold for 1 min and finally increase the temperature to

335°C @ 6°C and hold for 6 min.

f) FID condition: Temperature 370°C

g) Other parameters: Make up flow30mL/min, H2 flow 40mL/min, Air flow-400mL/min

2.3 Triglyceride standard: Prepare a solution of the standard in hexane to get the concentration of 5mg/ ml as in

case of ghee. Use Supelco TAG mix (C24, C30, C36, C42, C48) from Sigma Aldrich as standard.

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Fig1: Chromatogram of TAG mix ( C24,C30,C36,C42,C48)

RT=retention time in minute

Table 1: TAG (%w/w) profile of fat sample

Triacyl glycerol Cow ghee Buffalo ghee Lard

C24 0.17 .019 -

C26 0.25 .313 -

C28 0.324 .434 -

C30 0.855 1.008 -

C32 1.582 1.879 -

C34 4.022 4.3265 -

C36 9.613 9.2133 -

C38 13.323 14.0779 -

C40 11.80 7.353 -

C42 7.6 5.071 -

C44 6.315 7.927 .0626

C46 6.75 6.619 .155

C48 8.366 7.60 1.900

C50 11.312 11.92 15.542

C52 11.044 11.84 59.420

C54 4.960 4.3 22.918

RT 9.08 14.23 18.97 23.14 26.86

C24 C30 C36 C42 C48 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 min

0.0

2.5

5.0uV(x10,000)

0

100

200

300

kPa

Chromatogram

274

Communicating Science

Meena Malik

1. Introduction

Communicating science is part and parcel of a scientist’s daily life. Scientists give talks, write papers and

proposals, communicate with a variety of stakeholders, and also engage themselves in educating others.

Thus, scientists need to communicate effectively regardless of their specialization. In other words, to be a

successful scientist, one must be an effective communicator too. Increasingly, effective communication

skills are becoming part of the core professional skills every science student and professional should have.

2. Diverse Range of Stakeholders

Scientists have to interact with a diverse range of stakeholders in terms of their profile, needs and

expectations. Though many of the concepts scientists deal with on a daily basis are technical and

scientific in nature, yet these are often the basis of policies that affect the population at large. Broadly, the

scientists have to meet the expectations of the following divergent categories of people:

Peers

Donors

Policy makers

Students

Extension Professionals

Farming Community

General Public

In fact science thrives when scientists communicate more effectively. Science is increasingly becoming

interdisciplinary and the ability to communicate more effectively across disciplines fosters collaboration

and innovation. Effective communication of scientists’ ideas and innovations can enhance their ability to

secure funding or brighten their career prospects. It allows them to write better and more comprehensible

research papers. It also allows them to be better teachers and mentors for next-generation scientists.

Effective communication enables scientists to reach out to broad and diverse categories of stakeholders. It

helps build up a broad base to their scientific endeavours, becomes more relevant to society, and

encourages more informed decision-making at all levels. Strong and effective communication helps

bridge the gap between them and the stakeholders and makes new developments and innovations

accessible to the target group that traditionally remains excluded from the process of science. Thus, it can

help make science more diverse and inclusive.

3. Communicating Science

Science education is screaming for transformation. Research scholars and scientists need to be trained not

only in their subjects, but also in terms of their soft skills and other life skills such as critical thinking,

problem solving and communication proficiency. In Institutions of higher learning, the scientific

communication is mostly perceived as short cuts, bullet points, technical terms, scientific equations and

http://compassblogs.org/blog/2013/04/01/gradscicomm-how-compass-is-answering-the-national-demand-for-science-communication-training/

275

formulas. This is more of information processing rather than the knowledge sharing. There is no place for

creativity or criticism. To move from information to knowledge, from experimental facts to rationale

understanding of facts, we need language to express. Incomplete sentences only reflect inadequate

learning and incomplete interpretation/ representation of facts. For creating new knowledge in science,

we need to lay emphasis on the art of scientific communication. Effective communication depends a lot

on our linguistic empowerment, which in turn, enhances our critical thinking and leads to cognitive

empowerment.

Effective communication means transmitting the message clearly and concisely so that it is understood.

Communicating science is as important to the scientific process as designing, conducting and analyzing

the experiment itself. A scientific experiment, irrespective of its spectacular results, is not completed until

the results are communicated. In fact, the foundation of science is based on the premise that original

research must be communicated. This is the only way by which new scientific knowledge can be

authenticated and then added to the existing data base that we call science. Any branch of knowledge

requiring a systematic study involves the use of scientific communication for the purpose of recording

and reporting information. Science writing is different from creative writing as it deals with scientific

facts and does not present an imaginary view of reality. Scientific reporting is objective in content and

systematic in form. It is always precise, exact, and to the point so that it may have the desired effect on

the reader and lead to the required action.

4. Oral Communication Skills

Effective communication skills are one of the important core professional skills for every science

professional. The goal should be to communicate clearly through oral and non-verbal communication.

First step towards effective oral communication is to connect and be aware of how others feel when they

are around us or are talking with us.

Making eye contact and acknowledging someone else's presence by looking at them in the eye is

very important. Constant focused attention is must to help connect with the target audience.

Effective communication is the combined harmony of verbal and nonverbal actions. Body

movements, mostly facial expressions, volume of voice, intonation and rhythm do play an important

part in oral communication. It does not matter so much what we say. It matters a lot how we say it. A

set of various behaviors and methods of relaying information also has a great impact on our lives.

Developing communication skills also demands constant practice on all the four fundamental skills

of listening, speaking, reading and writing. Volunteering to give presentations within smaller groups

initially helps improving both conversational speaking and public speaking.

5. Platforms for Written Communication

In the field of education and research, journals publish technical material on specialized fields and are

circulated amongst the scientists and scholars. There are several platforms for scientific written

communications as listed below:

Research papers in journals

Review papers

Research report

Research notes

http://www.nature.com/scitable/topicpage/effective-communication-13950970

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Letters to journals

Books and book chapters

Book reviews

Scientific magazines

Special publications

Annual reports

Newsletters

Consultancy reports

Newspaper articles

Conference abstracts

Proceedings

Conference posters

Extension literature

Writing for web

Writing for social media

All these writings must conform to the rules of scientific and technical reporting so that they are properly

understood and appreciated. All types of articles such as Technical Articles; Semi-technical Articles;

Popular Articles; Research Papers; Dissertations and Theses, and Technical Bulletins are covered under

the ambit of Scientific Writing.

The nature of the subject, the purpose of the scientific reporting and the reader for whom the report is

meant determine the form and structure of the communication. Every written communication has a

specific purpose and a specific audience. It should be carefully planned and constructed to fit both. Every

scientific communication has one certain clear purpose: to convey information and ideas accurately and

efficiently. The objective requires that the communication be: (1) as clear as possible; (2) as brief as

possible; and (3) as easy to understood as possible.

Scientific communication, if it is to be effective and efficient, must be designed for the needs and the

understanding of a specific reader or group of readers. One must have adequate knowledge of the

educational and professional background of the readers. The language and style of the communication

depends, to a great extent, on the academic and professional background of its readers. We need to have

an idea of what the reader expects from the report and his level of understanding. Writing should be

aimed at the average reader, but should also cater to those at either extreme of the range. It should interest

the more knowledgeable reader and be intelligible to the reader who is less familiar with the subject.

There is no precise formula for the organization of scientific presentations and reports. The material in

any report should be presented in an order that leads logically towards a conclusion or conclusions. The

various sections of the report are organized so that each of them has its logical conclusions. Almost every

scientific communication should have three functional elements. This does not mean that it should be

divided by boundaries into three distinct parts. But functionally it should have a beginning, middle and an

end.

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6. Research Paper as a Form of Scientific Writing

A scientific paper is a written and published report describing original research results. A

research/scientific paper is primarily an exercise in organization. Each scientific paper should have, in

proper order, its Introduction, Materials and Methods, Results and Discussion (IMRAD). Early journals

published descriptive papers. IMRAD pattern slowly progressed and came to be adopted by most of the

journals in the latter half of the nineteenth century.

IMRAD pattern is an effective way to proceed to answer these four questions:

Introduction: What question was studied?

Materials and Methods: How was the problem studied?

Results: What are the findings?

Discussion: What do these findings mean?

7. Some Important Language Points and Skills

The ability to produce a clear, concise and professionally presented report or oral presentation is a skill

that one is required to develop to be successful both at the research/academic institution and the

workplace alike. A clear, concise and well written report saves a lot of time of the users, be it researchers,

students, teachers, managers or the clients. In other words, the value of accuracy and precision is not only

important for researchers in scientific education and research but also for professionals in all sorts of work

situations.

Successful communication depends upon the correct use of language and a good style of writing. One

may learn the correct use of language, but has to cultivate a good style of writing. The former concerns

grammar, usage, spelling, capitalization and punctuation, the latter concerns the organization of ideas

through proper choice of words, arrangement of words into sentences, grouping of sentences into

paragraphs, sections and chapters. The use of abbreviations, the approach to the reader, use of idioms, use

of visual aids, the format and layout of the report are all aspects of style. Following are some of the some

of the language skills that make scientific writing effective:

7.1 Choice of words

The primary objective of scientific communication is to transmit information briefly, clearly and

efficiently. This can be achieved only through simple, direct and plain style. The first step towards a

simple and clear style is to use simple language. One must choose a short word rather than a long word, a

plain and familiar word rather than a fancy or unusual word and a concrete word rather than an abstract

word.

7.2 Conciseness

Conciseness describes writing that is direct and to the point. Writing that is not concise is wordy. Wordy

and indirect writing irritates the readers. In contrast, concise writing appeals to readers because it is direct.

Hence, all efforts should be made to eliminate from the writing every word that does not contribute to the

meaning or clarity of the message. Conciseness makes the writing clear and effective.

7.3 Discreet Use of Jargons

Jargon is specialized vocabulary of a particular group- words that an outsider unfamiliar with this field

would not understand. Jargon encompasses all technical terms. Such terminology is useful and often

necessary in technical communication restricted to people working on the same or similar subjects.

Technical terms become jargon only when carelessly used for wider audience. Jargon is a special

278

language of a particular field or profession. We can’t expect lawyers to say habeas corpus in English just

because the rest of us don’t understand. The Jargon of any given field is often the most efficient means of

communication within that field. It becomes offensive when handy English equivalents are available or

people outside the field are expected to understand, what is said. In other words, using jargons

unnecessarily is pretentious, showy, and artificial.

7.4 Avoid Colloquial Diction

Colloquial diction is a language that reads like spoken English. In some contexts, colloquial diction is

perfectly appropriate. This is mostly used in fiction as conversational lines for the characters and is

considered as a private style. In public style or technical reporting - colloquial diction is not desirable.

7.5 The Verb ‘Be’

The verb ‘be’ is often a cause of stylistic problems. Eight basic forms of verb ‘be’ are: am, are, is, was,

were, be, being, been. Avoid verb ‘be’ followed by adjectives or nouns that can be turned into strong,

economical verbs.

7.6 Appropriate Use of Coordination and Subordination

A common failing of technical writers is the expression of ideas of unequal importance in constructions

that seem to give equal weight. Appropriate use of coordination and subordination should be made by

carefully examining which ideas are important and which are minor, reworking them into simple,

compound and complex sentences. Meaning can be grasped more quickly and more easily if subordinate

ideas are indicated and put in subordinating constructions. A sentence should express the main thought in

a principal clause. Less important thoughts should be expressed in subordinate clauses. Length of the

sentence should be kept as short as far as possible by using not more than one or two subordinating

conjunctions or relative pronouns in a sentence. There is a greater risk of grammatical error in longer

sentences.

8. Conclusions

Scientific communication helps create a linking base for scientific endeavours for making them more

relevant to society, and encourages more informed decision-making at all levels. Strong and effective

communication helps bridge the gap between inventor and the target user. To help make science more

diverse and inclusive, we need to lay emphasis on imparting skills in communicating science as well.

Research scholars and scientists need be trained not only in their subjects, but also soft skills and other

life skills such as critical thinking, problem solving and communication proficiency. Scientific

communication is objective in content and systematic in form. It has to be clear, simple and well ordered

communication to transmit the scientific facts. It has a specific purpose and a specific audience. It should

be carefully planned and prepared keeping the reader in mind. It is the art of making the subject

intelligible to others, which requires invaluable mental discipline and in turn enhances clear thinking.

9. Selected Reading

CBE Style Manual: A Guide for Authors, Editors and Publishers (1983). 5th

ed. Council of Biological Editors,

Bethesda, Maryland, USA.

Chicago Manual of Style (1996). 14th

ed. Prentice Hall of India.

Day R A (1998) How to Publish a Scientific Paper. 5th

ed. Oryx Press, Westport, Connecticut.

Gordon H M and Walter J A (1970) Technical Writing. 3rd

ed. Holt, Rinehart and Winston.

Joseph G (2000) MLA Handbook for Writers of Research Papers. 5th

Ed. Affiliated East-West Press. New Delhi.

279

Leggett G, Mead C D, Charvat W and Beal R S (1982) Handbook for Writers. 8th

ed. Prentice- Hall, USA.

Malik M and Malik R (2013) The Art of Technical Reporting and Writing, NDRI Publication No 93/2013, p. 1-117.

Malik M (2014) “Technical Writing in Dairy Research” In Compendium of AAddvvaanncceedd FFaaccuullttyy TTrraaiinniinngg ((CAFT) oonn

Advances in Technology, Quality and Safety of Functional Dairy Foods from 8th

July – 28th

July, 2014 at NDRI,

Karnal, 165-170.

Malik M (2013) “The Art of Scientific Writing: From Information Processing to Knowledge Sharing”, 21st Century

Learners: Learning Styles and Strategies: Proceedings of 8th

International and 44th

ELTAI Conference from July

18-20, 2013 at SRM University, Chennai, 256-258.

Malik M (2013) “Scientific writing: styles and strategies” In Compendium of National Training on Advances in

Production, Functional, Rheological &Quality Aspects of Traditional Indian Dairy Products from 8- 28 October

2013 at NDRI Karnal, 234-237.

Malik M (2012) “The Art of Scientific Writing and Communication” In Compendium of AAddvvaanncceedd FFaaccuullttyy TTrraaiinniinngg

CCoouurrssee oonn IInnnnoovvaattiivvee TTrreennddss iinn DDaaiirryy aanndd FFoooodd PPrroodduuccttss aanndd FFoorrmmuullaattiioonn”” ffrroomm 1100--3300 OOccttoobbeerr,, 22001122 aatt NNDDRRII,,

KKaarrnnaall,, 213-18.

Malik M (2011) “Concepts and Skills in Technical and Scientific Writing” In Compendium of Course in Advanced

Faculty Training (CAFT) on Advances in Processing and Quality Assurance of Dairy Foods (22nd

March 11th

April 2011), NDRI Karnal, 359-64.

Malik M (2011) “Essentials of Good Technical Writing” In Compendium of Advanced Course in Faculty Training

on Technological Developments in Cheese and Fermented Dairy Foods (5th

July to 25th

July 2011), NDRI

Karnal, 268-73.

Richard W S (1969) Technical Writing. Barnes & Noble, New York.

Sewak S N and Batra R K (2008). Scientific and Technical Writing: A Practical Approach. 3rd

ed., Kala Sanchar,

Ludhiana.

Troyka L Q (1987). Simon & Schuster Handbook for Writers. Prentice Hall, Inc. New Jersey.