OPTIMIZATION OF CULTURE PARAMETERS AND

PRESERVATION OF PROBIOTICS STREPTOCOCCUS PHOCAE

PI80 AND ENTEROCOCCUS FAECIUM MC13 FOR BACTERIOCIN

AND BIOMASS PRODUCTION

Thesis submitted to Pondicherry University for the Degree of

DOCTOR OF PHILOSOPHY

By

P. KANMANI., M.Sc., M.Phil

Department of Biotechnology School of Life Sciences Pondicherry University

Pondicherry-605 014 INDIA

APRIL- 2011

PONDICHERRY UNIVERSITY

DEPARTMENT OF BIOTECHNOLOGY SCHOOL OF LIFE SCIENCES

PONDICHERRY-605 014 INDIA

Dr. V. ARUL

Associate Professor

CERTIFICATE

Certified that this thesis entitled Optimization of culture parameters and

preservation of probiotics Streptococcus phocae PI80 and Enterococcus faecium

MC13 for bacteriocin and biomass production

is a record of research work done by

the candidate Mr. P. Kanmani during the period of his study in the Department of

Biotechnology, School of Life Sciences, Pondicherry University, Pondicherry, under my

supervision and that it has not previously formed the basis for the award of any degree,

diploma, associateship or fellowship.

Pondicherry

Date: (V. ARUL)

Phone: 91-413-2655715 (Off.) Fax: 91-413-2655715/2655265 91-413-2357492 (Res.) Email: varul18@gmail.com

DECLARATION

I hereby declare that the work presented in this thesis has been carried out by me under

the guidance of Dr. V. Arul, Associate Professor, Department of Biotechnology, School

of Life Sciences, Pondicherry University, Pondicherry, and this work has not been

submitted elsewhere for any other degree.

Pondicherry

Date: P. KANMANI

D ED I CATED TO MY

PARENTS AND WI FE

CONTENTS

CHAPTER PAGE No

1. GENERAL INTRODUCTION 1

2. REVIEW OF LITERATURE 8

3. GENERAL MATERIALS AND METHODS 44

4. OPTIMIZATION OF CULTURE PARAMETERS FOR 70

ENHANCED BACTERIOCIN AND VIABLE CELLS

PRODUCTION BY PROBIOTICS STREPTOCOCCUS PHOCAE

PI80 AND ENTEROCOCCUS FAECIUM MC13

5. FORMULATION OF LOW COST FERMENTATION MEDIA 107

FOR ENHANCED BACTERIOCIN AND VIABLE CELLS

PRODUCTION BY PROBIOTICS STREPTOCOCCUS PHOCAE

PI80 AND ENTEROCOCCUS FAECIUM MC13

6. CHARACTERIZATION AND AMPLIFICATION OF 143

BACTERIOCIN GENES FROM PROBIOTICS

STREPTOCOCCUS PHOCAE PI80 AND ENTEROCOCCUS

FAECIUM MC13

7. PRODUCTION AND PURIFICATION OF 160

EXOPOLYSACCHARIDE FROM PROBIOTICS

STREPTOCOCCUS PHOCAE PI80 AND

ENTEROCOCCUS FAECIUM MC13 AND ITS FUNCTIONAL

CHARACTERISTICS ACTIVITY IN VITRO

8. CRYOPRESERVATION AND MICROENCAPSULATION OF 193

PROBIOTICS AND PREBIOTIC IN ALGINATE-CHITOSAN

CAPSULES IMPROVES SURVIVAL IN SIMULATED

GASTROINTESTINAL CONDITIONS

9. SUMMARY 217

10. REFERENCES 221

ACKNOWLEDGEMENTS

It is my great privilege to express reflective gratitude to my guide Dr. V. Arul, Associate

Professor, Dept. of Biotechnology, Pondicherry University for giving me the fantastic

opportunity of my lifetime to work under his guidance. I am deeply beholden for the

constructive criticism, constant encouragement and concern to bring out my hidden ideas

and channelize them to perform my best.

I express my sincere gratitude to Prof. N. Sakthivel, Head, Department of

Biotechnology, School of Life Science, Pondicherry University, for extending lab

facilities and valuable suggestion in completing this successful project work.

I would like to thank Prof. S. Jayachandran, Former Dean, Department of

Biotechnology, School of Life Sciences, Pondicherry University.

I express my deep sense of gratitude to Doctoral Committee members, Dr. K. Prasanth,

Dept. of Biotechnology and Dr. C.R. Ramanathan, Dept. of Chemistry, for their

constructive criticism and valuable encouragement.

I would like to thank Prof. Priya Davidar, Dean, School of Life Sciences, Pondicherry

University.

I am thankful to Dr. S. Jayachandran, Dr. N. Arumugam, Dr. V. Balasubramanian,

Dr. V. Venkateswara Sarma, Dr. Sudhakar, Dr. A. Hannah Rachel Vasanthi and

Dr. Arun kumar Dhayalan who have been influential in all my academic pursuits. I

cherish my experience and expertise in experiments, which would be a treasure

throughout my life.

Words are not adequate to express my deep sense of gratitude to my lab colleagues Mr.

R. Satish kumar, Mr. N. Yuvaraj, Mr. K. A. Paari, Mr. V. Pattukumar and Mr. P.

Venkatesh kumar for all the advice, discussions and morale boosting that I so often

required.

My special thanks to seniors, Dr. A. Gopalakannan, Dr. S. Issac Kirubakaran, Dr.

Krishnaveni, Dr. P. Ravindra Naik, Dr. S. Vaithinathan, Mr. N. Badrinarayanan

and colleagues Mr. M. Perumal, Mr. G. Raman, Mr. Jean Cletus, Mr. Kennedy, Mr.

Saranathan, Ms. P. Lalitha, Ms. Moushmi priya, Ms. Revathi, Ms. Asha, Ms.

Pathma, and Ms. Veena for their immense co-operation and valuable support.

I also thanks to our office staffs, Mr. Ramalingam, Mr. C. Balakrishnan, Mr.

Kannayiram, Mr. Vadivel, Ms. Sarala, Ms. Vinothini and Ms. Muthammal for their

co-operation and help.

I gratefully acknowledge with thanks to Department of Biotechnology (DBT), New

Delhi, India for their financial supports.

I would like to thank Central instrumentation Facilities (CIF), Pondicherry University,

Pondicherry for providing assistance in using necessary instruments.

I wish to express my heart felt gratitude and reverence to my parents Mr. M. Paulraj,

Mrs. Ramalakshmi Paulraj, my brother Mr. P. Karuppasamy, my wife Ms. G.

Suganya, my sister Ms. P. Kalavathi and my son K. Sai Pranav Kamal for their

magnanimous help rendered during all phases of my research and helping me in all the

possible ways to complete my academic pursuit.

Date P. Kanmani

LIST OF ABBREVIATIONS

CFU Colony Forming Unit

AU Arbitrary Unit

TVC Total Viable Cells

CFS Cell Free Supernatant

cm Centimeter

DNA Deoxyribose Nucleic Acid

RNA Ribose Nucleic Acid

dNTP 2 deoxynucleotide 5 triphosphate

EDTA Ethylene Diamine Tetra Acetic acid

GTE Glucose Tris EDTA

MRS de Man Rogosa and Sharpe

SDS Sodium Dodecyl Sulfate

PBS Phosphate Buffered Saline

RPM Revolution Per Minute

TAE Tris Acetate EDTA

TE Tris EDTA

TEMED N, N , N - Tetramethylethylenediamine

APS Ammonium Per Sulphate

PCR Polymerase Chain Reaction

DSC Differential Scanning Calorimetry

FTIR Fourier Transform Infrared Spectroscopy

UV Ultra Violet

bp Base pairs

g/L Gram per liter

h Hours

kb Kilo Base

kDa Kilo Dalton

kV KiloVolt

L Liter

µ Micron

µl Microliter

µM Micromolar

mg/L Milligram per liter

min Minutes

mL Milliliter

mM Millimolar

mm Millimeter

nm Nanometer

OD Optical Density

w/v Weight per volume

v/v Volume per volume

N Normality

Sec Seconds

RSM Response Surface Methodology

CCD Central Composite Design

CHAPTER 1

GENERAL INTRODUCTION

CHAPTER 2

REVIEW OF LITERATURE

CHAPTER 3

GENERAL MATREI ALS AND METH OD S

CHAPTER 4

OPTI MI ZATI ON OF CULTURE

PARAMETERS FOR ENH ANCED

BACTERIOCI N AND VI ABLE CELLS

PROD UCTI ON BY PROBI OTI CS

STREPTOCOCCUS PH OCAE PI 80 AND

ENTEROCOCCUS FAECI UM MC13

CHAPTER 5

FORMULATI ON OF LOW COST

FERMENTATI ON MED I A FOR

ENH ANCED BACTERI OCI N AND VI ABLE

CELLS PROD UCTI ON BY PROBI OTI CS

STREPTOCOCCUS PH OCAE PI 80 AND

ENTEROCOCCUS FAECI UM MC13

CHAPTER 6

CH ARACTERI ZATI ON AND

AMPLI FI CATI ON OF BACTERI OCI N

GENES FROM PROBI OTI CS

STREPTOCOCCUS PH OCAE PI 80 AND

ENTEROCOCCUS FAECI UM MC13

CHAPTER 7

PROD UCTI ON AND PURI FI CATI ON OF

EXOPOLYSACCH ARI D ES FROM

PROBIOTICS STREPTOCOCCUS PH OCAE

PI 80 AND ENTEROCOCCUS FAECI UM

MC13 AND I TS FUNCTI ONAL

CH ARACTERI STI CS ACTI VI TY I N VI TRO

CHAPTER 8

CRYOPRESERVATI ON AND

MI CROENCAPSULATI ON OF

PROBI OTI CS AND PREBI OTI C I N

ALGINATE- CH I TOSAN CAPSULES

I MPROVES SURVI VAL I N SI MULATED

GASTROI NTESTI NAL COND I TI ONS

CHAPTER 9

SUMMARY

CHAPTER 10

REFERENCES

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1

CHAPTER 1

GENERAL INTRODUCTION

The Russian scientist, Elie Metchnikoff has given the concept of probiotics and he suggested that

the long life of Bulgarian peasants resulted from the consumption of fermented milk products.

The term probiotic was derived from the Greek language for life . The World Health

Organization (WHO) defined probiotics as live microorganisms when administered in adequate

amounts conferring a beneficial health effect on the host . The first use of the word to describe a

microbial feed/food supplement was by Parker in the year of 1974. He defined it as "organisms

and substances which contribute to intestinal microbial balance". Later, Fullar (1989) modified

as probiotics are live microbial feed supplements, which beneficially affect the host animal by

improving its intestinal microbial balance. Havenaar and Huis in t Veld (1992) proposed

probiotics as "a mono or mixed culture of live microorganisms which applied to animal or man,

affect beneficially by improving the properties of the indigenous microflora". During the

treatment, probiotic bacteria is able to survive gastric environment as well as exposure to bile

and pancreatic juice in the upper small intestine to exert beneficial effects in the lower small

intestine and the colon, however there are persuasive data on beneficial immunological effects

also from the dead cells (Mottet and Michetti, 2005). In addition, the probiotic bacteria colonize

the intestinal mucus layer where they can affect the intestinal immune system, displace enteric

pathogens, supply antimutagens and antioxidants and many other possible effects by cell

signaling process. Di Caro et al. (2005) reported that the intake of lactic acid bacteria (LAB)

influences multiple systems which were analyzed by microarray techniques. The up and down

regulation of 334 and 92 genes involved in inflammation, apoptosis, cell-cell signaling, cell

adhesion, differentiation, signal transcription and transduction was identified after one month of

2

LAB treatment. Moreover, probiotics may even activate macrophages directly (Tejada-Simonn et

al., 1999). In recent years, probiotics have been used for various treatments of diseases like

lactose intolerance, acute gastroenteritis, food allergy, atopic dermatitis, crohn s disease,

rheumatoid arthritis, and colon cancer (Marco et al., 2006).

Probiotics are now being applied in aquaculture, poultry and other livestock farming for

improving the health and growth (Gatesoupe, 1999). The use of beneficial bacteria i.e. probiotics

to displace pathogens by competitive processes are being used in the animal industry as a better

remedy than administering antibiotics and are now growing acceptance for the control of

pathogens in aquaculture (Havenaar et al., 1992). The concept of probiotics application started

and became successful in the world of aquaculture to overcome the problems in shrimp culture

due to high application of chemicals and sanitizers. The rapid growth of penaeid shrimp culture

has been accompanied by an negative impact of disease. Reports of infection and disease caused

by vibriosis have been far the most numerous of the reported bacterial agents of penaeid shrimp

and reported to constitute of bacteria present in the normal micro flora of cultured and wild

penaeid shrimp (Gomez et al., 1998; Singh et al., 1990). Vibriosis is traditionally controlled with

chemotherapeutic agents (Mohanty et al., 1990). Frequent use of chemotherapeutic agents,

especially antibiotics leads to the emergence of resistant strains pathogenic to animals (Aoki,

1975). Since many bacterial pathogens developed resistance to antibiotics, the use of probiotics

for sustainable and ecofreindly aquaculture is gaining importance to prevent disease incidence

and healthy pond ecosystem management. Although numerous probiotic formulations are

available already in the market for shrimps, animals and humans, new probiotics which can

effectively control pathogenic microorganisms are still in demand (Swain et al., 2009). We have

3

isolated new probiotics in our laboratory from shrimp and fish. (Swain et al., 2009; Goplakannan

and Arul, 2010)

Probiotic bacteria consists of species belonging to the families Bacteroides,

Saccharomyces cerevisiae, Bacillus subtilis, Nitrobacter spp., nitrosomonas spp., Streptococcus

faecalis, Rhodobacter spp., Fusobacterium, Butyrivibrio, Clostridium, Bifidobacterium,

Eubacterium, Lactobacillus spp., Enteroccocus spp., and Escherichia coli constitute less than 1%

of all intestine microorganisms. However, most of the probiotic bacteria comprise from Lactic

acid bacteria, such as Lactobacillus spp., Bifidobacterium spp., and Enterococcus spp., (Klein et

al., 1998). Lactic acid bacteria are the most important unique probiotic microorganisms normally

associated with the human gastrointestinal tract. These bacteria are Gram-positive, rod-shaped,

non-spore-forming and catalase-negative those are devoid of cytochromes and are of non-aerobic

habit, but are aero-tolerant, fastidious, acid-tolerant and strictly fermentative; lactic acid is a

major end-product of sugar fermentation (Axelsson, 1993). Some of the known LAB and

bifidobacteria are used as probiotic bacteria such as Lactobacillus plantarum, Lactobacillus

rhamnosus, Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus casei,

Lactobacillus lactis subsp lactis, Bifidobacterium bifidum, Bifidobacterium breve,

Bifidobacterium longum, Bifidobacterium adolescentis and Bifidobacterium animalis etc, (Anal

and Singh, 2007). Lactic acid bacteria are commonly found in foods, including fermented meat,

vegetables, fruits and dairy products, but also in respiratory, intestinal, genital tracts of humans

and animals, in sewage and in plant materials. Lactic acid is of paramount importance in food

and feed technology, where their major role is inhibition of growth of food spoiling

microorganisms (Dominquez et al., 2007). The production of high levels of lactic acid also

contributes considerably to taste formation and known to have positive effect on human and

4

animal health. LABs are capable of producing and excreting inhibitory substances other than

lactic acid and acetic acid. These substances are antagonistic to a wide spectrum of

microorganism, and thus can make significant contributions to their preservative action. They are

produced in very smaller amounts than lactic acid and acetic acid, and include formic acid, free

fatty acids, ammonia, hydrogen peroxide, diacetyl, becteriolytic enzymes, bacteriocins and

antibiotics as well as several well defined or completely undefined inhibitory substances (Sareela

et al., 2000).

Bacteriocin production has been described for several genera of lactic acid bacteria

(LAB), including Lactobacillus, Carnobacterium, Pediococcus, Lactococcus, Enterococcus,

Streptococcus, and Leuconostoc (Nes et al., 1996). Bacteriocins are heterogeneous group of

protein that are ribosomally synthesized by lactic acid bacteria, which can display broad

spectrum of antimicrobial activity against Gram positive and Gram negative pathogenic bacteria

(Klaenhammer, 1993). LAB bacteriocins have attracted growing interest in recent years because

of their potential usage as biopreservatives in the food industry to eradicate food spoilage and

foodborne pathogenic bacteria. Nisin is one of the most important bacteriocin produced by

bacteria such as Lactococcus lactis subsp. lactis, which is the most intensively studied

lantibiotics, due to their potential application in many areas. It has a broad range of inhibitory

activity against bacterial pathogens especially towards food spoilage bacteria and is heat stable at

very low pH of the surrounding environment (Choi et al., 2000). Nisin is commercially produced

by bacterial culture as they are known for its safety as rightly approved by FAD and is widely

used as a food preservative, especially in canned food and dairy product in most of the countries

(Deegan et al., 2006).

5

Generally, bacteriocin production is closely associated with growth of bacterial culture as

because bacteriocin is released during the growth of bacteriocin producing cultures and at end of

the bacterial growth, bacteriocin efficiency decreases very slowly due to the protease degradation

(Hur et al., 2000). Subsequently, bacteriocin production is also significantly affected by changes

in growth conditions such as culture medium, pH and temperature (Kim et al., 2006). Bacterial

growth phase can be prolonged to enhance the tenure of bacteriocin production and medium

components such as NaCl, ethanol, increasing concentration of carbon and nitrogen sources can

stabilizes the bacteriocin production (Leory and DeVuyst, 2003). Also, the pH of the medium

has shown to significantly affect the bacteriocin activity. Therefore, it is cleared that the

optimization of environmental factors is very essential for enhancing the bacteriocin production.

The effect of medium components on bacteriocin production, such as enterocin P from

Enterococcus faecium P13, bacteriocin from Enterococcus faecium ST311LD Enterococcus

mundtii (Todorov and Dicks, 2005; Todorov and Dicks, 2009) and thermophilin 1277 from

Streptococcus thermophilus SBT1277 (Kabuki et al., 2007) has been previously reported.

Exopolysaccharides are longchain polysaccharides which containing branched, repeating

units of sugars or sugar derivatives. These sugar units are mainly composed of glucose, fructose,

mannose, galactose, and rhamnose etc., (Welman and Maddox, 2003). Polysaccharides from

microorganisms with a novel functionality, reproducible physico-chemical properties, and

nontoxic substances with immunostimulatory, antitumour, antioxidant activity and became a

better alternative to polysaccharides of plant and algal origin (Wang et al., 2008; Pan and Mei,

2010). Some examples of microbial exopolysaccharides such as dextrans, xanthan, gellan,

pullulan, yeast glucans, and bacterial alginates are potentially used in many industries.

Furthermore microbial polysaccharides were developed as food additive including xanthan from

6

Xanthomonas campestris and gellan from Pseudomona elode. Microorganisms are more suitable

for EPS production than macroalgae and higher plants, since they display high growth rate and

amenable to manipulation of environment for influencing growth or EPS production (Freitas et

al., 2009a).

Lactic acid bacteria are generally recognized safe (GRAS). Hence EPS secreted by LAB

can be regarded as safe biopolymer and offer an alternative source of microbial polysaccharides

for use in the various food and pharmaceutical industries. Moreover, Sanni et al. (2002) reported

that the lactic acid bacteria have effectively produced exopolysaccharide which is a natural

promising candidate for industrial application. EPS from LAB have potential application in the

improvement of the rheology, texture and mouthfeel of fermented products (Welman and

Maddox, 2003). In gastrointestinal tract, EPS from LAB will remain stable in order to enhance

the colonization of probiotic bacteria. LAB polysaccharides have also been expressed

antitumour, immunostimulatory (Welman and Maddox, 2003), antibiofilm (Kim et al., 2009) and

antioxidant activity (Pan and Mei, 2010).

Due to the wide variety of industrial application, maintenance of high viability probiotic

cells is important. Thus, freeze drying and microencapsulation were carried out for preservation

of probiotic cells for long term. Freeze-drying is one the most significant, suitable and successful

techniques for long time storage of yeasts, bacterial and fungal strains (Zayed and Ross, 2004).

During the freeze drying process, survival of bacterial cells depends on various factors, including

growth conditions, protective agents, the initial cell concentration, freezing temperature and

rehydration conditions (Zhao and Zhang, 2009). Protective agents could provide cryoprotection

to the probiotic cells during the freeze drying process and also give a suitable matrix to allow

stability and ease of rehydration. Also the amount of probiotic cell viability after freeze drying

7

differs according to various factors, including type of bacteria and efficacy of the protective

agent used during freeze drying. Micro-encapsulation is a promising technique for bacterial cell

protection against adverse environmental conditions and also used for extending the bacterial

cells life during storage (Brinques and Ayub, 2011). In this method, freeze drying probiotic

strains are entrapped within the alginate matrix during the formation of spheres and subsequently

stored at suitable temperature for long term (Krasaekoopt et al., 2006).

Carbohydrate polymers including alginate have been used in various food applications.

The reversibility of microencapsulation, i.e. solubilizing alginate gel by sequestering Ca2+ ions

and the promising release of entrapped probiotic cells into the human intestinal region is another

important advantage (Prakash and Jones, 2005). Also a cross-linked gel matrix at low pH is

reported to undergo reduction in alginate mass causing a quicker degradation and release of

active ingredients (Krasaekoopt et al., 2006).

The present study was taken up to optimize the culture parameters, medium formulation,

bacteriocin, viable cell and exopolysaccharide production and preservation of probiotic bacteria

Streptococcus phocae PI80 and Enterococcus faecium MC13 for long term storage. The

optimization of various parameters will help in large scale production of the probiotics,

bacteriocin and EPS for application in shrimp culture and food processing industry.

8

CHAPTER 2

REVIEW OF LITERATURE

During the past two decades probiotic bacteria have been increasingly used as health promoting

bacteria in variety of food system, because of its safety, functional and technological

characteristics. It also widely used in aquaculture and live stock industry especially poultry.

Commonly, Lactobacillus spp., Bifidobacterium spp., Saccharomyces boulardii, Enterococcus

spp., Bacillus spp., and some other microorganisms have been considered as probiotic strains

(Patel et al., 2009). Possibly these bacterial strains exerted several beneficial effects into

gastrointestinal tract of host while administered with variety of food system. Lactic acid bacteria

(LAB) usually produce antimicrobial substances like bacteriocin which have broad spectrum of

antagonist effect against closely related Gram positive and Gram negative pathogens (Kanmani

et al., 2010b). LAB strains often produce polymeric substances such as exopolysaccharides

(EPS) which increase the colonization of probiotic bacteria by cell-cell interactions in

gastrointestinal tract (Welman and Maddox, 2003).

2.1. Probiotics in Aquaculture

Aquaculture is the fastest growing industry in the food sector (FAO). The demand for

fish shrimp, shell fishes, crabs, and other invertebrates growing at a pace where supply from wild

is diminishing. Hence aquaculture gains importance globally. Most of the species cultured both

onshore, offshore and fresh water system are affected by a variety of pathogens, for which cure is

not there or farmers resort to indiscriminate use of antibiotics. The prolonged use of

chemotherapeutic agents like antibiotics makes the pathogens to become resistant to these

agents. Hence, many developed countries importing aquaculture products reject the consignment

if it contains traces of antibiotic residues. This result in huge loss to the exporting countries and

9

subsequently the farmers end up in closing the farms. Vaccines are one of the prophylactic

treatment effectively controls pathogens. However they are available for fe pathogens. Thus

probiotics may provide an alternative method to control fish and shrimp pathogens. Many

researchers reported control of fish and shrimp pathogens by administering probiotics in feed and

ater (Rengpipet et al., 1998; Gomez-gill et al., 1998; Nikoskelainen et al., 2003; Gopalakannan

and Arul, 2011).

Probiotics are not only used for controlling pathogens but also to enhance growth,

stimulate enzyme synthesis and immuneresponse (Irianto and Austin, 2002, Nikoskelainen et

al., 2003, Rengpipat et al., 2003; Gatesoupe, 2005a; 2005b). Most importantly to keep the pond

water clean by controlling ammonia and decomposing of uneaten feeds, algae, dead planktons

and dead larvae in the pond bottom (Lightner, 1996, Ayyappan and Mishra, 2003; Jawahar

Abraham and Palaniappan, 2004). Numerous commercial probiotics are available in the market

but most of them were isolated from heterologous host. Probiotic from homologous host found to

be safer and effective. Farmers prefer less expensive, effective probiotics (Babu, 2004).

2.2. Bacteriocins of LAB

2.2.1. Production of bacteriocin

Bacteriocins are defined as small antimicrobial proteins or peptides which ribosomally

synthesized by lactic acid bacteria in to the growth medium. Bacteriocin production by LAB is a

cell survival mechanism which exhibit broad spectrum of inhibitory activity towards the closely

related bacteria due to the combined action of bacteriocin and the autolysin (Gollop et al., 2003).

Some of the bacteriocins are also active against more distant species (Jack et al., 1995).

Furthermore, bacteriocins enhance survival of LAB in complex ecological systems interest has

focused on prevention of growth of harmful bacteria in the fermentation and preservation of

10

dairy products. Therefore, this is more interesting with respect to probiotic strains that may

inhibit growth or adhesion of pathogenic bacteria by secreted products and not merely an effect

of acidic pH. Lorca et al. (2001) and Bach et al. (2003) reported that pathogen Helicobacter

pylori was inhibited by a protein secreted from L. acidophilus, as well as Escherichia coli

O157:H7 (EHEC) was eradicated in rumen fluid by S. cerevisiae subsp. boulardii. The

antimicrobial activity of bacteriocin is facilitated by inhibiting the cell wall formation or causing

pores in cell membrane which leads to cytoplasm leakage and death of target bacterial strains

(Cleveland et al., 2001; Deegan et al., 2006). Due to the biochemical and molecular biological

techniques development have led to the elucidation of several aspects of the regulation,

production, processing, secretion, structure and mode of action of bacteriocins. Bacteriocins

from LAB are cationic, hydrophobic and amphiphilic nature which is composed of 20 to 60

amino acid residues (Nes and Holo, 2000). Bacteriocin productions by LAB are classified into

three main classes based on their genetic and biochemical characteristics (Nissen-Meyer and

Nes, 1997).

The class I bacteriocins are lantibiotic, which is small (

11

to structural and functional characteristic features. Type A lantibiotics (e.g., nisin, subtilin, and

Pep5) are elongated molecules with a flexible structure in solution, which disrupts the membrane

integrity of the target organism. While type B lantibiotics adapt a more rigid and globular

structure which blocks enzyme functions (Jung, 1991). The class II bacteriocins is small (

12

Maqueda et al. (2004) was described first circular protein such as enterocin AS-48. However the

lactic acid bacteria mainly produced class I or class II bacteriocins (Zendo et al., 2005).

2.2.2. Factors affecting bacteriocin production by LAB

Environmental conditions such as temperature, pH, salinity and media components are

important external factors for higher amount of bacteriocin and biomass production by lactic acid

bacteria (Kanmani et al., 2010a). Moreover, bacteriocin production by LAB strains are growth

associated and is therefore induced by growth conditions including temperature and medium pH.

The temperature is one of the important culture parameter which can significantly enhance the

growth of bacterial strains by improving the fermentation conditions. Kim et al. (2006) reported

that temperature mainly affects the bacteriocin production and to a lesser extant inactivation. The

production of bacteriocin such as Enterocin RZS C5, Enterocin P from Enterococcus spp.,

Pediocin PD-1 from Pediococcus spp., and bacteriocin from Bacillus lincheniformis AnBa9 were

often regulated by optimum temperature (Kayalvizhi and Gunasekaran, 2008). Higher level of

bacteriocin activity was recorded under the suboptimal growth temperature. The strain

Streptococcus thermophilus SBT1277 has showed better bacteriocin and total viable cells

production at optimum temperature of 35oC which is much higher than the cells grown at 25oC

and 45oC (Kabuki et al., 2007). Bacteriocin production at higher volumetric concentration in

Lactobacillus strains was achieved by changing the growth temperature from 30oC to 25oC

(Anthony et al., 2009). Bacteriocin production of B. licheniformis AnBa9 with respect to growth

temperature was in accordance with the production of bacillocin 490 in B. licheniformis and

lichenin in B. licheniformis 26 L-10/3RA in which increase level of bacteriocin production was

obtained in suboptimal temperature (Anthony et al., 2009). Though, lichenin production occurred

strictly under the anaerobic condition. Also the bacteriocin-like substance (BLIS) production was

13

recorded at 30oC in B. licheniformis P40 (Cladera-Olivera et al., 2004). Aforementioned all the

cases, maximum bacteriocin production achieved with temperature which was optimum for the

bacterial cell growth.

Medium pH is another important culture parameter for bacteriocin and biomass

production by LAB strains (Kanmani et al., 2010a). During the fermentation period, LAB strains

were able to produce some organic acids such as lactic acid and acetic acid that were reduced pH

of growth medium. The low pH of the medium can cause the growth of bacterial cells and leads

to completely stop or slow the growth rate. Use of controlled pH fermentation was improved the

growth of bacterial cells and leads to increase the bacteriocin production (Anthony et al., 2009).

Usually, LAB growth is optimal in a medium pH range (pH-6.0-7.0) with lower and higher

values decrease the specific cell growth rate. Bai et al. (2004) reported that biomass and lactic

acid production by Lactobacillus lactis BME5 was significantly lower at pH 5.5 when compared

with pH range of 6.5-7.0. Optimum bacteriocin production by B. licheniformis strains were

between the pH 6.5 and 7.0 (Anthony et al., 2009). Maximum micrococcin GO5 production was

obtained at the growth pH 7.0 to 9.0. Also B. licheniformis AnBa9 was produced 50,000 AUml-1

of ABP (specific activity of 6933 AU/mg) in optimized medium at pH 8.0 (Anthony et al., 2009).

Moreover, Enterococcus faecium P13 was significantly increased Enterocin P activity at constant

pH 6.5 which was four fold higher when compared with uncontrolled pH fermentation (Herranz

et al., 2001). Ondaa et al. (2003) reported that eight fold increased bacteriocin activity was

achieved from Lactococcus spp., GM005 in pH controlled fermentation. Controlled medium pH

was also enhanced the bacteriocin stability in cultured broth. Bacteriocin activity was decreased

in cultured broth by absorption of bacteriocin producer cells, proteolysis and degradation

14

(Herranz et al., 2001). Zendo et al. (2005) indicated that absorption of bacteriocin to producer

cells was increased in higher medium pH.

Growth media and its components are most important another key factor for higher

amount of bacteriocin production and good fermentation process. Li et al. (2002) reported that

cell growth and metabolite production were greatly influenced by various carbon and nitrogen

sources, growth factors and organic salts. Bacteriocin-producing lactic acid bacteria are needed

complex nutritious media to grow and bacteriocin production. Various media are used to

cultivate the bacteriocin- producer such as CM, SM8, M17, M17S and MRS media (Li et al.,

2002). All of these culture media are great to neutralizing lactic acid and improving cell growth,

but do not consider the aggregation of bacteriocin (Li et al., 2002). Most of the growth media

were developed culture specific (M17 and MRS for Lactobacillus strains) and also provide

favorable environment for bacterial cell growth. However, these types of growth media were cost

expensive and leads to not suitable for large scale culture production in fermentation industry.

Bacteriocin production by LAB strains were also often regulated by optimum media components

and concentration. Therefore media optimization is essential for higher amount of bacteriocin

and biomass production by LAB and also reduced cost. The carbon and nitrogen sources are

most important nutrients in growth media, but in some cases of media these sources were not

sufficient for successful bacterial cell growth (Kayalvizhi and Gunasekaran, 2008). Avonts et al.

(2004) reported that L. johnsonii La1 and L. gasseri K7 could successfully grown in milk

medium if nitrogen source yeast extract was added. Sucrose, peptone, KH2PO4 and yeast extract

were gratefully enhanced the bacteriocin production and growth of L. lactis ATCC 11454 (Li et

al., 2002). Lactose and lactose rich substrates including sausage, whey and skim milk powder

were influenced the growth and bacteriocin production by lactic acid bacteria (Anthony et al.,

15

2009). During L. lactis fermentation, nisin production was influenced with increasing initial

concentration of glucose up to 25.0g L-1, but higher concentration of glucose didn t enhance the

nisin activity (Papagianni et al., 2006). Among the nitrogen sources, yeast extract has the most

significant impact on bacteriocin production (Bustos et al., 2004). Yeast extract is water soluble

content of autolyzed yeast cells and also it contains large quantity of free amino acids, short

peptides and growth factors (Anthony et al., 2009). High concentration of yeast extract (45g L-1)

has greatfully influenced the bacteriocin production by B. licheniformis AnBa9 (Anthony et al.,

2009). The level of biomass was increased form 3.0g.l-1 to 10g.l-1 when the concentration of

yeast extract shifted from 5.0g L-1 to 25g L-1 (Kulozik and Wilde, 1999). Moreover surfactant

Tween 80 is one of the most important components in the growth medium which greatly

influenced the bacteriocin and biomass production by Staphylococcus warneri (Prema et al.,

2006). Franz et al. (1996) reported that surfactant Tween 80 stimulates the protein or peptide

secretion through its influence in membrane fluidity. In general, NaCl concentration affects the

growth and bacteriocin production in several bacterial strains (Delgado et al., 2007). The

negative effect of NaCl on bacteriocin production could be due to its interference with the

inducer receptor interaction. However, Leal-Sánchez et al. (2002) reported that addition of NaCl

improved the plantaricin S production by Lactobacillus pentosus B96. Antibacterial bacteriocin

production was very low with improved specific activity if the NaCl concentration exceeded 1%

in the fermentation medium. Varying concentrations of NaCl in the fermentation medium did not

influence bacteriocin activity compared with centre point of all variables; hence, low level of

NaCl is required for higher bacteriocin production (Anthony et al., 2009).

16

2.2.3. Biosynthesis and regulation of Peptide bacteriocin synthesis

Bacteriocins are ribosomally synthesized peptides which are normally organized in

operon clusters encoding bacteriocin production and immunity genes (Sahl and Bierbaum, 1998).

The following bacteriocin gene clusters are mainly located on the chromosome such as

mersacidin, subtilin, sakacin, nisin and divergicin A etc. (Chen and Hoover, 2003). The

biosynthesis of lantibiotics operons normally contains genes coding for prepeptide (LanA-

homologous gene of different lantiobiotic gene clusters), enzymes responsibe for reaction

modification (LanB or LanM), the removal of leader peptide (LanP) by proteases, ATP biding

site, peptide translocation of peptide (LanT) by super family transport proteins and immunity

(Lan I). These informations were collected from the genetic analysis of various lantibiotics such

as nisin, lacticin, epidermin and subtilin (Chen and Hoover, 2003). Bacteriocins are mostly

synthesized as biologically inactive prepeptide molecule which carries an N- terminal leader

peptide which is attached to the C- terminal propeptide. Prepeptide formation, reaction

modification, proteolytic cleavage of the leader peptide, and the translocation of the modified

prepeptide or mature propeptide across the cytoplasmic membrane are the most important

biosynthetic pathway for lantibiotics. Prior to or during or after export from the cell, the cleavage

of the leader peptide may happen. There are two categories of genetic organization of

lantibiotics, such as group I and II that can be identified according to the biosynthetic pathway,

which can be either type A or type B lantibiotics (Guder et al., 2000; McAuliffe et al., 2001).

Three well known regulatory system signal producing proteins such as pheromone, membrane-

bound histidine protein kinase (HPK) and cytoplasmic response regulator (RR) were involved in

the regulation of lantibiotic and non lantibiotic biosynthsis (Parkinson, 1993; Nes et al., 1996).

The pheromone exists as peptides in the medium, activates the histidine protein kinase, which

17

autophosphorylates the conserved histidine residue in its intracellular domain when it senses a

certain concentration of bacteriocin. Subsequently, the phosphorylated group was transferred to

the aspartic acid residue on the RP receiver domain resulting intramolecular change that triggers

the response regulator to activate the transcription of the regulated genes including the structural

gene, the export genes, the immunity genes (Kuipers et al., 1993). However, most of the non

lantibiotics produce peptide with no antagonistic activity and are used as an induction factor (IF)

to activate the transcription of the regulated gene (Ennahar et al., 2000).

2.2.4. Targets-receptors for bacteriocins

Most bacteriocins are membrane active, causing permeabilization and eventual killing of

the target Gram positive and Gram negative bacteria (Stevens et al., 1991). Smith and Hillman

(2008) reported that the lantibiotics have multiple modes of bactericidal activity, which cause

transmembrane pore formation; lipid II mediated pore formation and lipid II abduction from

physiological domains. Some of the class II bacterioicns were also found to be membrane active

peptide which destroys the cytoplasmic membrane integrity by the formation of pores in

bacterial membrane. Subsequently, the aforementioned bacteriocins affect the membrane

permeability and leads to leakage of low molecular mass metabolites or dissipation of the proton

motive force. Konings et al. (1989) reported that the proton motive force is an electrochemical

gradient over the cytoplasmic membrane composed of membrane potential and the pH gradient

(pH), which drives ATP synthesis and accumulation or extrusion of ions and other metabolites.

Collapse of the proton motive force, induced by bacteriocin action and leads to cell death by the

termination of energy-requiring reactions. Low intracellular levels of ATP, the inability to carry

out active transport of nutrients, and the inability to maintain sufficient concentration of

cofactors such as K+ and Mg2+, are all a direct result of proton motive force collapse and leads

18

to growth inhibition and cell death (Venema et al., 1995; Montvile and Chen, 1998). The class II

bacteriocins are thought to form a barrel-stave like pore or a carpet mechanism whereby peptides

orient parallel to the membrane surface and interfere with membrane structure (Moll et al.,

1999). In contrast, the class I bacteriocin can induce the pores formation by a wedge like model

(Moll et al., 1999). The lateral oligomerisation of bacteriocin monomers or the complementation

of two class IIb peptides that form two-component poration complexes occurs with the

hydrophobic side of peptides facing the fatty acid chains of the membrane lipids. The

hydrophilic sides of -helical peptides form the inside wall of the water-filled pore (Ojcius and

Young, 1991). Amphipathic structures are important for this process and the -helix formed by

each bacteriocin monomer must at least consist of twenty amino acid residues in order to be able

to completely span the membrane (Lear et al., 1988). The diameter of the pores depends on the

number of bacteriocin monomers involved (Tahara et al., 1996).

2.2.5. Immunity proteins

The bacteriocin producing bacteria protect from their own bacteriocin activity through

the action of immunity protein, LanI, dedicated ABC transporter protein and LanFEG

(McAuliffe et al., 2001). The LanI is most probably closed with outside of the cyoplasmic

membrane and possibly confers immunity to the producer cells by preventing pore formation by

bacteriocin. While, LanFEG acts by bacteriocin transportation which is integrated in to the

membrane, back to the surrounding medium and maintaining the concentration of the bacteriocin

in the membrane. Most of the non lantibiotic immunity protein is encoded by a gene located

suddenly downstream of and in the same operon as the bacteriocin gene (Nes et al., 1996). The

bacteriocin immunity gene is located downstream of, but in the opposite orientation to the

structural gene of bacteriocin (Franz et al., 1999). Where as, the immunity of gene of CbnA is

19

also encoded in the opposite orientation, but not in close proximity to the structural gene (Franz

et al., 2000). The combined actions of both peptides are needed for complete bacteriocin activity

and they have only single immunity protein whose gene is linked to the structural gene of two

bacteriocins (Diep et al., 1996). Some of the bacterial strains produce multiple bacteriocins with

different immunity proteins which are specific to each of the bacteriocins (Diep et al., 1996). The

immunity protein is in cationic nature, size from 51 to 254 amino acids that provides immunity

against the bacteriocin. The interaction of immunity protein with membrane appears to protect

the bacteriocin producer against its own bacteriocin activity (Nes and Holo, 2000). Some of the

immunity proteins are devoid of apparent transmembrane helices which provide immunity to two

peptide bacteriocins that are larger and sized from 110 to 154 amino acids (Allison and

Klaenhammer, 1996). After that, the immunity protein from Lactobacillus plantarum C11

contains 247 -257 amino acids and predicted to span the cytoplasmic membrane (Diep et al.,

1996). Moreover, the immunity protein may allow some level of cross protection of the bacteria

against other closely related bacterioicns. For example, the immunity genes of CnbA and

enterocin B exchanged to confer cross protection against the related bacteriocins (Franz et al.,

2000).

2.2.6. Applications of bacteriocin in preservation of food products

The food industry is experiencing increased pressure by consumer demands to develop

natural preservatives, minimally processed food products with extended shelf life without usage

of chemical preservatives. Schnurer and Magnusson (2005) reported that world wide 5-10% of

food production was spoiled by fungal and bacterial pathogenic bacteria especially Listeria

monocytogenes. L. monocytogenes is gram positive and facultative anaerobic rod shaped

bacterium which is resistant against refrigeration condition, low pH (3.6) and high salt

20

concentration. These properties are responsible for 2500 illnesses and 500 fatal cases in the

United States (Deegan et al., 2006). Lactic acid bacteria are food grade microorganisms which

are widely used in the food industry for technological attributes and food preservative properties,

as because they produce antimicrobial compounds which inhibit a broad range of food spoilage

pathogenic bacteria especially Listeria monocytoges (O Sullivan et al., 2002). There is an

increasing demand from consumers for chemical free additives, ready to eat and minimally

processed foods with low salt, fat and sugar contents; bacteriocins are becoming an interesting

alternative for consideration as natural preservative for biopreservation of foods. Bacteriocins

from LAB offer several desirable properties which make them suitable for food preservation: (i)

bacteriocins are generally recognized as safe substances, and are non toxic to eukaryotic cells (ii)

become inactivated by digestive proteases and having some influence on the gut microflora (iii)

bacteriocins showed broad spectrum of inhibitory activity toward the many food born and

spoilage pathogens (iv) they reveal a bactericidal mode of action normally acting on the bacterial

cytoplasmic membrane (v) are usually plasmid encoded, facilitating genetic manipulation. In

recent years, many of the research works indicated the application of bacteriocins in food

preservation that offers several benefits: (i) bacteriocins can extend the shelf life of food and

exhibit protection in temperature abuse conditions (ii) can reduce the risk of food pathogens

transmission, chemical preservative usage and improve the economic losses due to food spoilage

(iii) they allow the use of less severe heat treatment without compromising food safety and allow

the marketing of novel food with less acidic, higher water and lower salt content (Thomas et al.,

2000). Schillinger et al. (1996) explained that the food supplemented with ex-situ produced

bacteriocins or by addition of bacteriocin producer strains under conditions that favour

production of bacteriocin in situ.

21

Biopreservation is defined as the use of lactic acid bacteria which has the ability to inhibit

the growth of pathogenic bacteria by synthesizing their own metabolites for enhancing the safety

of food. Nisin is one of the most promising bacteriocin approved by United States Food and

Drug Administration (USFDA) for use as food preservative and are allowed in more than fifteen

countries around the world (Delves-Broughton, 1990). Nisin is a small peptide molecule

produced by lactic acid bacteria Lactobacillus lactis sub sp. lactis, which showed broad spectrum

of inhibitory activity against food pathogenic bacteria such as L. monocytogenes, Staphylococcus

aureus and Clostridium botulinum (McMullen and Stiles, 1996). Moreover, nisin affects the post

germination stages of bacterial spore development by inhibiting pre-emergent swelling, out

growth and formation of vegetative cells (Davies and Delves-Broughton, 2000). The efficacy of

nisin was determined by Davies et al. (1997) for restrain of L. monocytogenes in ricotta type

cheese at 8oC. The growth of L. monocytogenes was significantly inhibited by the addition of

100 IUml-1 nisin at eight weeks of incubation period. In addition, Zottola et al. (1994) used nisin

containing cheddar cheese which is made by nisin producing lactococci as ingredient in

pasteurized process cheese and cold pack cheese spreads. Schillinger et al. (1996) reported the

pasteurized process cheese spreads with nisin to prevent the outgrowth of clostridia spores

including Clostridium tyrobutyricum. Furthermore, Pawar et al. (2000) determined the activity of

nisin (400 and 800 IU g-1) with sodium chloride (2%) against L. monocytogenes in minced raw

buffalo meat. At storage temperature 4oC, the growth of L. monocytogenes was significantly

inhibited in meat by nisin over a sixteen days incubation period. While, the higher L.

monocytogenes cell load was observed in control sample. The combination of bacteriocin with

NaCl (40mg ml-1) significantly reduced the growth of Salmonella spp. (2.84 log 10 CFU) in broth

(Ragazoo- Sanchez et al., 2009). Nilsson et al. (1997) evaluated the inhibitory effect of nisin

22

with carbon dioxide on the survival of L. monocytogenes in cold -smoked salmon at low

temperature. Addition of nisin (500 and 1000 IU g-1) with CO2 packed cold salmon resulted in 1

-2 log10 loss of L. monocytogenes over 20 days. Leistner (2000) described the concept of hurdle

(combined preservative factors) technology which began to apply in the food industry in a

rational way after the observation that survival of pathogens successfully decreased when they

confronted with multiple antimicrobial factors. The application of bacteriocin as part of hurdle

technology has received great attention in recent years; since the bacteriocin can be used in

combination of selected hurdles in order to enhance microbial inactivation (Deegan et al., 2006).

The application of chemical factors, natural antimicrobials, physical treatments (heat) and non-

thermal physical methods (pulsed electric field, high hydrostatic pressure (HHP) and modified

atmosphere packaging) combined with LAB bacteriocin increase the cell membrane permeability

of Gram positive and Gram negative pathogens (Galvez et al., 2007).

Notably, the presence of sodium chloride increased the antimicrobial activity of

bacteriocin (nisin, leucocin F10 and enterocin AS-48) against L. monocytogenes and

Staphylococcus aureus (Parante et al., 1998; Ananou et al., 2004). The combination of nitrate

with enterocin EJ97 and enterocin AS-48 increased antibacterial activity against L.

monocytogenes, Bacillus coagulans and B. cereus (Galvez et al., 2007). Moreover, the

combination of nisin with acetic acid and sorbate restrained the growth of L. monocytogenes in

Ricotta type cheese (Davies et al., 1997). Enterocin AS-48 with phenolic compound such as

carvacrol, eugenol, geraniol and hydrocinnamic acid were showed higher antibacterial activity

against B. cereus, B. macroides and Paenibacillus spp. in vegetable soups and purees (Grande et

al., 2007). The combination of bacteriocins with chelators such as disodium pyrophosphate,

EDTA, sodium citrate, sodium lactate, sodium polyphosphate and sodium triployphosphate

23

enhanced antibacterial activity against Salmonella, Escherichia coli, L. monocytogenes,

Arcobacter butzleri and B. coagulans in beef and chicken (Galves et al., 2007). The combination

of nisin with other bacteriocins such as pediocin AcH and leucocin F10 increase their

antibacterial activity against L. monocytogenes in broth (Parante et al., 1998). Yin et al. (2007)

reported that the combination of nisin with Pediocin ACCEL enhanced inactive ation of L.

monocytogenes in fresh fish fillets. Ananou et al. (2004) reported that the activity of enterocin

AS-48 was higher against S. aureus which are sub lethally affected by heat due to the lower

concentration of remaining viable cells and to the cell damage stimulated by thermal treatment.

Modified atmosphere packaging system was used for prolonging the shelf life of food products

in food industry. The growth of L. monocytogenes was inhibited on pork immersed in nisin and

packed in 80% CO2 and 20% air at 4oC (Fang and Lin, 1994). Furthermore, the bacterial cell

death or injury caused by high hydrostatic pressure (HHP) increases with pressure and the

synergism with bacteriocins. Farkas et al. (2003) reported that the combination of nisin with

HHP showed excellent synergistic activity against L. monocytogenes growth. Similarly, the

combination of HHP with nisin and lacticin also enhanced inactivation of S. aureus and L.

monocytogenes associated with milk and whey (Black et al., 2005; Morgan et al., 2000). The

pulsed electric field (PEF) is a non thermal method which produces high voltage pulses between

a set of electrodes for microbial inactivation. PEF could also be applied to enhance the

antimicrobial action of bacteriocins, since PEF disrupt the bacterial outer cell membrane and

allowing bacteriocin to reach the target bacterial cytoplasmic membrane (Galvez et al., 2007).

The combination of nisin (2.55ppm) with pulsed electric field (PEF) were increased inactivation

of E. coli K12 (1.5log reduction) and L. innocua (0.7log reduction) in orange juice (McNamee et

al., 2010). Martínez-Viedma et al. (2008) reported that the enterocin AS-48 with PEF showed

24

greater inhibition against pathogen Salmonella enterica in apple juice. In addition, the

combination of nisin with PEF inactivates of S. aureus in skim milk (Sobrino-Lopez and Martin-

Belloso, 2006). Recently, the bacteriocin from LAB was also been used as a medicine in

pharmaceutical industry. Most of the pathogenic bacteria were found to be resistant against the

antibiotics. For this sense, the discovery of bacteriocins is a possible solution to aforementioned

problems (Schnurer and Magnusson, 2005).

2.3. Exopolysaccharides of LAB

2.3.1. Production of exopolysaccharides

Exopolysaccharides (EPS) are renewable resource representing an important class of

polymeric materials of biotechnological value with a wide variety of potential applications

(Kumar et al., 2007). EPS are long-chain polysaccharides containing branched, repeating units of

sugars or sugar derivatives such as glucose, fructose, mannose and galactose etc, which are

secreted into their surrounding environment during the bacterial growth. Microbial

exopolysaccharides such as dextrans, xanthan, gellan, pullulan, yeast glucans and bacterial

alginates are potentially used in many industries as food additive including xanthan from

Xanthomonas campestris and gellan from Pseudomona elode. Microorganisms are more suitable

for EPS production than macroalgae and higher plants, since they display high growth rate and

manipulate the provided environment for their growth resulting in enhanced EPS production

(Parikh and Madamwar, 2006). Among the wide variety of polysaccharide producing micro-

organisms, lactic acid bacteria have gained special attention because of the remarkable property

of the polymers they synthesize and as they don t carry any health risk which are generally

recognized as safe (GRAS). In gastrointestinal tract, EPS from LAB will remain stable in order

to enhance the colonization of probiotic bacteria (Kanmani et al., 2011d). EPS produced by LAB

25

either capsular polysaccharides (CPSs) or slime polysaccharides. The CPSs strongly bind with

bacterial cell surface, while slime EPS are secreted in to surrounding environment. These

biopolymers are composed of one type of monosaccharide (homopolysaccharides) or repeating

units of different monosaccharides (heteropolysacchrides) with molecular weight ranges from

4.0×104 to 6.0 ×106 Da (Mozzi et al., 2003). The composition of monosaccharide is independent

on the sugar source used and the differences in polymer depends on the carbohydrate present in

the medium (Mozzi et al., 2001). Mozzi et al. (2003) reported that L. casei CRL 87 synthesizes

heteropolysccharides such as glucose, galactose and rhamnose and this monomeric composition

independent on the sugar source used. Tuinier et al. (1999) reported structural and functional

relationship of EPS from LAB reveal number of variation in composition, charge, spatial

arrangement, rigidity and interact with proteins and DNA. Moreover, the correlation between

EPS concentration and viscosities are important for functionally valuable polysaccharides

production.

2.3.2. Biosynthesis of EPS from LAB

Anabolic and catabolic pathways are involved in biosynthsis of EPS by LAB. Many of

the bacterial strains use UDP- glucose, UDP-galactose and dTDP-rhamnose as precursors for

EPS production (Whitfield and Palment, 2003). When glucose present in the medium, both the

anabolic and catabolic pathways of sugar degradation appears to glucose -6- phosphate, in which

the flux of carbon bifurcates between the formation of fructose- 6- phosphate toward the

products of glycolysis, biomass and ATP formation and toward the biosynthesis of sugar

nucleotides, the precursors of EPS (Welman and Maddox, 2003). Degeest and De Vuyst (2000)

reported that the enzyme -phosphoglucomutase, UDP- glucose pyrophosphorylase and UDP-

galactose 4- epimerase were important for biosynthsis of EPS from Streptococcus thermophilus

26

LY03. These enzymes were involved in the conversion of glucose -6- phosphate to glucose -1-

phosphate, glucose -1- phosphate to UDP- glucose and dTDP- glucose and UDP- glucose to

UDP- galactose. In addition, the enzymes RfbaA, B, C and D have also converted the glucose 1-

phosphate to dTDP-rhamnose. Moreover, the glucose 1- phosphate serves as hub for channeling

sugars to the formation of glycogen and links with glycolysis, gluconeogenesis and CO2 fixation

(Laws et al., 2001). Also Levander and Radstrom (2001) explained the role of enzyme

phosphoglucomutase play at the branching point between glycolysis and leloir pathway, in

glucose and lactoctose metabolisms of S. thermophilus and its relationship to EPS metabolisms.

Lactobacillus casei use two alternative pathways for galactose metabolisms such as lelior

pathway and tagatose 1, 6- biphosphate pathway (Chassy and Thompson, 1983). In the lelior

pathway, the exogenous galactose was transported though the ATP energized permease system

activated by ATP dependent galactokinase and released galactose 1- phosphate in to the cells for

the synthesis of sugar nucleotides. In another way, galactose 6-phosphate was released in to the

cytoplasm due to the transport of galactose though the galactose specific phosphotransferase.

Similarly, Mozzi et al. (2003) reported that the EPS producing strain L. casei CRL 87 displayed

higher galactose-P uridyltranferase, UDP-galactose 4-epimerase, UDP-glucose

pyrophosphorylase and dTDP-glucose pyrophosrylase activity (40.2± 5.1, 11.1±2.8, 48.2±1.7,

28.0±1.2 nmol min-1 mg cell protein-1) when cells grown in medium contain galactose as carbon

source, while the presence of glucose showed lower enzyme activity. These results indicated that

the amount of galactose was metabolized through leloir pathway for the synthesis of sugar

nucleotides. This flux is a outcome of sugar metabolism in L. casei, while growth on galactose

provides sugar directly to glycolysis aswell to precursor formation, where as cells growth on

glucose requires - phosphoglucomutase flux towards glucose 1- phosphate which compensate to

27

direct glucose from glycolysis to formation of precursors (Mozzi et al., 2003). Hence the number

of polysaccharides was released from aforementioned sugar nucleotides via the action

housekeeping enzymes.

2.3.3. Genes encoding EPS production by LAB

Genetic information of both Gram positive and Gram negative bacteria indicates that the

biosynthetic pathway of the hetero polysaccharides is controlled by several house keeping genes

and a cluster of EPS related genes, which contains four functional regions involved in (i)

regulation of EPS production, (ii) chain length termination, (iii) biosynthesis of EPS repeating

unit and (iv) polymerization and export of repeating units (Dan et al., 2009). EPS production by

mesophilc bacteria such as L. lactis subsp. cremoris and L. lactis subsp. lactis were usually

associated with plasmid and the genetic instability can be described by the loss of that plasmid.

On the other hand, the genes encoding EPS production by thermophilic LABs (L. delbrueckii

subsp. bulgaricus and S. thermophilus) were located in chromosome and the genetic instability

can be explained by the mobile elements or genomic instability such as deletions and

rearrangements (Stingele et al., 1996). In general, genes encoding EPS biosynthesis in both

mesophilic and thermophilic LABs strains are organized in four functional regions: a central

region containing genes for glyccosyltransferase which is essential for the assemblage of EPS

repeating unit, two regions flanking the central region which show similarity to enzymes

participated in polymerization and export, and a regulatory region located in the 59th end of EPS

gene cluster (Degeest et al., 2001). Also the chimeric structure of eps locus may involve in both

horizontal transfer and genome exchanges within L. lactis and S. thermophilus (Degeest et al.,

2001). Similarly, Liu et al. (2009) predicted horizontal gene transfer events between two strains

which includes the transfer of EPS biosynthesis genes from S. thermophilus to L. bulgaricus and

28

gene cluster cbs-cblB(cglB)-cys-E for the metabolisms of sulfur containing amino acids,

transferred from L. bulgaricus from S. thermophilus. Nga (2006) also explained horizontal gene

transfer system in S. thermophilus for EPS synthesis. A 32.5-kb variable locus of S. thermophilus

CNRZ368 chromosome, known as eps locus, contains 25 open reading frames (ORFs) and 7

mobile elements. The 17 ORFs are related to polysaccharide synthesis in many bacterial strains.

The end 13.6-kb regions encoded seven mobile elements and eps ORF which are relatively

similar to epsL of L. lactis NIZOB40. These results suggested that the 13.6-kb region may

acquired from L. lactis by horizontal transfer and that genetic exchanges within the S.

thermophilus strains would have accounted for the variation in the different eps ORFs.

Moreover, the genetic locus of EPS in S. thermophilus Sfi6 revealed a 15.25-kb region encoding

15 open reading frames. Expression of EPS in the non EPS producing heterologous bacteria,

Lactococcus lactis MG1363 showed that within the 15.25-kb region, a 14.52-kb region encodes

13 genes from epsA to epsM that were capable of directing EPS synthesis. In the Swiss-prot

database, homology searches of the predicted proteins showed high homology (40 to 68%

identity) for eps A, B, C, D, and E and the genes participated in capsules synthesis in S.

pneumonia and S. agalactiae. Homology (37 to 18% identities) was observed for epsB, D, F and

H with genes encoding capsules synthesis in S. aureus. Genetic locus of EPS in L. lactis NIZO

B40 encoded 12-kb regions which located on a single 40-kb plasmid (Welman and Maddox,

2003). Lamothe et al. (2002) determined that the similarity between gene cluster of other LABs

strains. Gene cluster of eps in L. delbrueckii subsp. bulgaricus encoded 18-kb regions containing

14 genes which are homologous to that of other LABs gene clusters. The EPS gene clusters of

four L. rhamnosus strains consists chromosomal DNA regions of 18.5kb encoding 17 ORFs

which are highly similar to each other (Peant et al., 2005). The central portion of the locus

29

contained six genes (WelF, WelG, WelH, WelI, WelJ and WelF) which encoding potential

glucosyltransferase. Dabour and LaPointe (2005) determined the complete nucleotide sequences

of 17.5-kb of chromosomal DNA regions encoding 19 ORFs in L. lactis subsp. cremoris SMQ-

461. Within 17.5-kb region, a 13.2-kb region encodes 15 ORFs containing 14 genes (epsA to

epsM and epsR) which involved in regulation (epsR), chain length determination (epsABC),

biosynthesis of repeating unit epsDEFG, epsI and eps K) polymerization (epsH and export

(epsM). These results showed that high (90%) level similarity to Lactococcal strains, L. lactis

subsp. cremoris NIZO B40 and HO2 (Dabour and LaPointe, 2005). Recently, Dan et al. (2009)

identified the eps gene cluster of L. fermentum TDS030603, revealing a 11,890 base pair

chromosomal DNA regions encoding 13 ORFs, within which six ORFs encodes six genes (epsB

to epsG) capable of directing EPS biosynthesis. These genes were homologous to genes in L.

delbrueckii subsp. bulgaricus. L. rhamnosus GG gene cluster contain 17 putative ORFs, of

which 16 ORFs encodes genes involved in the biosynthesis of polysaccharides and 1 encodes a

putative transposase (Lebeer et al., 2009). The genes from L. rhamnosus GG exhibited higher

similarity to other LABs such as L. rhamnosus ATCC 9595 and L. reuteri F275.

2.3.4. Applications of exopolysaccharides from LAB

Lactic acid bacteria are able to produce exopolysaccharides which are a natural promising

candidate for industrial application. In nature, bacterial EPS fulfills a variety of diverse functions

including cell protection, adhesion of bacteria to solid surfaces and involved in cell to cell

interactions. Addition of EPS (or) EPS producing LAB strains in dairy foods can provide

viscofying, stabilizing, gelling and water binding function. In this sense, a growing interest has

developed in use of EPSs produced by LAB which have been awarded as Generally Recognized

As Safe (GRAS) status. Moreover, EPS from LAB have potential application in the

30

improvement of the mouth feel, rheology, texture and taste perceptions of fermented products

(Welman and Maddox, 2003). EPS may play a role in the probiotic activity of several LABs.

During the fermentation of milk with EPS + LAB exhibits a ropy (or) viscous texture and also

enhances the viscosity and reduces syneresis in yoghurt. Similarly, EPS produced by S.

thermophilus is predominant, suggesting an important role played by this EPS for the

improvement of yoghurt texture (Stingele et al., 1996). High consumer demand for smooth and

creamy dairy products, which is typically met by increasing the content of fat, sugars, proteins or

stabilizers. Demand for low fat, sugars content products and low levels of additives and low cost

factors make EPS a viable alternative (Welman and Maddox, 2003). In recent years, many

researchers have reported EPS + LAB can increase the functional properties of low fat

mozzarella cheese. Because LAB producing EPS have potential water binding properties and

moisture retention in low fat cheese, as well as EPS producing LAB starter culture can also

improve the moisture and melt properties of mozzarella cheese. Similarly, EPS producing S.

thermophilus has been shown to improve the high moisture content and melt properties of low

fat mozzarella cheese without deleterious affecting whey viscosity (Broadbent et al., 2001). Zisu

and Shah (2005) reported that the EPS producing S. thermophilus starter culture shown to

increase the meltabilty and strechability of the low fat mozzarella cheese, as well as the moisture

content of cheeses increased to 55.82% in the presence of EPS (41.18mg g-1). Moreover, EPS

decreased the hardness, springiness and chewiness of cheeses made with pre-acidified milk. The

higher moisture (56.67%) content was observed in pre-acidified low fat mozzarella cheese

containing capsular and ropy EPS (30.42 and 30.55 mg g-1). The low fat mozzarella cheese

containing EPS producing S. thermophilus starter improved shred fusion, meatability and a

reduction in surface scorching (Zisu and Shah, 2005). Saija et al. (2010) have used EPS present

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dairy ingredients and EPS producing S. thermophilus for the manufacture of mozzarella cheese

with better moisture and meltability. Recently, the increased demand for natural polymers for

diverse industrial applications has led to a renewed interest in EPS production by

microorganisms. The EPS from microoragnisms are widely used in the food, cosmetic, textile,

pharmaceutical and chemical industries and functions as biofloccculants, bioabsorbants,

emulsifiers, heavy metal removal agents, drug delivery etc (Wang et al., 2008). Physiological

function of EPS is believed to act as the first line of biological defense against phogacytosis,

phage attack, antibiotics, toxic metal ions and physical stresses including desiccation and

osmotic stress. Moreover, several EPS exhibit bioactivities beneficial to health including

prebiotic and anti-inflammatory effect (Fukuda et al., 2010). The EPS have also been reported to

show antiulcer, antiviral, immunomodulatory, antioxidant and antibiofilm activity (Kodali and

Sen, 2008; Kim et al., 2009). The antioxidant compounds play a role in preventing and curing

chronic inflammation, atherosclerosis, cancer and cardiovascular disorders. So, most of the

research work are focused on the production of natural antioxidant polysaccharides which have

potential applications in food industry. Free radicals such as superoxide anion radical (O2-),

hydroxyl radical (OH) and reactive oxygen species (ROS) are highly reactive molecules derived

from metabolisms of oxygen. The ROS play a role in cell physiology as well as damage of cell

membrane and DNA, inducing oxidation which cause membrane lipid peroxidation and

decreased membrane fluidty (Pan and Mei (2010). EPS of L. lactis sub sp. lactis displayed

stronger superoxide and hydroxyl radical scavenging activity and significantly decreased the

level of malondialdehyde (MDA), while increasing the activity of catalase (CAT) and superoxide

dismutase (SOD) in mice in a dose dependent manner (Pan and Mei, 2010). Kodali and Sen

(2008) found that the EPS of Bacillus coagulans RK-02 had stronger superoxide, hydroxyl and

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1, 1 diphenyl -2-picrylhydrazyl (DPPH) radical scavenging activity and significantly enhanced

lipid peroxidation inhibition in mice liver microsomes. Formations of biofilm by pathogenic

bacteria are considering as an important cause of chronic and recurrent infections, specifically

because of their capability to form and persist on medical surfaces and indwelling devices. Kim

et al. (2009) found that the EPS of Lactobacillus acidophilus A4 had stronger antibiofilm activity

against the growth of enterohemorrhagic E. coli O157: H7, Salmonella enteritidis, S.

typhimurium KCCM 11806, Yersinia enterocolitica, Pseudomonas aeroginosa KCCM 11321, L.

monocytogenes ScottA and B. cereus.

2.4. Production of folate

Folate is a most essential nutritive component in human diet. It is involved as a cofactor

for the transfer of one carbon units from donor molecules into important biosynthetic pathways

leading to methionine, purine and pyrimidine biosynthesis. Also, folate mediates the

interconversion of serine and glycine and role in histine catabolism. It is a water soluble B group

vitamin including normally present food folate (polyglutamates) and synthetic folic acid in

supplements and fortified foods. Deficiency of folate in human may create several health

disorders including cancer, cardiovascular diseases, growth retardation, megaloblastic anaemia,

congenital malformations and neural tube defects in newborn infants (Iyer et al., 2010;

Gangadharan et al., 2010). The human body needs daily 200-400 g of folate, while pregnancy

woman are advised to take double doses (Muhamad Nor et al., 2010). Recently, high level intake

of chemically produced folate have been shown to cause severe health defects such as vitamin

B12 deficiency, potential unknown risks for pregnant woman and induce cancers. Thus, the

rationale would suggest focusing on naturally produced folate for fortification. Many research

works have focused on the biosynthesis of folate by LAB strains. Iyer et al. (2010) reported that

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the high level of folate (58 g l-1) produced by S. thermophilus RD102 in optimum culture

condition. LABs such as Lactococcus lactis NZ9000, L. lactis MG1363, L. plantarum I-UL4 and

L. johnsonii DSM 20553 have capacity to produce folate. Among them, the higher folate

(60.39mg ml-1) production was observed in L. plantarum I-UL4 (Muhamad Nor et al., 2010).

Dana et al. (2010) recorded increasing folate production by Lactobacillus spp., in skim milk

fermentation. Folate producing LAB isolates Lactococcus sub sp. cremoris CM 22 and

Lactococcus sub sp. lactis CM 28 showed highest level of folate production (12. 5 and 14.2 mg

ml-1) after 7 h of fermentation in skim milk (Gangadharan et al., 2010). Tomar et al. (2009)

reported that the optimum medium components enhance the folate production by folate

producing S. thermophilus.

2.5. Production of biosurfactants

Biosurfactants are amphiphilic compounds which produced by microorganisms with a

marked surface activity. These molecules show a distinct tendency to accumulate at the interface

between fluid oases that exhibit different degrees of polarity and hydrogen bonding such as oil

and water, air and water, reducing the surface and interfacial tension (Van Hamme et al., 2006).

Moreover, they can exhibit a vast variety of chemical structures such as glycolipds, lipopeptides,

lipopolysaccharides, phospholipids, fatty acids, neutral lipids, polysaccharide-protein complexes

and with several advantages over chemical surfactants including low toxicity and higher

biodegradability and effectiveness at specific temperature and pH (Rodriguez et al., 2010).

Biosurfactants were also contained diverse properties and exhibited several physiological

functions to the producer strains such as enhancing solubility of hydrocarbon/water insoluble

compounds, heavy metal bindings, bacterial pathogenesis, cell adhesion and aggregation and

biofilm formation. Some of them are showed to possess antibacterial, antiviral and antifungal

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proprieties (Gudina et al., 2010). The interest in these biosurfactants has increased considerably

in recent years, as they are potential candidates for many commercial applications in the food,

pharmaceuticals, petroleum and biomedical industries (Moldes et al., 2007). The biosurfactants

are used as emulsifiers in food industry especially bakeries, where they play a significant role in

the rheological characteristics of flour and meat products. Also they have been shown to promote

the biodegradation and bioremediation of hydrocarbons. Antiadhesion is another important

properties of biosurfactants against bacterial pathogens (Gudina et al., 2010). Biosurfactants

from LABs have been shown to reduce adhesion of bacterial pathogen in glass, silicone rubber,

surgical implants and voice prostheses. Previous adsorption of biosurfactants was used as a

preventive event to delay the biofilm formation by pathogens in catheters and medical insertional

materials, decreasing the use of synthetic drugs and chemicals (Gudina et al., 2010). Number of

bacterial strains produced a wide variety of biosurfactants. Among the biosurfactant producing

bacteria, LABs are a most promising biosurfactant producing strains because of their diverse

functional properties. Lately, The LAB strain L. pentosus have produced biosurfactant with high

stability and good emulsifying capacity using liognocellulose as growth substrate (Portilla Rivera

et al., 2008). Recently, Gudina et al. (2010) isolated biosurfactant from L. paracasei sub sp.

paracasei A20. The biosurfactant (between 25-50mg ml-1) showed better antibacterial (

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2.6. Production of antioxidant

Oxidation is important to many living things for the production of energy to fuel

biological process and oxidative stress that can damage many biological molecules. It is well

established that oxygen centered free radicals and other reactive oxygen species are continuously

produced in vivo during the passage of nutrients through gastrointestinal tract (Lin and Chang,

2000). Oxidative damage plays an important pathological role in human diseases including

cancer, emphysema, cirrhosis, atherosclerosis and arthritis. Human and other organisms possess

antioxidant defense and repair system which have evolved to protect them against oxidative

damage, these systems are not enough to completely prevent the damage. Furthermore,

antioxidant supplements may help to reduce the oxidative damage in human body (Lin and

Chang, 2000). Several LAB strains possess antioxidant activity which are able to decrease the

risk of accumulation of ROS while ingestion of food. LABs also decreased the superoxide anion,

hydroxyl radicals and hydrogen peroxide (Virtanen et al., 2007). Said and Gilland (2005)

evaluated the antioxidant activity of L. delbrukeckii sub sp. bulgaricus, L. acidophilus and L.

casei by free radical absorbance capacity. When soymilk ferment with LAB strains including L.

acidophilus CCRC 14079, S. thermophilus CCRC 14085 and Bifidobacterium infants CCRC

14633 and B. longum showed significant antioxidant activity. It was measured by the inhibition

of ascorbate autoxidation and scavenging of free anion radicals (Wang et al., 2006). Klayraung

and Okonoki (2009) reported that the LAB strains L. fermentum FTL2311 and FTL10BR

displayed antioxidant activity which expressed as trolox equivalent antioxidant capacity (TEAC)

and equivalent concentration (EC) for free radical scavenging activity. The L. fermentum

FTL2311 revealed TEAC and EC values of 22.54±0.12 and 20.63±0.17 M mg-1 respectively,

while L. fermentum FTL10BR showed TEAC and EC values of 24.09±0.12 and 21.26±0.17 M

36

mg-1 respectively. L. casei 114001 and L. fermentum ME-3 exhibited excellent antioxidant

activity by the suppression of luminol oxidation and microsomal lipid peroxidation (Uskova and

Kravchenko, 2009). Leuconostoc mesenterioides subsp. cremoris, L. jensenii and L. acidophilus

showed higher antioxidant activity in fermented milk whey. The antioxidant activity was

measured by analyzing the radical scavenging activity using a spectrometric decolorization assay

and lipid peroxidation inhibition was assyed using liposomal model system peroxide (Virtanen et

al., 2007). Capcarova et al. (2010) reported that the presence of L. fermentum and E. faecium

significantly increased the antioxidant activity in broiler chicken. Moreover, antioxidant enzymes

such as superoxide dismutase and hydroperoxidase have provided protection against ROS

(Bruno-Barcena et al., 2010). Acid stress has been shown to be associated with the induction of

Mn superoxide dismutase in Lactococcus lactis.

2.7. Production of siderophores

Iron is an essential element for most of the living things in order to run their number of

crucial biochemical reactions including reduction of the oxygen for ATP synthesis, reduction of

ribotide precursors of DNA, formation of heme and detoxification of oxygen radicals. Iron (0.4-

1.0 M) is required for the optimum growth of microorganisms in culture medium (Patel et al.,

2009). Some of the environmental restrictions and biological imperative have tempted microbes

to produce specific molecules which can compete with hydroxyl ions for the state of ferric iron, a

nutrient which is abundant but biologically lacking. Iron requiring microbes usually produce

siderophores during the growth of microbes under iron starvation conditions which are specific

iron (III) chelating agent (Neilands, 1981). Siderophores are low molecular weight (500-

1000Da) iron chelating agent which bind ferric ions with high affinity (Neilands, 1981).

Previously, Streptococcus faecalis and L. plantarum ATCC 14917 have been well studied in

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relation to siderophores production (Pandy et al., 1994). Pandy et al. (1994) studied the iron

requirement and siderophores production in LAB strains belonging to the genera of

Lactobacillus, Lactococcus, Pediococcus, Leuconostoc and Carnobacterium. Recently, Patel et

al. (2009) found siderophores (154.03 MW) from potent probiotic stains of Bacillus spp. and

chemically characterized that siderophores containing 2, 3-dihydroxy benzoic acid.

2.8. Preservation of probiotic bacteria

2.8.1. Freeze drying

Freeze drying is one the most important, convenient and successful techniques for long

term preservation of probiotic bacteria, yeasts and sporulating and non-sporulating fungal and

bacterial strains (Zayed and Ross, 2004). In this method, cells are first frozen at -196oC and then

dried by sublimation under high vacuum conditions (Santivarangkna, et al., 2007). The

processing conditions were usually associated with freeze-drying which was milder than spray-

drying and high cell viability were achieved in freeze-dried powders. Surprisingly, this method

has been shown that cellular inactivation occurs typically at the freezing step (Meng et al., 2008).

To and Etzel (1997) found that 60-70% of cells survived at the freezing step and can live at the

subsequent dehydration step. During the freezing process, the extra-cellular ice formation causes

an increase in extra-cellular osmolality, so as rapidly as ice forms outside of the cell in solution

and the cell begins to dehydrate. Two types of freezing methods involved etc. slow freezing and

fast freezing. The slow freezing process gradually dehydrating the cell as ice is slowly formed

outside the cell leads to widespread cellular damage, while fast freezing can avoid solute effects

and excessive cellular shrinkage (Fowler and Toner, 2005). During the freezing step, the higher

cell surface area could increase the cell membrane damage due to extra-cellular ice crystal

formation (Fonseca et al., 2000). Therefore, cell size is an important factor on survival of

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probiotics during freeze-drying, with small spherical cells such as Enterococci spp., are being

more resistant to freezing and freeze-drying than larger rod shaped Lactobacilli spp., (Fonseca et

al., 2000).

2.8.2. Spray drying

Spray drying is an important alternative inexpensive method which retaining higher cell

viability than freeze drying process (Morgan et al., 2006). The spray-drying approach involves

the injection of medium at high velocity at temperatures up to 200oC and produces which

produces dry granulated powders from a slurry solution by atomizing the wet product at high

velocity within a chamber (Meng et al., 2008). Accordingly, this process results in exposure of

the drying medium to high temperatures for a short time, which can be detrimental to the

integrity of live bacterial cells. Bacterial cells encounters heat stress, dehydration, oxygen

exposure and osmotic stress at time spray drying process (Teixeira et al., 1997). During the

spray-drying, the cell membrane can increase cell permeability which may result in the leakage

of intracellular components from the cell into the surrounding environment (Meng et al., 2008).

Type of probiotic strains, outlet temperature and drying medium were important factors for

retaining the high relative cell viability during the spray drying method. Recently, many research

works have focused on the pos