STUDIES ON PRESERVATION OF SEA FOOD

PENAEUS MONODON BY PHYSICAL, CHEMICAL

AND BIOLOGICAL METHODS

Thesis Submitted to Pondicherry University for the Degree of

DOCTOR OF PHILOSOPHY

By

K. A. PAARI, M.Sc.,

Department of Biotechnology

School of Life Sciences

Pondicherry University

Puducherry 605014

INDIA

AUGUST 2012

PONDICHERRY UNIVERSITY

DEPARTMENT OF BIOTECHNOLOGY

SCHOOL OF LIFE SCIENCES

PUDUCHERRY-605014

INDIA

DR. V. ARUL

Associate Professor

CERTIFICATE

Certified that this thesis entitled “Studies on preservation of sea food Penaeus monodon by

physical, chemical and biological methods”is a record of research work done by the

candidate Mr. K. A. Paari during the period of his study in the Department of

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

that it has not previously formed the basis of the award of any degree, diploma, associateship

or fellowship.

Puducherry

Date: (V. ARUL)

Phone: 91-413-2654429 (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.

Puducherry

Date: K. A. PAARI

Dedicated to my

Beloved parents and teachers

CONTENTS

S.NO. CHAPTERS PAGE NO.

1 GENERAL INTRODUCTION

2 REVIEW OF LITERATURE

3 MATERIALS AND METHODS

4 BIOPRESERVATION OF PEANAEUS MONODON USING

PROTECTIVE CULTURE STREPTOCOCCUS PHOCAE PI 80

ISOLATED FROM MARINE SHRIMP PENAEUS INDICUS.

5 POTENTIAL FUNCTION OF A NOVEL PROTECTIVE CULTURE

ENTEROCOCCUS FAECIUM-MC13 ISOLATED FROM THE GUT

OF MUGHIL CEPHALUS: SAFETY ASSESSMENT AND ITS

CUSTOM AS BIOPRESERVATIVE

6 COMBINED EFFECT OF ANTIOXIDANT PACKAGING AND

GAMMA IRRADIATION ON SHELF LIFE EXTENSION OF

PENAEUS MONODON

7 EVALUATION OF PROCESSING METHODS LIKE GAMMA

IRRADIATION AND HEAT TREATMENT ON ANTIOXIDANT

PROPERTIES OF FRUIT PEEL EXTRACTS AND ITS

PROSPECTIVE APPLICATION IN PRESERVATION OF TIGER

PRAWN PENAEUS MONODON

8 STUDY ON THE EFFECT OF MODIFIED CHITOSAN ON THE

PRESERVATION OF TIGER PRAWN PENAEUS MONODON

9 SUMMARY

10 REFERENCES

11 PUBLICATIONS

ACKNOWLEDGEMENTS

Foremost, I would like to convey my heartfelt gratitude to my supervisor Dr. V. Arul,

Associate Professor, Department of Biotechnology for the continuous support of my Ph.D.,

research, for his patience, motivation, enthusiasm and immense knowledge. His guidance

helped me throughout my research.

Besides my supervisor, I would like to thank my Doctoral committee members: Dr. N.

Arumugam and Dr. N. Thirunavukarasu, for their encouragement, insightful comments and

suggestions during my research.

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

extending lab facilities and valuable suggestions.

I am thankful to Prof. S. Jayachandran, Dr. Sudhakar, Dr. Hanna Rachel Vasanthi, Dr.

Prashant, Dr. Arun Kumar Dhayalan, Dr. Venkateswara Sarma, and Mr. V. Balasubramanian

who have been influential in my research. I am also thankful to my school teachers who

motivated and groomed me in my academic pursuits. I am thankful for the support provided

by the EPR lab of the Chemistry Department, Pondicherry University.

I thank my fellow lab mates: Dr. P. Kanmani, Dr. R. Satish, Mr. N. Yuvaraj, Mr. V.

Pattukumar and Mr. Venkatesh, for their immense co-operation, emotional support and for all

the fun that we had during my course which really helped me a lot. Will be missing them. you

guys gave a homely feel at our lab.

I genuinely thank my seniors, Dr. A. Gopalakannan, Dr. S. Isaac Kirubakaran, Dr.

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

colleagues Mr. G. Raman, Mr. Jean Cletus, Mr. Kennedy, Mr. Saranathan, Mr. Abhijith, Mr.

Aezaj, Mr. Wasim, Mr. Anand, Mr. Suresh, Ms. M. Revathi, Ms. P. Lalitha, Ms. Moushmi

Priya, Ms. Asha, Ms. Pathma, Ms. Veena, Ms. Shubashree, Ms. Gokulapriya, Ms. Upas, Ms.

Mamta, Mr. Rahul, Mr. Naresh, Mr. Veeresh for their immense co-operation and valuable

support. Sabari anna, vasanth anna and Rajesh supported me in many ways that made me feel

light at hard times of my life. It won’t be nice if I forget my school buddies Er. Ezhilarasan,

Er. Ajay, Er. Madhan, Er. Hari, Er. Pressana. They were eager to see me as a researcher. I

also thank our office staffs, Mr. Ramalingam, Mr. Balakrishnan, Mr. Vadivel, Ms. Sarala,

Ms. Vinothini, Mr. Meiappan, Ms. Chandra and Ms. Muthammal for their co-operation and

help. Dr. Osmali and Dr. Thayan flooded me with lot of new ideas and boosted my

confidence whenever I felt down.

I gratefully acknowledge the monetary support given by CSIR, New Delhi in the form of

senior research fellowship and Department of Biotechnology, New Delhi for project funding.

I would like to thank Central Instrumentation Facility (CIF), Pondicherry University for

providing assistance in using necessary instruments.

Though there is no need to thank my parents, as whatever I have with me is from them, I

wish to convey my gratitude to my mother Ms. Valarmathi and father Dr. K. Alagesan for

showering me with love and care throughout my life. To them I dedicate this thesis. My

father taught me how to fight back hard times of life. Thanks pa... my mother believed in me

more than I believed myself. Thanks ma... I wish to thank my brothers Mr. Kumanan and Dr.

Ramkumar ceyar for providing a loving environment. They happily and readily overtook my

responsibility and played my role many a times during this study... thanks you guys.

I remember the blessings of my grandmother Shri. Anjutham. She showered me lot of

positive energy. She will be happy now I feel. Also I remember the blessings of my

grandfather Shri. T.R.K, who used to feel pride when I joined for this degree, both are not

live now, but your blessings made this happened...

Finally, I would like to thank everybody who is important for the successful realization of my

thesis, as well as expressing my apology that I could not mention personally one by one.

Date: K. A. Paari

LIST OF ABBREVIATIONS

APS - Ammonium per sulphate

bp - Base pairs

BHA - Butylated hydroxyl anisole

CFCS - Cell free culture supernatant

CFU - Colony forming units

Cm - Centimeter

DNA - Deoxyribose nucleic acid

dNTP - 2’ deoxynucleotide 5’ triphosphate

EDTA - Ethylene diamine tetra acetic acid

g/l - Grams per lit

H - Hours

kGy - Kilo gray

kDa - Kilo dalton

MRS - de Man rogosa and sharpe

Ml - Milliliter

mM - Millimolar

mg/l - Milligram per liter

N - Normality

Nm - Nanometer

OD - Optical density

PV - Peroxide value

PBS - Phosphate buffered saline

PMMA - Poly methyl methacrylate

PCR - Polymerase chain reaction

Rpm - Revolution per min

SDS - sodium dodecyl sulphate

TCBS - Thiosulfate citrate bile salts

TEMED - N,N,N-tetramethylethylenediamine

TMA - Trimethyl amine

TVB - Total Volatile base

TBARS - Thiobarbituric acid reactive substances

UV - Ultra Violet

V - Volt

VRBA - Violet red bile agar

V/V - Volume per volume

W/V - Weight per volume

1

INTRODUCTION

The processing of food products is no longer as simple or uncomplicated as it was in the

earlier period. Due to the lack of appropriate preservation methods, majority of fish

processing industries in the developing countries, endure major losses (loss of 25% from

their production) (Venugopal et al., 1999). Even in developed countries like USA and

European countries, which follow extremely advanced technology in food preservation,

food borne illness occurs sporadically leading to deaths. One such unpleasant incident

happened in the recent listeriosis outbreak in Santiago-de-chile in the years 2008 and

2009, which affected more than hundred people. The mortality rate allied to recent listeria

contaminated cantaloupe food borne pathogen outbreak in USA rose to 29, leading to

fierce ill effects to a large group of consumers demonstrating the poor and deprived

sanitary practices, services and facility. In the same way salmonella outbreak also caused

severe illness and deaths in United States in the year 2009. Salmonella enterica serotype

Kentucky was detected in various regions of Australia and India as an emerging food

pathogen. A recent report says that nearly 100,000,000 illnesses and 155,000 deaths occur

each year due to non-typhoidal Salmonella infection. Thirty one percent of food related

deaths is due to Salmonella related food borne illness, followed by Listeria (28%),

Campylobacter (5%), and Escherichia coli O157:H7 (3%). Centers for Disease Control

and Prevention (CDC) found that one in six Americans become ill, almost 128,000 are

hospitalized for severe food borne infections, 3,000 die owing to food borne diseases and

nearly 5 to 6 billion dollars are spend for hospital payments caused by means of food

spoilage and poisoning.

Food deterioration generally happen due to various factors like harvesting technique,

presence of food spoilage causing or food poison causing pathogenic microbes, change in

2

biochemical parameters during food harvesting and storage (Poli et al., 2006). Mounting

awareness and knowledge of consumers concerning the probable health risks associated

with a number of chemical and antibiotic food additives has led research groups to

instigate a hunt for natural, safe alternatives against food borne pathogens. Emergence of

pathogenic strains that are resistant to commonly used antibiotics has forced farmers and

aquaculture workers to dump more loads of antibiotics and chemicals to combat

aquaculture related diseases which resulted in the occurrence of antibiotic residues in

many sea foods (Aoki, 1975). Usage of antibiotics is reported to be unsafe to animal and

human health as more handling of antibiotic residues possibly will function as procreation

foundation for the emergence of resistant food borne pathogens (Cabello, 2006; Zhou et

al., 2009). Food and Drug Administration (FDA) in the year 2003 issued a circular to all

importers to register with their country‘s food product regulations. European Union too

framed a ―Zero tolerance‖ factor for the limiting the usage of antibiotic residues in the

imported aquatic food products from India. Firm standards for microbial counts for the

presence of food borne pathogens such as salmonella and coliform were also framed by

the U.S. and EU import regulations. Marine products from India had faced automatic

detention for shrimp products for not maintaining microbial standards. Similarly

European Union too banned Indian sea food due to poor hygienic and sanitary measures

but thankfulness to the agreement made between the Government of India and FDA,

which reinitiated the exports between the nations that were considered as vital for the

economy of the country. So, finding a suitable alternative preservation method is the need

of the time for marine food exporters and aquaculture society to enhance the trade and

production for national and international consumers.

3

Scientific communities have proposed human and eco- friendly alternatives such as

vaccines (Kurath, 2008), antibiotic substitutes (Dorrington & Gomez-Chiarri, 2008)

probiotics or Lactic acid bacteria (Kesarcodi et al., 2008) for food preservation. The word

―probiotics‖ is derived from the greek word termed ―for life‖ (Vasiljevic & Shah, 2008).

In 1908, Elie Metchnikoff, a Noble Laureate proposed the concept of probiotics.

Metchnikoff postulated that probiotics are accountable for maintaining the microbial

balance in the gut of living. He also proposed that consumption of Lactic acid bacteria is

the cause for healthy life style of Bulgarians which neutralize the ill effects of pathogenic

microorganisms. The World Health Organization (WHO) defined probiotics as ‗‗live

microorganisms when administered in adequate amounts conferring a beneficial health

effect on the host‖. Parker in 1974 defined probiotics as ―substsances that contribute to

microbial balance‖ in the intestine. Fuller in 1989 defined probiotics as ―live feed

elements‖ which improves the microbial balance of the host. Havenaar and Huis in‘t Veld

(1992) defined probiotics as ―mono or mixture of live cultures that enhance the properties

of indigenous microflora‖. 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 (Kanmani, 2011a). Probiotic bacteria have the ability to bind to the

mucus layer in the intestine and have the potential to survive the gastric and bile

environment. They bind, survive and have an effect on the intestinal immune system.

However, a study carried out by Mottet & Michetti (2005) proved the beneficial role of

dead cells of probiotic bacteria. Vibrio infection is mostly reported as causative agent for

diseases in Penaeid shrimp (Gomez et al., 1998; Singh et al., 1990). Chemical agents

were by and large used to control infection caused by vibrio, which leads to the

4

development of resistant strains (Aoki, 1975). Drawbacks caused due to chemical

additives can be minimised by finding a suitable alternative which can potentially carry

out the function of chemical preservatives. Safe natural microbial cultures are now

brought in to action for preserving foods. Although few commercial probiotic products

can be obtained in market, new protective cultures effectual of controlling food borne

pathogens are still in demand (Swain et al., 2009). Lactic acid bacteria isolated from

certain products would be the more appropriate choice as biopreservative (protective)

cultures for biopreservation of such same products, as they will be more competitive than

lactic acid bacteria isolated from some other sources (Jeppesen & Huss, 1993; Vignolo et

al., 1993). The antagonistic mode of action by the probiotic protective culture is by the

secretion of lactic acid, free fatty acids, ammonia, hydrogen peroxide, diacetyl,

becteriolytic enzymes, bacteriocins as well as by several completely indefinite inhibitory

substances (Saarela et al., 2000). Bacteriocins are proteinaceous secretory molecules that

exhibit inhibitory activity against sensitive strains of bacteria. Bacteriocins are

ribosomally synthesized proteins that confer defence system to the host which generally

react on the membrane of pathogens forming pores resulting in the leakage of metal ions

K+, Na

+ and other cellular contents. Bacteriocins secreted by Lactic Acid Bacteria are of

high significance owing to their eventual use in the food processing industry as safe food

preservatives (O ‗Sullivan et al., 2002). Probiotic cultures are graded as bacteriocin

secreting and bacteriocin non-secreting protective cultures. However, Bacteriocinogenic

strains are considered as an admirable candidate for preserving foods as because their

bacteriocin could be used like an antibiotic substitute (Joerger, 2003; Gillor et al., 2008).

Usually lactic acid bacteria (LAB) have been utilised to maintain microbial stability in

fermented foods and now reported to have various optimistic effect with non fermented

food products. Instead of using a heterolactic strain isolated from a different source, we

5

have attempted to use a homolactic strains i.e., LAB, isolated from the gut of shrimp is

applied for controlling diseases when during preservation of sea foods. Probiotic potential

of LAB strains Streptococcus phocae PI80 and Enterococcus faecium MC13 isolated

from the gut of Penaeus indicus and Mugil cephalus respectively was characterized in our

lab. Laboratory studies revealed the potential of these two cultures against shrimp and

fish pathogens (Swain et al., 2009; Pattukumar et al., 2012; Gopalakannan & Arul, 2011).

Bacteria belonging to the group LAB are graded with two safety status ―Generally

recognised as safe‖ and ―Qualified presumption of safety‖. Although, infection or

bacterimia due to consumption of LAB are reported very rarely (0.05%–0.4%), a

commission named ―International Platform For Lactic Acid Bacteria‖ designed few rules

to warrant the safety of LAB culture ahead of employing it for food allied applications.

But as the incidence of bacterimia are rising with respect to LAB treatment, Donohue et

al. (1998) listed few methods for analysing the safety of LAB by using in vitro studies,

animal models and indicated that the safety standards of probiotic strains have to be

established by analysing the metabolic activities, toxicity studies and the properties of the

microbe. Nonexistence of pathogenicity is a fundamental factor of probiotic safety,

therefore it is vital to authenticate the safety of any new strain by acute oral toxicity tests

for probiotic safety assessment (Duangjitcharoen et al., 2009). Japan started encouraging

the use of probiotic products. Nearly 53 products containing cultures of probiotic

properties have been marketed (Vasiljevic & Shah, 2008).

Radurization is a irradiation process carried out to sterilize the fishery products and this

process has been developed for various fishery products (Chwla et al., 2003; Mahrour et

al., 2003; Poole et al., 1994). The success rate of irradiation in increasing the shelf life of

food products normally depends on the following factors, initial quality of the fish, dose

6

selected for irradiation, packaging conditions for irradiated foods and storage

temperature. The prospective potential of this technique for extending the shelf life and

the keeping quality of fishery products is supported by research worldwide. Irradiation

can put a stop to the growth of pathogenic microorganisms in foods but spoilage due to

physical and chemical path may occur. The free radicals formed through the process of

food irradiation can counter with different muscle components leading to damage. Free

radicals are reported as the cause for carcinogenesis, mutagenesis and cardiovascular

disease (Seifried et al., 2003; Valko et al., 2007). Antioxidant combinations are generally

used for quenching the free radical generated damages in irradiated pork (Ahn & Nam,

2003), turkey meat (Ahn et al., 2006), fishery products (Pazos et al., 2005). Both

chemical and natural antioxidants are engaged to scavenge the radicals generated in

irradiated food (Ahmad, 1996). However, due to the increase of toxicological concerns

regarding the use of synthetic antioxidants and increasing customer fondness for foods

stabilized with natural antioxidants (Löliger, 1989), there have been mounting comfort in

picking out plant extracts to control oxidation of lipids in lipid-based products (Ahn et al.,

1998) as they are considered to be safe and no special approval for their application is

necessary. Antioxidant properties of rosemary (Vareltzis et al., 1997), ginger extracts

(Fagbenro et al., 1994), grape seed extracts (Gokoglu & Yerlikaya, 2008), pomegranate

extracts (Gokoglu et al., 2009) and citrus peel (Kang et al., 2006) was employed in

preservation of foods. Irradiation of fishery products results in the production of

malonaldehyde that reacts with nitrogenous substances in the food ending up in the

discolouration of fish surface. Polyphenolic compounds from natural sources are now

employed to control melanosis development. Gokoglu & Yerlikaya (2008) have

demonstrated the inhibitory activity of monomeric phenolic compounds of grape seeds on

the inhibition of melanosis in shrimp. Finding natural antioxidants which can quench the

7

free radical mediated damages is the current demand of food industries which use

irradiation as mode of preserving foods. Also the antioxidant potential of natural

antioxidants can be enhanced by various techniques like heat and irradiation treatment.

Chitosan, a natural polysaccharide comprising glucosamine and N-acetylglucosamine, has

been used widely in food processing, medicine and biotechnology fields (Harish

Prashanth & Tharanathan, 2007). In the recent past, chitosan became an appealing

molecule due to its antibacterial, film forming property, antioxidative and biodegradable

ability (Fan et al., 2009; Rhoades & Roller, 2000). Enrichment of antioxidant activity of

chitosan by gamma irradiation at three different doses were reported by Feng et al.

(2008). Suitability of irradiated chitosan in inhibiting oxidative rancidity in meat sample

has been reported (Kanatt et al., 2004). In this study, the antioxidant and antimicrobial

activity of chitosan was enhanced using irradiation. The effect of modified chitosan

derivatives were analysed during the preservation of Penaeus monodon. The spoilage of

some seafood is well understood from our previous research and this understanding has

enabled the development of new efficient natural preservation techniques. Our research

will shed light on those factors responsible for food spoilage and will provide the

overview of the new technology on extending the shelf life of sea foods by preventing the

growth of food pathogens.

8

REVIEW OF LITERATURE

The processing of fishery products is no longer as undemanding as it was in the earlier

period. Plenty of sophisticated techniques are being developed to indulge the demands of

customer contentment. In majority of developing countries like India, sea food processing

industries suffer major losses (about 25%) due to lack of satisfactory refrigeration

services (Venugopal et al., 1999). Schnurar & Magnusson (2005) reported that almost 5-

10 % of worldwide food production is ruined by food spoiling pathogenic

microorganisms. Requirement for fishery products are getting higher. Minimising the loss

that occur during post harvest handling and enhancing the quality and safety of fishery

products will be able to play a primary role in satisfying this demand. Improved

understanding and management of the successful food preservation techniques could ease

food researchers to develop potent preservation methods. Knowing the unique attributes

of each preservation technique has subsequently turned out to be vital for opting methods

to preserve food products. Seafood spoilage is governed by various parameters like

improper handling practices, inappropriate postharvest usage conditions, the subsistence

of food degrading microbiological agents and at last due to unrestrained physiological

changes that cause spoiled biochemical reactions (Gram & Huss, 1996; Ward & Baj,

1988; Venugopal, 1999; Poli et al., 2006). The spoilage processes of few food

commodities are currently well understood and this knowledge has enabled the food

technologist to improve common conventional preservation techniques in to novel food

based specific preservation techniques. Due to the country‘s enormous marine water

potential, fishery products cannot only be considered simply as a food resource, but also

as a source of exportation revenues. Blue revolution in India has really augmented the

production of sea food products from 0.75 to 6.1 million metric tons during the period

from 1951 to 2003. The stretched lengthy coastal line (8129 Kms), two million km2 of

9

fishing economic zone, 1.2 million hectors of brackish water bodies are at present being

appropriately utilised to meet the demands of local and international consumers. Fishery

products are the major export item in case of quantity (38.42%) and the second largest in

earnings for the countries trade (20.42% in US $ earnings). In this food production,

shrimp products are the important product in the countries total marine export,

contributing 66.97 per cent. India is budding as one of the top ten producer of marine

products as nearly 85% of the products are exported to United States, European Union,

Japan and China. Also it is reported that the export rate is getting increased by a margin

of 20- 25%. As on data, India produces almost nine million tonnes of sea food and makes

trade worth nearly $ 4 billion in 2011. The business line of a magazine stated that shrimp

exports had increased by 16 % in quantity and 43% in dollar earnings during last year. A

recent study analyzed that production of fishery products would reach 1.2 crore tonne by

2015 and the revenue will be around Rs. 23,500 crore by 2013. However, with the

increase in the sea food production, there is also an increase in the demand from national

and international consumers. The mounting demand for sea food products will not be

compromised by today‘s commercial fish stocking and preserving method. And so

enhanced management of sustainable methods, for preserving the quality of food products

are the need of time. New programmes/process for enhancing the security of food from

production to consumption is now promoted by World Health Organisation. This review

provides the overview of the new technology, changing demands of food quality, safety,

and preservation methods for inhibiting spoilage of foods.

Quality of fishery products:

Sea foods harvested from marine sources undergo various handling practices from the

harvesting site until it is supplied to customer. Utmost freshness for sea food products can

be noticed immediately after the harvest. Beyond that, the qualities of fishery products are

10

directly dependent on the handling system, physical and biochemical treatments. Quality

loss happens due to microbial and chemical contamination and the product values are

generally compromised. Global fish losses due to quality deterioration are estimated to be

20-40% and were estimated to be 3 million to 12 million tonnes (FAO, 2005). The

deterioration rate is getting increased due to poor harvesting technology, processing

technology, preservation technology and marketing systems. The quality of fishery

product is influenced primarily by the following factors: inherent class of fish, interaction

between the product and the environment, quality loss during handling and storage and

the microbial quality of stored fish. Discolorations of fish resulting in melanosis,

oxidation of lipids resulting in poor sensory attributes and uncontrolled autolytic changes

are the major quality problems that occur in fishery products (Ashie, 1996; Bremner,

1988; Whittle, 1990). Lipid oxidation is a main factor responsible for deteriorating the

quality of fish products which happens due to the high lipid content and the magnitude of

polyunsaturation in fishery products (Azhar & Nisa, 2006; Ackman, 1989). Colour

change is another damage that happens with fish during quality loss. The translucent

appearance of the product alters to opaque form when stored in inappropriate storage

conditions (Colby, 1993). The colour of flesh changes to yellow from its natural colour

due to lipid oxidation and reactions with carbonyl amines. In addition to lipid oxidation,

myetoperoxidase from fish leukocytes and the free radicals formed during storage react

with the β-carotene resulting in the colour changes. With respect to texture, the firmness

of the fish becomes mushy due to myofibrillar disintegration caused due to the action of

proteases. Few marine fishes and fresh water fishes contain trimethyl amine oxide

(TMAO), which is broken down as trimethyl amine. The level of trimethyl amine is an

index of chemical spoilage. The major food spoilage causing amine trimethylamine oxide

is transformed to trimethylamine and consequently to di-methylamine and in the end to

11

formaldehyde which produce off-odours and a fishy smell. Release and secretion of

volatile compounds from fishery products are responsible for the changes in odour. Few

regular classes of bacteria accountable for fish spoilage includes Pseudomonas,

Aeromonas, Yersinia enterocolitica,, Shewanella, Salmonella sp., Staphylococcus aureus,

different species of Clostridium botulinum, Bacillus cereus, Escherichia coli 0157:H7,

Vibrio parahaemolyticus and Listeria monocytogenes (Dillon,1992; Doyle, 1989;

WHO,1994; Chou. et al., 2006; Hassan et al., 2012). Many of above mentioned spoilage

bacteria are psychrotrophic in nature which can grow at low temperature under

refrigerated condition. These spoilage causing bacteria degrades the nitrogen sources in

the fish muscle resulting in the evolution of volatile compounds. Secreted volatile

compounds have an negative effect on the sensory worth of the fishery products. Food

spoilage potentials of predominant pathogens in fishery products have been determined

by Alur et al. (1995; 1989). A lot of studies have reported the association between levels

of trimethyl amine and other biogenic amines to predict the invulnerability of sea foods

(Veciana- Nogues et al., 1990; Al Bulushi, 2009). Food spoiling pathogens carry out few

chemical modifications which are associated with the production of trimethyl amine, total

volatile base, hydrogen sulfide, dimethyl sulfide and methyl mercaptan, indole, skatole,

putrescine, and cadaverine etc. (Crawford, 1996; Alur, 1995; Ashie, 1996; Venugopal,

1990). These volatile amines are formed by pathogenic microorganisms due to

decarboxylation of free amino acids. Food spoiling pathogens are also reported for

production of biogenic amines (Ordonez et al., 1999; Ozogul & Ozogul, 2007). Lactic

acid bacteria are reported for reducing the production of biogenic amines in fishery

products (Yongjin et al., 2007; Zhong-Yi et al., 2010). Preserving the quality of fishery

products will undoubtedly contribute to enhanced food security and better income.

Progressing study is now required to develop low-cost, non-destructive, potential methods

12

to reduce the post harvest damage which will be useful for maintaining the standards of

quality of fishery products and is vital to warrant sea food safety.

Natural antimicrobials from microbial sources

Lactic acid bacteria

Lactic acid bacteria (LAB) are Gram-positive, resistant to acid conditions, have the ability

to survive lower pH, have low GC profile, strongly fermentative, usually non-motile, non-

sporulating bacteria that secrete lactic acid as the end product through fermentation of

carbohydrates. Different species of lactic acid bacteria belonging to the group

Streptococcus, Leuconostoc, Pediococcus, Aerococcus, Enterococcus, Vagococcus,

Lactobacillus, Carnobacterium grow generally in diverse environmental environment like

gastrointestinal tract, dairy food products, seafood, plant surfaces (Ring & Gatesoupe,

1998). Microorganisms belonging to Lactobacillus spp., Bifidobacterium spp.,

Saccharomyces boulardii, Enterococcus spp., Bacillus spp., and a few other

microorganisms are considered as probiotic strains (Patel et al., 2009). The word

‗probiotics‘ find its origin from the Greek word ‗for life‘. The word ―probiotics‖ was

initially coined by Kollath, a German bacteriologist and briefed them as ―microbially

derived factors‖. According to definition from the World Health Organisation ‗probiotics

are denoted as ―live micro organisms which when administered in sufficient quantity

offer a health profit on the host‖. Elie Metchnikoff, a Russian Noble laureate in the

ground of medicine conceded out a study on probiotics and proposed that these microbes

are accountable for maintaining microbial balance in the gut by replacing the proteolytic

bacteria and other harmful pathogens (Gordon, 2008). German professor Alfred

Nissle isolated E. coli from a soldier to treat shigellosis when antibiotics were not

accessible. Roy fuller in 1989 defined probiotics as supplements that play the role of

13

enhancing microbial balance. Used by tradition around the globe for promoting

healthiness and for therapeutic purposes, probiotics that are chiefly from the LAB group

are at the moment engaged in various fields. Probiotics are generally used to cure acne

vulgaris (Bowe, 2011), preventing intestinal barrier dysfunction (Lutgendorff et al.,

2009), hypertension (Huey-Shilye et al., 2009), allergic rhinitis (Ouwehand et al., 2003),

preservation of foods (Paari et al., 2011c; 2012), anti cancer properties (Satish et al.,

2011).

During the past two decades protective probiotic cultures are used in huge quantities as

health promoting bacteria in diverse food system, due to its safety and functional

characteristics. These live microorganisms are of food grade and exert beneficial effects

to the host when supplied along with the food system and are graded as GRAS (Generally

Recognised As Safe) by the United States Food and Drug Administration (Rodgers,

2001). They are extensively used in food industries for their food preserving properties

and for their skill to contain the growth of food borne pathogens (O‘ Sullivan et al.,

2002). Lactic acid bacteria usually guarantee for selection and implementation as

protective cultures for bio preservation of foods. Lactic acid bacteria make use of the

carbohydrate accessible in the food and secrete lactic acid as metabolite which lowers the

pH ensuring the safety of preserved food product.

Antimicrobial compounds like organic acids, bacteriocins, exopolysaccharide, hydrogen

peroxide secreted by the microorganisms of LAB group bring them success in food

industries. Potent microorganisms having food preserving properties are employed for

preserving food. According to Ross et al. (2002) biopreservation refers to the process of

preservation of food employing beneficial microorganisms or their secretory metabolites.

As consumers claim for chemical free additives, minimally processed foods, bacteriocins

14

and bacteriocin secreting starter cultures are becoming an appealing candidate for

contemplation as natural preservative for biopreservation of foods.

Safety/ Regulatory issues of probiotics

Government policies deviate and diverge amongst different countries, where a standard

status of probiotics as an component in food is not acknowledged universally on an

international basis. Regulatory establishments need to implement a ‗standard of identity‘

for grading cultures as probiotics with proper documentation system (Reid, 2005). For the

most part, Probiotics are in general considered as dietary supplements moderately than as

biological products and so assessment of the safety of the probiotics, their purity and

―potency of action‖ before marketing is not made mandatory (Boyle et al., 2006). Lactic

acid bacteria encompass an extensive record of use as probiotic microorganism and

acquire an excellent safety record (Borriello et al., 2003). Lactobacilli, Pediococcus and

Leuconostoc have been far and wide used in food industries for a long phase in human

history and have been taken as food stuffs for a long time (Mäyrä-Mäkien & Bigret,

1993). Approval by Food and Drug Administration is not required for marketing dietary

supplements but endorsement and complete pre-market review is made mandatory for

products marketed for disease treatment. Probiotic products in Australia are considered as

―complimentary medicine‖ and require prior market review by the Board of Therapeutic

Goods Administration. Europe and Japan require proper market review and should satisfy

the regulations of the legal requirements (Commission of the European Communities,

1996: FAO/WHO, 2001). The Joint FAO/WHO (Food and Agricultural Organisation and

World Health Organisation) consultation recommends the labelling of employed probiotic

strain; also they recommend that identity of the strain must be disclosed at species level

as the effects provided by the strains are species specific. Reid et al. (2001) reported few

15

regulatory issues; the label should contain the information regarding the viable

concentration of each and every protective probiotic culture. It is vital to substantiate the

safety of any new strain by oral toxicity tests that have been advocated as the fundamental

test for probiotic safety assessment studies (Duangjitcharoen et al., 2009). Though,

International Platform for Lactic Acid Bacteria (LABIP) assures the safety of lactic acid

bacteria based on the research outputs conceded out by different research groups,

appraising the riskiness of probiotic culture, in front of its usage as a biopreservative has

turned out to be essential to puzzle out few food safety problems. Often new isolates do

not hold any history concerning their safety and may not share the Generally Recognised

As Safe (GRAS) status of long-studied lactic acid bacteria. The safety of lactic acid

bacteria has been doubted in few studies which developed rare infections (Boulanger

&Lee, 1991; Harty et al., 1994) and so confirming the safety of protective culture ahead

of incorporating it in to the food products is recommended (Donohue et al., 1998). The

regulations of ―food law‖ of European parliament certify the safety of food additives for

consumer agreement. The European food safety agency has formulated an approach

towards the safety of food additives named as ―qualified presumption of safety‖ (Jamet &

Chamba, 2008). French Food Safety Agency (AFSSA) confirms the safety of ingredients

of new starter cultures in France (AFSSA, 2002)

Properties and mechanism of probiotics:

A potent probiotic bacterium is expected to have the following properties for efficient

function;

- Acid and bile tolerance

- Adhesion to mucosal service

16

- Safe for food and clinical usage

- Reduction of cholesterol

- Antimicrobial action against food borne pathogens

- Zero pathogenicity to the host

- Safe for clinical trials and usage

- Enhancement of barrier properties in the intestinal epithelial cells.

These probiotic microorganisms carry out the pathogen inhibition process by blocking

adhesion sites for pathogens (Resta-Lenert & Barrett, 2003). Adhesion to epithelial cells

is a principal means of access for pathogen survival and colonisation in the

gastrointestinal tract which leads to synthesis of toxins for initiating the necrotic process

(Satish et al., 2011). The binding sites in the intestinal epithelial surfaces are adhered by

the probiotic strains thus preventing the binding of pathogens like Listeria monocytogenes

(Samir et al., 2005; Sinead et al., 2007), Escherichia coli (Resta-Lenert & Barrett, 2003),

Salmonella spp (Casey et al., 2004). The epithelial barrier in the gut serves as a wall and

selects the beneficial substances from the gut preventing the entry of toxins and

pathogenic microorganisms. The lumen of the gut is populated by a small amount of

valuable microorganisms that carry out the following function like maturation of

epithelial cells, stimulation of immune system in the mucous, competitive inhibition of

pathogens. Along with stimulation of immune system probiotics also normalize the ion

transport function in epithelial cells (Resta-Lenert & Barrett, 2009). Tolerance to bile and

acid factors are considered vital for the survival of the probiotic organism in the gastro

intestinal region and also for maintaining their adhesion to epithelial cells and metabolic

function. These probiotic organisms are now employed in the field of food preservation

17

(biopreservation). De Martinis et al. (2001) defined biopreservation as the method to use

non pathogenic microorganisms or their secretory metabolites for increasing the storage

ability of food products. Lactic acid bacteria able of synthesising antimicrobial peptides

are at present the candidate of interest for food research (Deegan et al., 2006; Jack et al,

1995). Nisin is one of the most hopeful bacteriocin approved by the United States Food

and Drug Administration (USFDA) for use as food preservatives (Delves-Broughton,

1990). Nisin, as well as several other antimicrobial compounds from Lactic acid bacteria,

is effectual in inhibiting pathogenic spoilage bacteria in food and other nutrient

containing systems. Nisin belongs to the lantibiotic group of class-I bacteriocins that are

in general synthesized by Lactococcus lactis. Reduction of bacterial contamination by

nisin treatments has been reported in different studies (Guerra et al., 2005; Mauriella et

al., 2005). Nisin either alone or in combinations with other antimicrobial substances have

found to be efficient as a hurdle for pathogen survival (Bari et al., 2005). Nisin is a

positively charged antibiotic peptide that is able to bind to negatively charged

cytoplasmic membranes of pathogens (Bonev et al., 2000). Nisin has been approved as a

food preservative and is effective in suppressing Gram-positive bacteria such as L.

monocytogenes and has been approved as a food additive by WHO (WHO, 1969). The

antimicrobial action of bacteriocin secreted by the probiotic microorganisms is generally

through the formation of pores in the cell membranes of pathogen and is now widely used

in various food applications (Breukink &De Kruijff, 2006; Delves-Broughton, 2005;

Joerger, 2007; Neetoo, 2007). The efficacy of nisin was determined by Davies et al.

(1997) for restrain of Listeria monocytogenes in ricotta type cheese at 8oC. Pawar et al.

(2000) determined the activity of nisin (400 and 800 IU g-1

) with sodium chloride (2%)

against L. monocytogenes in buffalo meat. At storage temperature 4oC, the growth of L.

18

monocytogenes was significantly inhibited in meat by nisin over a sixteen days incubation

period. whereas, the higher L. monocytogenes cell load was observed in control sample.

Bacteriocins can be applied as an element of hurdle technology that has received great

attention for microbial inactivation (Deegan et al., 2006). Bacteriocin activity can be

improved when treated in combination with other treatments such as heat treatment, high

pressure and irradiation treatment. Better membrane permeabilisation was noted in the

bacteriocin action when treated in combination with chelating agents (Fang &Tsai, 2003).

Diverse combinations of bacteriocins have also been tested. Combination of nisin with

Pediocin (Hanlin et al., 1993) and nisin with Leucocin (Parente et al., 1998) provides

enhanced antimicrobial activity. The combination treatment with bacteriocins was found

to be advantageous in following ways as these combinations may prevent the growth of

resistant microorganisms in a better way compared to the way explored by a single

bacteriocin and the concentration of bacteriocins can be condensed when treated in

combination with other bacteriocins. Bacteriocins when used along with heat treatment,

the intensity of heat can be reduced (Ueckert et al., 1998). Bacteriocin treatments are also

compatible when combined with pulse electric field and modified atmosphere packaging.

Calderón-Miranda et al. (1999) reported that the combination of nisin with pulse electric

field reduced the L. innocua count. 100% CO2 atmosphere with nisin reduced the Listeria

monocytogenes count (Nilsson et al., 1997; Szebo & Cahill, 1999).

Bacteriocin secreted by the lactic acid bacteria generally possess the following properties

(Galvez et al., 2007)

- They are not found to be toxic to eukaryotic cells

19

- They are reactive to proteases which describes the proteinaceous character of

bacteriocins. Bacteriocins are commonly digested by the digestive proteases and

made inactive so that the normal gut microbiota will not be disturbed

- Bacteriocins are in general tolerant to heat and pH

which facilitates the survival of

the proteinaceous substance in the intestinal pH.

- Bacteriocins exhibit wide scale of antimicrobial activity against food spoiling

microorganisms. They generally act by forming pores in the cellular membrane of

pathogens.

- Bacteriocins are supposed to be resistant to the digestion of gastric and bile

secretions. This asset of bacteriocin is essential for survival, adhesiveness and

metabolic functioning of probiotic organisms that secrete bacteriocins in gut.

- Genetic determinants of bacteriocin coding genes are normally coded in plasmids

that generally aid genetic manipulation.

Bacteriocin secreting probiotic microorganisms illustrate stable antimicrobial action

against foodborne pathogens in the environment of sea foods; this property increase our

interest in employing bacteriocins producing lactic acid bacteria as biopreservatives.

Foods can be preserved either with pure bacteriocins or by inoculating the bacteriocin

secreting protective culture (Stiles, 1996; Schillinger et al., 1996). Immobilized

preparations of Ex-situ secreted bacteriocins can be supplied as immobilized preparation,

that can be bound to a carrier like silica (Dawson et al., 2005), liposome (Degnan &

Luchansky, 1992), calcium alginate (Fang & Tsai, 2003). Selection and improvement of

bacteriocinogenic cultures are vital for improved production of bacteriocins (Ross et al.,

2000; Työppönen et al., 2003; Peláez & Requena, 2005; Leroi et al., 2010).

Bacteriocins secreted by the probiotic cultures are classified in to three groups Class-I, II,

III based on biochemical and genetic characteristics (González-Martínez et al., 2003).

20

Class-I bacteriocins are generally heat stable and undergo translational modification.

Nisin, a well established bacteriocin belongs to this group of bacteriocins. Based on the

charge and mode of action, class-I bacteriocins are further grouped as Class-Ia and Class-

Ib, the former one is positively charged and they act by forming pores on the membrane

whereas the later one is negatively charged and act by enzymatic reactions (Parada et al.,

2007). Class-II bacteriocins are generally small and are found to be less than 10 kDa.

They are further grouped in to three classes as Class-IIa, Class-IIb, Class-IIc based on

their activity. Pediocins, Sakacin bacteriocins belong to this group of bacteriocins. Classs-

III bacteriocins are high molecular weight proteins (more than 30 KDa).

Bacteriocin produced by Streptococcus sp and Enterococcus faecium

Streptococci bacteriocins belong to be in the cationic type A-lantibiotic group of

bacteriocins (Nes et al., 2007). Bacteriocin from Streptococcus salivarius was the primary

analyzed lantibiotic that limited the pathogen Streptococcus pyogenes (Ross et al., 1993).

Krull et al. (2000) reported the secretion of mutacins I, II and III by S. mutans which

encompass structural similarity to the lacticin 481. Nisin U produced by Streptococcus

uberis showed identity to Nisin A secreted by Lactobacillus lactis. Nisin U is considered

to be a variant of native nisin (Wirawan et al., 2006). Whitford et al. (2001) reported

secretion of bovicin 255 by Streptococcus bovis JB1 from rumen. Two dissimilar

bacteriocins BHT-A and BHT-B were isolated from Streptococcus rattus (Hyink et al.,

2005). Streptococcus rattus secretes a class I two-component lantibiotic and a class II

bacteriocin. Macedocin, a food grade bacteriocin produced by S. macedonicus was

isolated from artisan cheese (Georgalaki et al., 2002). Thermophilin 13, consisting of

peptide ThmA and Thmb bacteriocin is secreted by S. thermophilus, isolated from

yoghurt (Marciset et al., 1997). Our lab reported the secretion of antilisterial bacteriocin

21

phocaecin PI80 produced by S. phocae PI80, which was isolated from the gut of marine

shrimp Penaeus indicus (Satish & Arul, 2009).

Enterococci are gram-positive, cocci-shaped bacteria that are able to secrete bacteriocin

with antimicrobial activity towards a broad spectrum of pathogens. Enterococcus spp. like

Enterococcus faecium and Enterococcus faecalis exert admirable valuable effects and are

considered as safe, reliable probiotic bacterium. Amongst enterococci, E. faecium are

well-studied bacteria since they have produced most of the bacteriocins (enterocins).

Enterococcus faecium MC13 (AY751463) strain was previously isolated from the gut of

marine fish Mughil cephalus. Enterocin A and B secreted by E. faecium T136 were found

to be active against a broad range of Gram-positive bacteria (Casaus et al., 1997). Heat

and pH resistant Enterocin P showed inhibition against L. monocytogenes, S. aureus,

Clostridium perfringens and C. Botulinum. (Cintas et al., 1997). Enterocin A, enterocin B

and enterocin P like bacteriocins were reported to be secreted by E. faecium JCM 5804

which limited the growth of vancomycin resistant Enterococcus (Park et al., 2003).

Ananou et al. (2005) reported the inhibition of L. monocytogenes and S. aureus in

sausages by enterocin like bacteriocins. E. faecium EK13 produces enterocin A, which

showed excellent probiotic characteristics (Laukova et al., 2006). Enterocins secreted by

Enterococcus faecium MC-13 was studied for its biopreservative potential during

preservation of tiger prawn Penaeus monodon (Paari et al., 2012)

Biopreservation of sea foods:

Lactic acid bacteria were nominated as protective preservative culture to extend the

storage life, microbial control, chemical stability of fishery products during preservation

(Daeschel, 1989; Jeppesen & Huss, 1993; Helander et al., 1997; Gelman et al., 2001).

Quality changes during preservation of minced fish was analysed when biopreserved with

22

three lactic acid bacteria: L. plantarum, L. mesenteroides, P. pentocaseus (Gelman et al.,

2001). Though the concentration of cell inoculums for biopreservation was identical for

all three protective cultures, the quality changes mediated by these protective cultures

varied in fish products. L. plantarum, P. pentocaseus did not exhibit significant control

over microbial and chemical contamination, L. mesenteroides treated samples exhibited

lowest TVB-N, histamine, malonaldehyde values and lowest total bacterial counts.

Challenge studies performed employing three lactic acid bacteria (LTH 2342, LTH 4122,

LTH 5754) in single or in combinations inhibited Listeria monocytogenes in salmon

slides. All bacteriocin producing L. sakei strains limited the counts of Listeria by 2 log

CFU. Sensory properties of the cold-smoked salmon were not affected by the action of

the three lactic bacteria LTH 2342, LTH 4122, LTH 5754 (Weiss & Hammes, 2006).

Jiang et al. (2007) studied the biopreservative effect of pediocin secreted by the

Pediococcus pentosaceus ACCEL. The study analysed two diverse concentrations (1500

IU/mL and 3000 IU/mL) of pediocin for the preservation of skinless blue shark steak and

demonstrated that pediocin was found to be comparatively better than nisin in controlling

the food pathogen Listeria monocytogenes. Total aerobic plate count estimated in the

same research showed lower Log CFU in biopreserved samples compared to the control.

Nisin treatment resulted late toxin production in cod, herring fillets artificially inoculated

with food born pathogen clostridium. The mode of action of nisin is supposed to be by the

creation of pores in the cytoplasmic membrane that cause exhaustion of proton motive

force and loss of cellular ions, amino acids, and ATP (Crandall & Montville, 1998). The

application of nisin as a food preservative has been studied by various research groups

(Hurst & Hoover, 1993; Montville et al., 2001; Cleveland et al., 2001). Bacteriocins for

23

food applications are secreted by strains of Carnobacterium, Lactobacillus, Lactococcus,

Leuconostoc, Pediococcus, and Propionibacterium (Chikindas & Montville, 2002).

Many secretory peptides are found to be effective in in vitro media but have reduced

activity in food products. Therefore, an expanded study on activity of antimicrobial

peptides against spoilage microorganisms in food products is needed. The proficiency of

lactic acid bacteria on fish is useful, as they can grow on chilled meat and discourage the

growth of pathogenic organisms. Lactococcus piscium CNCM I-4031 was used for

improving the shrimp quality. Brochothrix thermosphacta was inhibited by 4 Log CFU

when co-inoculated with the protective culture Lactococcus piscium at a concentration of

106CFU. Increase in volatile bases was not recorded in the study. However, a shelf life

extension of about 10 days was noticed in shrimps treated with Lactococcus piscium (Fall

et al., 2010a). The same protective culture was analysed for anti listerial properties in

cooked peeled shrimp Penaeus vannamei. 3.4 Log reduction of Listeria monocytogenes

was noted in biopreserved samples compared to the uninoculated control shrimps (Fall et

al., 2010). Bacteriophage LISTEX P100 was investigated for its anti-listerial property

during the preservation of salmon fillets. Anti-listerial property was studied both in broth

and animal model system. In broth, Listeria monocytogenes was inhibited at the three

investigated concentrations (104, 10

6, and 10

8 PFU/ml) at three considered temperatures (0°C,

4°C, 10°C). Similar reductions were noticed in salmon fillets, resulting in minimized

pathogen count that was proportional to the concentration of phage. Though significant control

was not obtained in the 104 PFU/g dose of phage P100, 10

7PFU/g dose and 10

8PFU/g

dose yielded 2 and 3.5 log reduction of Listeria monocytogenes respectively. The study

also proved the stability of phage P100 on the salmon fillet tissue during storage

Mode of action:

24

Binding of bacteriocins to the cell membranes of food pathogens is the primary factor for

potent functionality of bacteriocins. Lipids in the membrane of gram positive food

pathogens are generally anionic in nature. The cationic character of bacteriocins shows

high affinity towards the anionic binding region of the cell membrane. The charge of the

nisin plays a crucial role in binding of bacteriocin. Van kraaij et al. (1997) reported that

the positive charge provided by the carboxy-terminal region are crucial for potential

binding of bacteriocin to the membrane of pathogens. The amino-terminal region of the

bacteriocin also plays a critical role on bacteriocin activity. Lys-Tyr-Lys residue sequence

in amino terminal region, occurrence of disulphide loop and the tryptophan residue in the

carboxy terminal region also determine the activity of bacteriocin Mesentericin (Fleury et

al., 1996). The N-terminal region of the bacteriocin plays a crucial role during the process

of membrane insertion. When Isoleucine from N-terminal region was replaced by a tryptophan,

it decreases the potential of membrane penetration by the bacteriocin. The bacteriocin

binds to the peptidoglycan precursor called Lipid –II layer after membrane insertion

forming ion-conducting pores which initiates the process of depolarizing the transmembrane

electric potential of the cells which propose that this peptidoglycan precursor molecule

might act as a docking molecule for bacteriocin binding, leading to pore formation. Other

than the role of amino terminal and carboxy terminal region of bacteriocins in membrane

binding and membrane insertion, the cell surface markers too play crucial role in the

mode of action. Interaction of cell wall markers like teichoic acid and lipoteichoic acid

also plays significant role towards initial interaction and for target specificity (Jack et al.,

1995). Contribution of receptor like factor was also reported by Fregeau Gallagher et al.

(1997) for Pediocin and Leucocin bacteriocins. Following membrane binding and

insertion the sensitive cells and the vesicles of the cells cause uncontrolled efflux of

essential cellular materials like cations and amino acids. Disruption of transmembrane

25

potential cause depleted proton motive force which interrupts the normal cellular

biosynthesis. Previous studies have indicated that the bacteriocins from Lactic acid

bacteria mediate the potassium ions (Mantovani et al., 2002; McAuliffe et al., 1998). A

study from our research confirmed the efflux of potassium ions by bacteriocin Phocaecin

secreted by Streptococcus phocae against E. coli DH5α, Listeria monocytogenes, and

Vibrio parahaemolyticus. The study confirmed the resultant leakage of potassium ions is

due to the formation of pores in the membrane resulting in the loss of intracellular

substances (Satish kumar & Arul, 2009).

Irradiation

Food irradiation is the practice of preserving food using ionising irradiation. High energy

radiations employed for food preservation is generally produced by radioisotopes Cobalt-

60 and Cesium-137. Cobalt-60 is advantageous and preferred over Cesium-137 as the

latter is water soluble, which possibly will be risky to environment (Venugopal et al.,

1999). Raw pork was sterilised using Schwartz in 1921 using x-rays. Commercial

application of x-rays for sterilising food products initiated in the year 1957. Joint

FAO/IAEA/WHO Expert Committee endorsed the technique of food irradiation to the

limit up to 10 kGy and resolved toxicity or safety suspicions on the wholesomeness of

Irradiatied Food. The universal ideologies or ―policy of practise‖ was outlined by the

Codex Alimentarius for the role of radiation services in the year 1984. Though, the

technique irradiation has got a history of 100 years and has been approved as a

scientifically recognized technology, the feedback of food industries towards this tool is

poor. The FAO (Food & agricultural organisation) and IAEA (International Atomic

energy agency) has accepted the custom of this method for preservation of food products

by implementing agreements globally. Scientific Committee on Food (SCF) encourage

the custom of this technique for eight different food products. Codex Alimentarius which

26

explains the globally acknowledged standards and guidelines removes the upper dose or

threshold boundary for irradiating foods. The pictogram ―Radura‖ was employed to

specify the ―symbol of quality‖ for processed food commodities by irradiation.

Radiation tolerance:

Dosage for irradiating foods can be divided in to three groups

A) Low dose

B) Medium dose

c) High dose

Low dose has the dose boundary up to 1 kGy. Medium dose is between 1-10 kGy and

high dose is above 10 kGy. Low dose is the dose usually applied to resources of plant

origin and medium dose is the dose used for supplies of animal origin and higher doses

are applied to spices. The oxygen in the air around the irradiation source gets oxidized

and so foods that are irradiated will also be oxidised. The oxidation rate depends on few

factors like dose level and dosage time. Therefore for this reason, radiotolerance of each

food has to be determined. The UN committee (Joint international committee on food

irradiation) allowed dosage up to 10 kGy for irradiating foods for human consumption.

Also Joint FAO/IAEA/WHO group accepts the dose up to 10 kGy for human

consumption. As lipid content and the oxidation rate of lipids are high in foods of animal

origin, the radiotolerance rate must be determined for improved marketability ahead of

irradiation of sea food.

Safety of irradiated foods:

The central shortcoming of irradiation technology is its name (Diehl, 1993). Consumers

think that food irradiation has got alliance with radioactivity and customers have nuclear

fear for irradiated foods. Despite lots of research promoting the benefit of this technology

for preserving foods, recognition and acknowledgement of this practice by food industries

27

and consumers develop little by little. Majority of feeding studies carried out to evaluate

the toxicity of food products were irradiated up to 10 kGy as most part of the applications

necessitate a radiation dose of not extra than 10 kGy. Furthermore, no toxicological

hazards were recorded for samples irradiated up to a dose of 10 kGy (WHO, 1981). Even

samples irradiated up to 58 kGy did not showed any harmful consequence in a study

carried out by Thayer et al. (1987). Scientific committee on food (SCF) recommended the

European community regarding the safety of irradiated foodstuff and authenticated that

no further animal feeding toxicological research are essential to measure the safety of

irradiated food up to10 kGy (Scientific Committee for Food, 1987). Even after these

many promises from the scientific community, the niche for irradiated food stuffs is still

little in the food market. Few opponents of food irradiation claim this technique as unsafe,

not nutritious, causing danger to consumer. However, Diehl (1991) reported that these

claims are fictitious and the European parliament is misinformed about this effective

technique. Experts from 38 countries formed an society named International Consultative

Group of Food Irradiation (ICGFI) recognized under the guidance of FAO, IAEA, and

WHO to aid governments in approving irradiation and marketing of food products. A

study carried out by FAO/IAEA/WHO assured that foods irradiated at higher dose

(between 25-60kGy) are safe to eat and nutritionally adequate (WHO, 1999; Diehl, 2002).

The state members of the United Nations adapted the codes for practise of irradiation and

the permits were revised by the Codex Alimentarius programme (Codex, 2003). An

international scheme named International Facility for Food Irradiation Technology

(IFFIT) was conducted to help out the developing countries to guide them in using

irradiation technology for food products (Farkas, 1985). Psychological factors, political

factors, propaganda and misinformation proposed by different protester groups and the

reluctance to put into service the irradiation process by food industries are reason for the

28

poor advancement of this method. Despite the fact that lot of projects and commission

take effort on efficiently propagating the practice of this technology, this technique is

underused and so, responsible execution from government sector is expected at the

moment to uphold this technology to make available hygienic nutritious foods to common

public (Ehlermann, 2005). The awaiting prospect of food irradiation will be based on the

awareness and understanding of the customers regarding the task of this practice in

controlling foodborne pathogens.

Status of food irradiation:

Market of irradiated food is in high note compared to non irradiated frozen meat in the

retail stores of USA ever since 1999 (Deeley, 2006). In Asian region, an agreement

named the Regional Cooperation Agreement (RCA) in association with the Food and

Agriculture Organization (FAO) and International Atomic Energy Agency (IAEA) inform

the prominence of irradiation for every two years. Quantities of irradiated food are high in

United States. A total of nearly 116,400 ton of irradiated foods is used in the USA,

Canada and Brazil which includes 101,400 ton of spices, 7000 ton of fruits and 8000 ton

of meat, representing 116,400 ton in total. The magnitude of irradiated food in Brazil was

23,000 ton in total. A total of 3,111 ton of vegetables, meats, sea foods, spices were

irradiated in France. Of this 2,789 ton of sea foods was sterilised using irradiation in

France. Nearly 183,309 tons of food commodities are irradiated in the Asian oceanic

region among which 15,208 tons were from meat and sea foods. In China, the amount of

irradiated food materials was 146,000 ton in totality which includes vegetables and

functional foods. In India, 1,500 ton of spices and vegetables and 100 ton of onion were

irradiated. A latest agreement with United States of America for irradiated mango and

other fresh fruits endorse the practice of this technique in India. Based on the last survey

29

data available, the total irradiated food quantity all over the world was 405,000 ton.

Spices and vegetables irradiated in large quantities in USA (80,000 ton) followed by

China (52,000 ton) and Brazil (20,000 ton). Fruits are irradiated in huge quantity in

Ukraine (70,000 ton). Vietnam (14,000 ton), USA (8000 ton) and Belgium (5500 ton)

were front runners in using irradiation for sterilizing meat and sea foods. China leads

other countries in using irradiation as a sterilisation method compared to USA and

Ukraine which stands next to China. In USA, spices are not labelled with irradiation mark

but fruits and meat products are sold with a label in the market. In Canada, if the

irradiated materials constitute more than 10% of the total food, a proper declaration is

advertised. Regulatory proposals for various food stuffs are at present under

consideration. The statistical values for irradiated foods were reported by Kume et al.

(2009). Nearly twenty five food products were permitted for irradiation and marketing in

hungary. The Hungarian food law promoted the usage of this technology through test

marketing. The response to irradiated foods for thirteen years revealed that consumers did

not averse irradiated foods (Kiss et al., 1990). Gamma irradiation was made successfully

a commercial process for decontamination of food products in Japan. The

commercialisation of irradiation technique was made successful by an national project in

Japan (Kume, 1985). Marketing for irradiated food stuffs is in a increasing note (Deeley,

2006).

Besides traditional preservation methods tried to stretch the storage life of fishery

products, irradiation is currently far and wide accredited as an precious tool for

inactivating pathogenic bacteria from food (Paari et al., 2011b; Manisha &Warrier, 2002;

Andrews & Grodner, 2004). Gamma irradiation has been engaged for decontamination of

dehydrated vegetables, fruits (Yu et al., 1993), meat products (Kanatt et al., 2007) and

animal foods (Kamat et al,. 2000). Application of gamma-radiation up to a dose level of

30

10 kGy can be used to get rid off or wholly diminish the numbers of food spoilage micro-

organisms in food products without compromising the nutritional or sensory quality

(Abu-Tarboush et al., 1996). Currently more than 40 countries lawfully recognized the

process of irradiation treatment for about sixty food commodities. Even if abundant

research groups admitted that gamma irradiation in low doses kills food spoiling

pathogenic micro organisms devoid of worsening food quality (Mendes et al., 2005;

Thayer et al., 1995), customers still have some suspicions relating to this physical

treatment. An extra benefit of this practice is that the processed food commodity in its

final wrap up pack can be sterilised using high penetration power by which re-infection

by food spoiling pathogens can be avoided. In spite of various research findings and

copious publications on the safety of engaging ionizing radiation to protect food, it is a

mostly underused technology.

The exposure of fishery products to lower doses has found to be an efficient method to

extend their shelf-life (Andrews & Grodner, 2004; Paari et al., 2011b). Radiation

treatment at doses of 2–7 kGy can effectively get rid of pathogens such as Salmonella and

Staphylococcus aureus, Listeria monocytogenes or E. coli O157:H7 (Farkas, 1998). A

transportation trial study proved the feasibility of irradiation processing for commercial

sliced dried Pollack via both sea and air transportation. Irradiated samples showed higher

acceptability than the non irradiated control in this trial study (Kwon et al., 2004).

Reduced chemical spoilage for irradiated samples were reported in various studies

(Mendes et al., 2005; Kyrana & Lougovois, 2002; Al-Kahtani, 1996; Jo, 2004;

Jeevanandam, 2001). Chouliara et al. (2004) reported reduced trimethyl amine by 1 and 3

kGy irradiation dose in refrigerated Sparus aurata fillets. Mendes et al. (2005) recorded

the uppermost TMA-N in non irradiated horse mackerel than the irradiated equivalent

during a 24 day study period. Ahmed et al. (1997) reported that formation of TVB-N and

31

TMA-N is limited in irradiated fish compared to non-irradiated. In contrast, a different

study carried out by Mendes et al. (2005) and Van Cleemput et al. (1980) showed no

adverse effect on trimethylamine content of Atlantic horse mackerel and shrimp at a dose

of 3 and 5 kGy.

Variation in amino acid content was not observed when haddock fillets were irradiated at

53 kGy by Proctor& Bhatia (1950). Thiobarbituric acid values were not significantly

affected in catfish fillets when treated at 0.5 kGy irradiation dose (Przybylski, 1989).

However, the influence of gamma irradiation up to 5 kGy on lipid oxidation showed

increased TBA values in both control and irradiated fish, particularly in mackerel and seer

meat (Ghadi &Venugopal, 1991).

Reduction of microbial spoilage in irradiated samples was reported in various studies

(Poole et al., 1994; Lakshmanan et al., 1999a; Sinanoglou et al., 2007; Ozden et al.,

2007; Cozzo-Siqueira et al., 2003). Minimised mesophilic count (Mendes et al., 2005),

reduction of H2S-producing bacteria (Abu-Tarboush et al., 1996) were reported in

irradiated samples. Lakshmanan et al. (1999) used 2 kGy irradiation dose to expand the

storage capacity up to four days for irradiated anchovy compared to the non irradiated

counterpart. Improved sensory shelf life was attributed to irradiated Trachurus trachurus

at 1 and 3 kGy and has reported lower total viable count (TVC) and amines (Mendes et

al., 2005). Extension of shelf life of cod fillets for 21 days was achieved by exposure to

1.5 kGy irradiation dose by Ronsivalli et al. (1968). Ozden et al. (2007) reported lower

Psychrotrophic bacteria and Enterobacteriaceae count for non irradiated compared to 2.5

and 5 kGy irradiated sea bass.

Antioxidants from natural sources for preservation of sea foods;

32

As fishery products are highly perishable due to the existence of fatty acids, irradiation

may cause radical generation. Unrestrained oxidative reactions are most important

culprits associated with the loss of sea food freshness. The resulting free radicals formed

due to the uncontrolled lipid oxidation respond with muscle components and cause food

deterioration. To lessen the loss that occur due to lipid oxidation, fish foods contains

endogenous antioxidant system (Pazos et al., 2005). Incorporation of antioxidants has

been reported for limiting the damages caused due to lipid oxidation (Guillen & Montero,

2007). Identifying potent antioxidants from natural sources rather than from artificial

synthesis is the growing interest (Peschel et al., 2006). The optimised custom of plant

antioxidants to preserve sea food is still in its infancy and a lot of research is vital in this

field for enhanced preservation of fishery products. Phenolic compounds are responsible

for the antioxidant potential of plant material (Pellegrini et al., 2000; Shahidi, 2000;

Sachetti et al., 2005). The antioxidant nature of phenolic compounds is due to the electron

(Hydrogen) donating ability of the plant polyphenols. Admirable oxidative stability in

mackerel fish was noted when treated with green tea extracts compared to the effects

from samples treated with synthetic antioxidants like butylated hydroxyl anisole and

butylated hydroxyl toluene (He & shahidi, 1997). An array of anthocyanins present in

fruits and vegetable materials were reported for protection against oxidative degradation

in the fishery products. Polyphenols from grape extracts have been comprehensively

studied for their antioxidant potential in various studies (Sa´nchez-Alonso et al., 2007;

Pazos et al., 2005a; Pazos et al., 2005b; Gonza´lez-Parama´s et al., 2004; Yilmaz

&Toledo, 2004; Escribano-Bailo´ n et al., 1995; Mazza, 1995). The potency of rosemary

extract in stabilizing dried sardine (Wada & Fang, 1994) and rainbow trout (Akhtar et al.,

1998) against oxidation was well studied. A study carried out by Selmi & Sadok (2008)

confirmed high radical scavenging potential of ethanol extracts of thyme Thymus

33

vulgaris. The authors reported better radical quenching potential than the standard

antioxidant BHT. The antioxidant potential of thyme during preservation of tuna fish was

exposed by the lower TBARS value and the unaltered fatty acids composition in tuna

fish. Utility of citrus peel powder to minimize oxidation of lipid was evaluated in salmon

meat homogenate by Kang et al. (2006). The authors noted that the salmon treated with

citrus peel provided considerable shield to lipid oxidation which was eminent by the

lower TBARS value in treated samples. Mushroom extracts were tried as a colour

stabilizer for processed fish meats by Bao et al. (2010). The same study compared the

phenolic content, ergothioneine level and DPPH radical quenching activity of various

mushrooms such as Flammulina velutipes, Hypsizigus tessellates, Pholiota nameko

Lentinula edodes, Pleurotus eryngii, Hypsizigus tessulatus, Grifola frondosa Pleurotus

cornucopiae, Flammulina populicola, Grifola frondosa and found that the extract

obtained from Flammulina velutipes was highly effective in stabilizing the colour of big

eye tuna and yellowtail meats. Inhibition of Lipid oxidation and met myoglobin

development in fish throughout storage was however noticed with all mushroom extracts.

Owing to awareness of the problems and side effects caused by chemical additives to

human health, natural antioxidants from herbs have received much attention for fish

preservation (Smid & Gorris, 1999; Attouchi & Sadok, 2010). A study carried out by

Attouchi and Sadok (2010) proved that treatment with 1 % (w/w) thyme powder reduced

TVB-N, TMA-N, TBA and LHC evolution in both the wild and farmed sea bream fillets

and extended their shelf life by about five days. The antibacterial activities of 10 different

plant polyphenols were evaluated against 96 bacteria [Staphylococcus aureus (20 strains),

Salmonella (26 strains), Escherichia coli (23 strains), Vibrio (27 strains)] which cause

food borne diseases (Taguri et al. 2004). The study found that the activities of plant

polyphenols against the bacterial groups are based on the structures of the polyphenols.

34

Epigallocatechin, epigallocatechin-3-O-gallate, castalagin and prodelphinidin showed

relatively elevated antimicrobial activities against the food spoilage causing

microorganisms. Shelf life extension of rainbow trout fillet was evident when treated with

essential oils of oregano, a perennial herb. The study found controlled level of pathogens

(H2S producing, Pseudomonas, Enterobacteriaceae) and controlled chemical

contamination in treated samples compared to the untreated counterparts (Pyrgotou et al.,

2010).

Extracts of oregano and cranberry were tested for antimicrobial nature against Vibrio

parahaemolyticus in vitro and in cod fillets and shrimps by Lin et al. (2005). Their

research declared that the utilization of combination of extracts limited vibrio count by 4

log CFU. Treatment with combined extracts exhibited better antimicrobial activity than

with the samples treated with single extract which may be attributed due to the action of

multiple phenolics for disrupting the cell membrane of pathogens. The same research

group reported the control of Listeria monocytogenes in cod fillets treated with

combination of oregano with cranberry. They compared the antimicrobial effect of

individual extracts along with the combination of extracts and concluded that the mixtures

of oregano (75%) and cranberry (25%) had the best inhibition in broth system. A study by

Gokoglu and Yerlikaya (2008) demonstrated the delaying of melanosis in Parapenaeus

longirostris by the usage of grape seed extract at a concentration of 2.5, 5.0, 10 and 15 g

L1. The study found that at application of 15 gL

-1 better sensory score, yellowness values

were observed.

Chitosan, a biopolymer has a extensive application in the food industries as a natural food

preservative (Rudrapatnam & Farooq ahmad, 2003; Shahidi et al., 1999). Chitosan is

derived from the deacetylation of chitin composed of glucosamine and N-acetyl

35

glucosamine linked by β 1–4 glucosidic bonds. The antioxidant, antimicrobial,

biocompatible, biodegradable property of chitosan attracted unique attention as food

preservative (Majeti & kumar, 2000; Fan et al., 2009; Kim & Thomas, 2007; Rhoades &

Roller, 2007). Shelf life of fishery products was extended by inhibiting the growth of

Pseudomonas and Shewanella by the addition of chitosan (Cao et al., 2009). Irradiated

chitosan displayed enhanced antioxidant activity in meat than autoclaved chitosan

(Kannat et al., 2004). Darmadji &Izumimoto (1994) projected that the antioxidative

nature of chitosan by the measuring the lipid oxidation products and oxidation rate. The

mechanism of action of chitosan biopolymer against foodborn pathogen is projected due

to the alterations in the permeability. The initial interaction between the positively

charged chitosan and negatively charged cell membrane of food pathogens resulted in the

formation of pores that caused leakage of protenaceous substances and other cellular

components (No et al., 2007). The antibacterial nature of chitosan depends on the

molecular weight (Jeon et al., 2001; No et al., 2002), degree of dacetylation (Tsai et al.,

2002) and type of target bacterium (No et al., 2002). The rate of oxidative stability was

compared with commercially available synthetic antioxidants like BHA, BHT. Inhibition

of lipid oxidation was found to be high in samples treated with chitosan in herring (Kamil

et al., 2002). The antioxidant nature of chiosan was also visualised by Kim & Thomas

(2007) during preservation of salmon. The study found that 30 kDa chitosan was efficient

in controlling negative shades of oxidation. Shelf life extension of fresh fillets of atlantic

cod and herring was analysed by Jeon et al. (2002). The study found that chemical

spoilage in the chitosan preserved samples was lower compared to the control sample.

The antibacterial nature is due to the cationic nature of the chitosan which is provided by

the NH3 group of glucosamine that interacts with negatively charged cell membrane of

36

pathogen and cause destruction of microbial activities (Je & Kim et al., 2006; Helander et

al., 2001).

Combination of treatments methods for preserving foods may cause synergistic effects of

microbiological barriers or hurdles, which reduce the intensity of treatment methods

(Leistner &Gorris, 1995). Kwon et al. (1995) combined gamma irradiation with air-tight

packaging to preserve anchovies but they noticed few alterations in the contents of

polyunsaturated fatty acids. Chouliara et al. (2004) achieved a shelf life of 27–28 days for

vacuum-packaged, salted marine sea bream (Sparus aurata) fillets irradiated at 1 or 3

kGy under refrigeration (4 ± 1◦C), compared to a shelf-life of 14–15 days for the non

irradiated vacuum-packaged, salted sea bream. Lee et al. (2002) and Savvaidis et al.

(2002) investigated the shared consequence of gamma irradiation and vaccum packaging

on the microbial control of sea foods. The bacterial load of the non irradiated control was

higher than the irradiated samples and the results were more definite at lower

temperature. Considering the demand for sea foods, finding a suitable factor which

possess food preserving properties will gratify the raising demands of marine based food

industry

37

MATERIALS AND METHODS

3.1 Materials

All culture media and standard enzymes for microbial analysis were purchased from

Himedia, India. TCBS was used for enumerating Vibrio parahemolyticus (MTCC-451).

Palcam agar supplemented with Listeria-selective supplement was used for the

enumeration of Listeria monocytogenes (MTCC-657) at 37°C. The compositions of

media are given in the segment 3.1.3.

Study culture

Penaeus monodon were procured from retail centers near Pondicherry University. The

strain Streptococcus phocae PI80 was isolated previously from the gut of the Indian white

shrimp and the strain Enterococcus faecium- MC-13 was isolated from the gut of Mughil

cephalus (Gopalakannan, 2007).

Bacterial strains

All bacterial strains were procured from Microbial Type Culture Collections (MTCC),

IMTECH, Chandigarh, India. The details of microbial cultures procured from MTCC are

listed in the following table (Table- 3.1.2).

Ethical permission

Animal experiments were performed in an accredited establishment (Reg.No. 1159 /c/07 /

CPCSEA; Animal facility of the Pondicherry University, India) as per the guidelines of

the ethical committee. Experimental animals were purchased from approved enterprises.

Male Wistar rats were purchased from Raghavendra Enterprises, Bangalore, India.

38

3.1.1. Instruments

Instruments company/firm

Autoclave - York scientific instruments, India

Deep freezer - Forma scientific, USA

Deionized water system - Millipore water corporation, USA

Electron paramagnetic

Resonance - Jeol, Japan

Fermentor - Supertech instruments, India

Fluorescent microscope - Nikon, Japan

Freeze drier - Virtis, USA

Gas chromatography - GC6890/MS5973

mass spectrometry

Gel electrophoresis - Amersham biosciences, USA

Gel documentation system - Bio-rad laboratories, USA

Gamma chamber - IAEA, Atomic energy of India.

High speed centrifuge - Sigma, USA

Ice flaker - Scottman, Italy

Incubators - Scigenics, india

Laminar flow hoods - Kirloskar, India

PCR thermal cycler - Eppendorf, Germany

39

3.1.1. Instruments (cont…)

Instruments company/firm

pH meter - STL instruments, India

Scanning electron

Microscope - Hitachi, Japan

Table top centrifuge - Remi laboratories, India

UV visible spectrometer - Hitachi, Japan

UV transilluminator - Fotodyne Inc, USA

Water bath - Matri instruments

Weighing balance - Sartorius, Germany

40

3.1.2. Bacterial strains

Bacterial strains Source

Aeromonas hydrophila 646 MTCC, Chandigarh, India

Aeromonas salmonicida 1945 MTCC, Chandigarh, India

Lactobacillus acidophilus 447 MTCC, Chandigarh, India

Lactobacillus rhamnosus 1408

Listeria monocytogenes1143

Proteus vulgaris 426

MTCC, Chandigarh, India

MTCC, Chandigarh, India

MTCC, Chandigarh, India

Salmonella typhi 734 MTCC, Chandigarh, India

Vibrio harveyi

Vibrio anguillarum

Hatchery water

MTCC, Chandigarh, India

41

3.1. 3. Microbiological Media

3.1.3.1. MRS agar

Components g/liter

Ammonium citrate 2.0 g

Beef extract 10.0 g

Dextrose 20.0 g

Dipotassium phosphate 2.0 g

Distilled water 1000 ml

Magnesium sulphate 0.05 g

pH 6.5

Polysorbate 80 1.0 g

Protease peptone 10.0 g

Sodium acetate 5.0 g

Yeast extract 5.0 g

3.1.3.2. Tryptone Soy Agar

Components g/liter

Agar 15.0 g

Casein enzymatic hydrolysate 17.0 g

42

Dextrose 2.5 g

Dipotassium phosphate 2.5 g

Distilled water 1000 ml

Papaic digest of soyabean meal 3.0 g

pH 7.5

Sodium chloride 5.0 g

3.1.3.3. Brain Heart Infusion Broth

Components

Beef heart infusion

g/liter

250 g

Calf brain 200 g

Dextrose 2.0 g

Disodium phosphate 2.5 g

Distilled water 1000 ml

pH 7.5

Protease peptone 10.0 g

Sodium chloride 5.0 g

3.1.3.4. Violet Red Bile Agar

Components g/liter

43

Agar 15.0 g

Bile salts 1.5 g

Crystal violet 0 .002 g

Lactose 10.0 g

Neutral red 0.03g

Peptic digest of animal tissue 7.0 g

Sodium chloride 5.0 g

Yeast extract 3.0 g

3.1.3.5. TCBS agar

Components g/liter

Agar 15.0 g

Bromo thymol blue 0.04 g

Ferric citrate 1.0 g

Oxgall 20.0 g

Proteose peptone 10.0g

Sodium chloride 10.0 g

Sodium citrate 10.0 g

Sodium citrate 8.0 g

Sodium thiosulphate 10.0 g

Sucrose 10.0 g

Thymol blue 0.04 g

Yeast extract 5.0 g

3.1.4. Reagents

3.1.4.1. Hydrochloric acid

44

1.08 ml of HCl was made up to 50 ml with distilled water.

3.1.4.1.1. Thiobarbituric acid (TBA)

0.375 g of TBA was dissolved in 100 ml of distilled water.

3.1.4.1.2. Trichloroacetic acid (TCA)

15 ml of TCA from 100% was made up to 100 ml with distilled water.

3.1.4.1.3. TCA: TBA: HCl (1:1:1)

Equal volume of above three solutions was mixed to make 1:1:1 ratio of TCA: TBA: HCl.

3.1.4.1.4. α, α-Diphenyl-β-Picrylhydrazyl (DPPH) radical scavenging assay

DPPH solution 0.2 mM

3.1.5. Genomic DNA isolation

3.1.5.1. GTE stock solution (Lysis buffer)

EDTA 10 mM

Glucose 50 mM

pH 8.0

Tris 25 mM

Water 100 ml

3.1.5.2. SDS stock solution (20%)

SDS 20.0 g

Water 100 ml

SDS was mixed with water at warm condition to proper dissolving.

45

3.1.5.3. EDTA stock solution (0.5M)

EDTA 14.61 g

Water 100 ml

To proper dissolving, the EDTA was mixed with water at warm condition or autoclaving.

3.1.5.4. 10 mM Tris (pH 7.6)

Tris 0.12 g

Water 100 ml

3.1.5.5. Sodium acetate (3M)

Distilled water 25 ml

Sodium acetate 10.2 g

3.1.5.6. Tris-Saturated Phenol

To prepare buffered phenol, distilled phenol is equilibrated first with equal

volume of 1M Tris-HCl (pH 8.0) and then with equal volume of 0.1M Tris-Cl (pH 7.5),

8-hydroxyquinoline is added to a final concentration of 0.1% and stored at 4oC in dark

bottle.

3.1.5.7. Phenol: Chloroform

Buffered phenol and chloroform were mixed in the ratio 24:1 and stored in a

brown bottle at 4C along with 0.1 M Tris-Cl in aqueous phase.

3.1.5.8. RNase

46

RNase (10 mg/ml) was dissolved in 10 mM Tris-Cl (pH-7.5) and kept in boiling

water bath for 15 min and cooled. It is stored at -20oC.

3.1.5.9. TE buffer

0.5M EDTA (pH 8.0) 0.2 ml

1M Tris HCl (pH 8.0) 1.0 ml

Distilled water 100 ml

3.1.6. Agarose gel electrophoresis

3.1.6.1. TAE Buffer (50X)

0.5 M EDTA (pH8.0) 100 ml

Glacial acetic acid 57.1 ml

Tris-base 242 g

The final volume was made up to 1000 ml using distilled water.

3.1.6.2. Ethidium bromide (EtBr) stock solution

Ethidium bromide 10.0 mg

Distilled water 1.0 ml

3.1.6.3. Gel loading dye (6X)

Bromophenol blue 0.25 g

Distilled water 100 ml

47

Glycerol 30 ml

Xylene cyanol 0.25 g

3.1.6.1. Phosphate buffered saline (PBS)

Disodium hydrogen phosphate 0.02 g

KCl 0.02 g

NaCl 0.8 g

pH 7.0

Sodium dihydrogen phosphate 1.4 g

Water 100 ml

3.2. Methods

3.2.1. Agar well diffusion method

Agar well diffusion method described by Lyon & Glatz (1993) was used for the analysis

of bacteriocin activity. The wells of 6 mm were made using well borer and bottom of the

wells were sealed with a few drops of MRS agar media. 100 µl of culture free supernatant

was added to the wells and kept at 4oC. After 2 h of incubation, the agar base was

loosened from edge of the petri dish with spatula and filled into the petri dish lid. 10 ml of

soft agar containing indicator strains were overlaid on the agar base. After 24 h of

incubation period, zone of inhibition was measured and tabulated.

3.2.2. Molecular biology assay

3.2.2.1.Total genomic DNA isolation (Marmur, 1961)

48

Lactic acid bacteria strains were grown in MRS broth at 37 °C. After 12 h of incubation,

1.5 ml of cultured broth was taken and centrifuged at 8,000 g for 6min. The pellets was

resuspended with 330 µl of GTE solution and incubated for 30 min. A pinch of lysozyme

was added to the same solution and incubated at 37 °C for 1 h. 10 µl of 20% SDS was

added and incubated at 37 °C for overnight. RNase (0.1 mg/ml) was added to the solution

to remove the RNA from solution and it was kept at 37 °C. After 3 h of incubation 17 µl

of EDTA (0.5M) was mixed and incubated at 50oC for 10 min. Proteinase K (10 µl) was

added and incubated at 37 °C for 3 h. After incubation, 200 µl of phenol: chloroform

(24:1) was added, mixed slowly and centrifuged at 16,000 g for 15 min. After

centrifugation, the aqueous phase layer was collected and mixed with equal volume of

isopropanol. It was slowly Shaked up and down until the pool of DNA was visibly seen, later

centrifuged at 16,000 g for 15 min. After centrifugation, the pellet was air-dried and

dissolved in 40 µl of 1X TE buffer. It was confirmed by running the agarose gel

electrophoresis.

3.2.2.2. Agarose gel electrophoresis

The isolated DNA sample was separated on 0.8% agarose gel. 1X TAE buffer was

prepared by appropriate concentration of 1 ml of 50X TAE buffer and mixed with 49 ml

distilled water. Followed a 0.4g of powdered agarose was added and mixed well. They

were allowed to boil until agarose dissolved completely. Then 3 l of ethidium bromide

(0.5 g/ml) was added from stock solution of 10 mg/ml and mixed well. The warmed

agarose solution was poured into the gel casting tray and allowed to set for 30-45 min at

room temperature. The gel was mounted in the electrophoresis tank. Electrophoresis

buffer was added to cover the gel to a depth of about 1mm. The isolated DNA sample was

mixed with loading buffer and loaded into the well of the submerged gel using a

49

micropipette. The voltage of 50-60 was applied. After 1-2 h the gel was taken out from

the buffer and was examined under UV illumination. The clear band was observed as red

orange fluorescence. The molecular weight was measured by using appropriate DNA

marker.

3.2.2.3 Photography

Agarose gels and polyacrylamide gels were photographed using gel documentation

system and digital camera.

3.3 Safety assessment of the protective culture

Animal experiments were carried out in a certified establishment in our university

(Reg.No. 1159 /c/07 / CPCSEA; Animal facility of the Pondicherry University, India)

according to guidelines of the ethical committee. The Central animal house is registered

with the Committee for the Purpose of Control and Supervision of Experiments on

Animals (CPCSEA). Experimental animals were purchased from approved enterprises.

Wistar rats were acclimatized for a week in experimental conditions following which they

were assigned for safety assessment studies. The treatments lasted for 28 days during

which activity, behavior, and hair luster of each mouse were observed. On day 29, all

animals were euthanized humanely by chloroform overdose and their blood and tissue

samples were collected for the following analysis.

3.3.1. Organ indices

Collected internal organs including liver, heart, kidney, spleen, testis, epididymis, lung

and pancreas were weighted immediately. The organ index values were obtained from the

weight of internal organ of each wister rats over its final body weight (g).

50

3. 3.2. Hematology

Blood samples were collected by cardiac puncture and the marker enzymes of liver,

kidney functions, blood cell count, hemoglobin, mean corpuscular haemoglobin, glucose

concentration, urea and creatinine levels were analyzed for both control and probiotic

protective culture fed group. Blood cells counts and haematological parameters were

determined with an automated hematology counter (Beckman Coulter Hematology

Analyser LH750).

3. 3.3. Histology

At the end of respective experimental periods, spleen, kidney and liver of the treatment

group and control group were removed and the samples were processed by hematoxylin

and eosin method (Ramiah et al., 2009). Briefly, at the end of the respective experimental

period, the spleen, kidney and liver of the treatment and the control group were fixed in

Bouin‘s fluid (Humasong, 1979). Samples were dehydrated in ascending grades of ethyl

alcohol. Alcohol was removed by the hydrophobic clearing agent xylene followed by

paraffin wax and was mounted at 50-60°C. Samples were cut at 5 µm thickness in rotator

microtome (Leica, Germany) and stained in Ehrlich‘s hematoxylin and eosin for

observation in a light microscope (Olympus CX41RF, Japan). The images were recorded

and then processed using corel draw software.

3. 3.4. Bacterial translocation

After feeding with test strains for 28 days, animals were humanely euthanazed by

isofluorane overdose. Blood was drawn via cardiac puncture of the right ventricle and 100

µl was spread-plated onto Mann Rogosa Sharpe agar (MRS) and Brain Heart Infusion

agar (BHI). The plates were incubated at 37°C for 24 h and examined for microbial

growth. Bacterial translocation was analysed in blood, liver, kidney and spleen (Zhou et

51

al., 2000b). Fifteen microlitres of blood were cultured in Mann Rogosa Sharpe agar and

Brain Heart Infusion agar and incubated at 37°C. Tissue samples were homogenized in

buffered peptone water and 100 µl of the resulting homogenates were cultured in Mann-

rogosa-sharpe and Brain Heart Infusion agar as aforementioned. After 48 h of incubation,

CFUs were counted and results were expressed as incidence of translocation. Positive

growth on agar plates was defined by the presence of any micro-organisms (including

single colony).

3. 3.5. Erythrocyte staining

The micronucleus test was performed to investigate whether the supplied protective

culture induced micronuclei in polychromatic erythrocytes in murine peripheral blood.

Animals that are exposed to protective culture were sacrificed at appropriate times after

treatment. Peripheral blood was obtained from the tail vein. Blood was collected on the

day following the last protective culture administration. Blood cells were stained

immediately after smear preparation. Whole blood smears were prepared on clean

microscopic slides, air-dried, fixed in methanol and stained with acridine orange

(125µg/ml in pH 6.8 phosphate buffer) for 1 min just before the evaluation with

fluorescence microscope (Paari et al., 2011c). The proportion of mature and immature

erythrocytes was determined for each animal by analysing the erythrocytes. Nearly 1000

erythrocytes were scored during the staining by manual evaluation. Data was eventually

developed to show that mouse peripheral blood is an acceptable cell population for

detection. The occurrence and pattern of erythrocytes were investigated. Immature

erythrocytes were identified by their orange–red colour, mature erythrocytes by their

green colour and micronuclei by their yellowish colour as stated by Celik et al. (2005).

3.4. Determination of Total volatile base nitrogen (TVBN)

52

Total volatile base was measured using microdiffusion method. Concisely, 1 ml of

sulphuric acid was pipetted into the inner chamber of the conway unit along with a drop

of Tashiro‘s indicator. In the outer chamber, 1 ml of 20% trichloroacetic acid extract and

potassium carbonate was added with gentle rotation. After absorption, the contents of the

inner chamber were titrated against sodium hydroxide (0.1 N) till the change of colour. A

blank was conducted simultaneously using 2% TCA solution instead of sample.

3.5. Microbial load analysis

Penaeus monodon was inoculated with the protective cultures at about 6 log CFU/g and

were wrapped in a sterile transparent bag. Uninoculated samples served as control. After

inoculation, naturally occurring spoilage microbial populations such as Vibrio and

Coliform counts were determined in both control and biopreserved samples. Briefly,

samples weighing 10 gm each were taken aseptically, homogenized for 2 min in 90 mL of

0.1% peptone saline in a stomacher, serially diluted tenfold in the same diluents, and

plated onto appropriate plates as described by Vanderzant & Splittstoesser (1992). Spread

plate technique was used to enumerate Vibrio and Coliforms using Thiosulfate citrate bile

salts sucrose agar and Violet red bile agar media. Lactobacillus Mann Rogosa Sharpe agar

was used for the enumeration of LAB. Microbial counts were made on selective media

and are expressed as log CFU/g

3.6. Measurement of peroxide value

Peroxide value (PV) was determined using titration method described by Sallam (2007).

Briefly, sample (5 g) was heated to 60°C for 3 min to melt the fat and then thoroughly

agitated for 3 min with 30 ml acetic acid–chloroform solution (3:2 v/v) to dissolve the fat.

Saturated potassium iodide solution (0.5 ml) was added to the filtrate. The titration was

53

run against sodium thiosulfate solution. Peroxide Value was reported as milliequivalent

peroxide per kg of sample:

PV (meq / kg) = S ×N/W* 1000

Where S is the volume of titration (ml), N is the normality of sodium thiosulfate solution

(N =0.01), and W is the sample weight (kg).

3.7. Irradiation treatment

Prawn samples were procured from landing centers near Pondicherry University. Samples

were degutted and segmented into four batches: non-irradiated (control), irradiated,

irradiated plus antioxidant packed, irradiated without antioxidant wrapping. Samples were

drawn at regular intervals for bacteriological, biochemical analyses and electro

paramagnetic resonance investigations.

3.7.1. Chemical analysis

Total volatile base was measured using microdiffusion method. Briefly, 1 ml of sulphuric

acid was pipetted out into the inner chamber of the conway unit containing a drop of

Tashiro‘s indicator. In the outer chamber, 1 ml of 20% trichloroacetic acid extract and 1

ml of potassium carbonate was added with gentle rotation. Succeeding absorption, the

contents of the inner chamber were titrated against sodium hydroxide until the

transformation of colour. A blank was conducted simultaneously using 2% trichloroacetic

acid solution instead of sample extract. Same procedure of total volatile base was

followed for estimation of TMA, except the addition of 1% formaldehyde to the outer

chamber of the conway unit. TVB and TMA concentrations were expressed as mg N/100

gm fish.

54

Lipid oxidation was determined by the TBA method. One gm of tissue was blended with

9 ml of 0.25 M sucrose. The homogenized sample was filtered using filter paper

(Whatman No. 4) and to 0.2 ml of this solution 0.2 ml of SDS, 1.5 ml of TBA was mixed

and final volume was made to 4 ml using distilled water before heating it at 80oC for 60

min. Absorbance at 532 nm was measured against a blank using a spectrophotometer. The

thiobarbituric acid reactive substances (TBARS) value was expressed as mg of

malonaldehyde (MDA) per kg.

3.7.2. Electron paramagnetic resonance analysis

EPR assessments were carried out employing an EPR spectrometer (JEOL, JES FA200)

at room temperature. All the spectra of EPR was recorded at the X-band (9.3GHz).

100mg sample was deposited in thin wall quartz EPR tubes (internal diameter of 3 mm,

length of 150 mm, and wall thickness about 0.1 mm) to produce cylindrical samples with

identical dimensions (sample column height 5.2 ±0.2 cm). Spectrum was recorded with

the following spectrometer settings: microwave frequency, 9.5 GHz; microwave power,

0.63-31.73 mW; centre field, 250 mT; Sweep width, 500 mT. The spectra were inscribed

following exposure of samples to gamma irradiation.

3.8. Antioxidant assays for plant extracts:

3.8.1. DPPH radical Scavenging Activity

The capacity to scavenge the ‗‗stable‘‘ free radical DPPH was monitored based on Blois

method (1958). Methanolic extracts from fruit peels (0.1 ml) were mixed with 0.9 ml of

methanolic solution containing DPPH radicals (0.041 mM). The mixture was mixed

strongly and left to stand for 60 min in the dark. The reduction of the DPPH radical was

determined by measuring the absorption at 517 nm. The radical-scavenging activity

(RSA) was calculated as a percentage of DPPH discoloration using the equation:

55

100

A

AARSA%

DPPH

sDPPH

Methanol was used as blank. AS is the absorbance of the solution when the sample extract

has been added and ADPPH is the absorbance of the DPPH solution.

3.8.2. Reducing Power

The reducing power of prepared extracts was determined following the protocol of

Oyaizu (1986). Methanolic extract (1 ml) was mixed with 1ml of 0.2 M phosphate buffer

(pH 6.6) and 1 ml of potassium ferricyanide (10 mg/ml). The mixture was incubated at

50°C for 20 min. After 1 ml of trichloroacetic acid (100 mg/ml) was added, the mixture

was centrifuged at 13400g for 5 min. The upper layer (1.0 ml) was mixed with 1 ml of

deionised water and 0.1 ml of ferric chloride (1.0 mg/ml) and the absorbance was measured

at 700 nm. Blank was prepared by adding every other solution but without extract and ferric

chloride (0.1%). Higher absorbance value of the mixture denotes stronger reducing power.

3.8.3. β-carotene bleaching assay

β-carotene bleaching assay was done in accordance with Matthaus method (2002). A solution

of β-carotene was prepared by dissolving 1 mg of β-carotene in 1 ml of chloroform. After

the chloroform was removed at 40°C under vacuum, 40 mg of linoleic acid, 100 ml of

distilled water and Tween 80 emulsifier were added to the flask with vigorous shaking.

Aliquots (5 ml) of this emulsion were transferred into different test tubes containing 0.2

ml of extracts. The tubes were shaken and incubated at 50°C in a water bath. The

absorbance at zero time was measured at 470 nm. Absorbance readings were then

recorded at 30 min intervals until the control sample had changed colour. A blank, devoid

of β-carotene, was prepared. Antioxidant activity was calculated using the following

equation

56

100contentcaroteneInitial

assayofmin120aftercontentcaroteneactivitytAntioxidan

3.8.4. Nitrite scavenging activity

This assay was carried out as described by Saha et al. (2004) with some modifications.

Methanolic extracts from fruit peels (3 ml) were mixed with 2 ml of citric acid buffer (pH

3.0) and 0.1 ml of 200 µg /ml NaNO2. 100 ml of distilled water were added to the flask

with vigorous shaking. After incubation for an hour at 37°C equal volume of Griess

reagent (1% sulfanilamide, 0.1% N-(1-naphthyl) - ethyline diamine hydrochloride, 2.5%

H3PO4) was added to the above mixture. The absorbance was determined after 10 min at

538 nm. BHA was used as the positive control. NaNO2 scavenging activity was

calculated using the following equation:

100A

)AA(AactivityscavengingNitrite%

dardtans

controlsampledardtans

Where AStandard is without NaNO2 and without extract. Acontrol is without NaNO2.

3.8.5. Hydroxyl radical scavenging activity

Hydroxyl radical scavenging activity was determined according to the method of

Smirnoff & Cumbes (1989) with a few modifications. 0.3 ml of 5 mmol/l phenanthroline,

0.8 ml of 0.75 mol/l phosphate buffer (pH 7.4) and 0.3 ml of 7.5 mmol/l Feso4 were

added to 0.5 ml of extract. To this mixture 0.2 ml of 1 % H202 was added and incubated at

37°C for 60 min. The absorbance of the resulting solution was measured

spectrophotometrically at 532 nm and the activity was calculated using the following

formula:

100A

A1activityscavengingradicalHydroxyl%

control

sample

57

Phosphate buffer was used as blank. A control is the absorbance of control without the

tested samples and A sample is the absorbance in the presence of tested samples.

3.8.6. Electron Paramagnetic Resonance spectroscopy

EPR investigations on RSA of extracts against a stable radical were measured using the

method described by Nanjo et al. (1996). A methanol solution of 60 µl each sample was

added to 60 µl of DPPH (60 µmol l-1

). After mixing vigorously, the solutions were

transferred into an aqueous cell quartz tube and fitted into the cavity of the EPR

spectrometer. The spin adduct was measured on EPR spectrometer exactly 2 min later in

an X-band EPR spectrometer at room temperature using standard rectangular cavity

operating at 9.4 GHz with a 100 kHZ modulation frequency. The microwave power and

modulation amplitude were 4 mW and 1 G, respectively.

3.8.7. Gas Chromatography-Mass Spectrometry analysis of natural Extracts

Dry powder of selected material showing maximum antioxidant properties (10 kGy

irradiated sample) was subjected to column chromatography employing silica gel (60-120

mesh size) and eluted stepwise using a linear gradient of chloroform: methanol. Fractions

after analysis by TLC were pooled into their complementary groups. The active fraction

which showed maximum DPPH radical scavenging activity in each extracts was

subjected for GC-MS analysis. Extracts were dissolved in ethanol (200 mg/ml) and

centrifuged at 13400g for 5 min to precipitate undissolved materials. The supernatant was

mixed with 4 volumes of BSA [N,O-bis(trimethylsilyl)acetamide] and derivatized in a

water bath (70°C) for 15 min. The compounds in extracts were identified using a gas

chromatograph-mass spectrometer. A split inlet (100:1) was used to inject sample (5 µL)

into an HP-5 column (30 m, 0.32 mm i.d., 0.25 µm film; Hewlett-Packard Co.). A ramped

oven temperature was used (100°C for 2 min, increased to 270°C at 10°C/min, and held

58

for 6 min). The inlet temperature was 250°C, and the carrier gas was He at a constant

flow of 1.5 ml/min. The ionization potential of the mass selective detector was 70 eV.

Identification of compounds detected was achieved by comparing mass spectral data of

samples.

3.9. Statistical analysis

One-way analysis of variance were conducted to determine significant differences

between treatment means. Differences at p<0.05 were considered significant.

Experiments were replicated thrice on different occasions with different fish samples.

Data were expressed as mean±standard deviation. Analysis was performed using a SPSS

package (SPSS 7 for windows, SPSS Inc, USA).

59

Introduction

Sea foods like prawns are rich in abundant nutrients that have been found to aid cardiovascular

health for which they are chiefly caught in Indian seas. From harvest to consumption, fishery

products are easily contaminated by pathogenic microorganisms that limit the shelf life of fishery

products. During the course of storage, certain pathogens like Listeria monocytogenes, Vibrio

parahemolyticus can grow from contaminated fish as well as from the processing environments

(Moharem, 2007). The ban and limitation of chemical additives and antibiotic preservatives has

driven the food research towards the search for natural antimicrobial compounds for food

preservation. Against this background, numerous antimicrobial agents that exist in

microorganisms are now relied and current approaches are intended towards the promise offered

by the technique biological preservation using protective cultures which are also known as

―natural inhibitors‖(Luchansky, 1999). Awareness in the role of biopreservation in assuring food

safety has augmented. An array of bacteria especially Lactic acid bacteria (LAB), occurring

commonly on foods have been evaluated for their potential to control spoilage organisms.

Approval by the United States Food and Drug Administration (FDA) for usage of nisin to

preserve cheese products paved way for commercialization of this technique. This natural way of

preservation has gained increasing attention for extending the shelf life of food products as they

grow instinctively on chilled meat, modify the environment to discourage the growth of other

organisms by producing antimicrobial compounds (Duffes, 1999). Protection of food is expected

due to the production of organic acids, carbon dioxide, ethanol, hydrogen peroxide and diacetyl

(Leroi, 2010). Also the anionic lipids in the cytoplasmic membrane of the pathogens act as targets

for the lantibiotic bacteriocins resulting in the formation of pore like structures (Moll et al., 1999).

Chemical indices of fish spoilage like, total volatile base and peroxide value may be measured as

a quality catalogue of a fish, which is correlated to the activity of spoilage bacteria. Reduction of

MDA content in foods is prudent, since MDA is known to be mutagenic and carcinogenic.

Inhibition of lipid peroxidation and chemical damages in foods by LAB has been observed before

in cheese and tuna mince (Songisepp et al., 2004). As consumers claim for chemical free

60

additives, bacteriocins and bacteriocin secreting starter cultures are becoming an appealing

candidate for contemplation as natural preservative for biopreservation of foods (Kanmani, 2011).

A handful of these cultures are previously being used in preservation of shrimp, meat, beef and

fish fillets (Fall et al., 2010; Cho et al., 2010; Ercolini et al., 2006; Jacobsen et al., 2003).

Previously, potent probiont from fermented food products were used as an efficient

biopreservative for shelf life extension of cheese product in our laboratory (Satish kumar et al.,

2010) but little is known about the possibility to use protective cultures for shelf life extension of

fishery products. Various researchers have reported the application of protective culture for food

preservation eg. Leuconostoc carnosum for preservation of meat products (Jacobsen et al., 2003),

Lactococcus lactis ssp. lactis for extending shelf life of fishery products (Wessels & Huss 1996).

Strains of Pediococcus family like Pediococcus acidilactici and Pediococcus pentosaceous were

favoured strains in USA for fermented sausages whereas, in European nations strains like

Lactobacillus plantarum and Lactobacillus curvatus were used for fermentation of meat. The

latest studies from our laboratory recognized the inhibitory activity of a probiotic culture

Streptococcus phocae PI 80 against Vibrio sp (Swain et al., 2009) and Listeria monocytogenes

(Satish & Arul 2009). Defying the protective culture S. phocae PI 80 belonging to the LAB group,

there has been controversy over their impregnability in food as preservatives. Acute oral toxicity

tests have been advocated as the fundamental test in protective culture safety assessment studies

(Duangjitcharoen, 2009). Appraising the riskiness of S. phocae PI 80, ahead of its usage as a

biopreservative has turned out to be important to puzzle out some food safety problems. The

intention of our study was to substantiate the safety of the protective culture S. phocae PI 80 in

the rat model and to investigate the usefulness of S. phocae PI 80 in prolonging the shelf life of

Penaeus monodon in realistic conditions.

61

4.2. Materials and methods

4.2.1. Samples and Microorganisms

Penaeus monodon were procured from fish landing centres near Pondicherry University. The

LAB strain Streptococcus phocae PI80 was isolated previously from the gut of Indian white

shrimp (Gopalakannan, 2006) and was characterized by Kanmani (2011). TCBS was used for

enumerating Vibrio parahemolyticus (MTCC-451). Palcam agar supplemented with Listeria-

selective supplement was used for the enumeration of Listeria monocytogenes (MTCC-657) at

37°C. All strains were procured from Microbial Type Culture Collections (MTCC), IMTECH,

Chandigarh, India. The complete detail regarding the microbial cultures used in this study is

mentioned in Chapter 3 (Table- 3.1.2) methodology section.

4.2.2. Safety assessment of the protective culture

Animal experiments were performed in an accredited establishment (Reg.No. 1159 /c/07 /

CPCSEA; Animal facility of the Pondicherry University, India) according to guidelines of the

ethical committee. The Central animal house is registered with the Committee for the Purpose of

Control and Supervision of Experiments on Animals (CPCSEA). Experimental animals were

purchased from approved enterprises. One month old Wistar rats were acclimatized for a week in

experimental conditions following which they were assigned to two different groups (control,

group supplemented with S. phocae PI 80). S. phocae PI 80 was inoculated in MRS broth medium

and incubated at 37°C for 24h. The cells were pelleted by centrifugation at 6000xg for 10 min at

20°C. Pellets were washed in phosphate Buffered Saline (PBS, pH 7.4) twice. Finally S. phocae

PI 80 was adjusted to 1x 109 cells in 10% reconstituted sterile skim milk. The

mice in the control

group were fed with skim milk under the same conditions as the test groups. With a sterile pipette,

animals of the treatment group were orally inoculated with 100 µl of cell suspensions containing

1x 109 cells of S. phocae PI 80. Control group was fed with skim milk powder. The treatments

lasted for 28 days during which activity, behavior and hair luster of each mouse were observed.

On day 29, all animals were euthanized humanely by chloroform overdose and their blood and

tissue samples were collected for chemical analysis.

62

Small pieces of liver and kidney samples were immediately excised and rinsed with ice-cold

physiological saline for histological studies. The tissues settled in 10% buffered formalin were

implanted in paraffin, cut into 6 µm sections and stained with haematoxylin and eosin (H&E).

Morphological indications were examined using an eyepiece micrometer on a light microscope.

Whole blood smears were collected on the day following the last administration of cells. Smears

were air-dried, fixed in methanol and stained with acridine orange (125 mg/ml in pH 6.8

phosphate buffer) for 1 min just before evaluating with a fluoresence microscope. The occurrence

and pattern of erythrocytes were investigated in both the control and S. phocae PI 80 fed groups.

4.2.3. Bacterial translocation

Blood samples and organs were analysed for detecting the presence of bacterial translocation.

Collected internal organs including heart, liver, kidney, spleen, testis, epididymis, lung and

pancreas were analysed for organ indices. The complete protocol for bacterial translocation and

organ indices are explained in the materials and methods section (Chapter-3).

4.2.4. Hematology

Blood samples were collected by cardiac puncture and the profile of marker enzymes of liver,

kidney were measured. Blood cell count, hemoglobin, hematocrit, packed cell volume, mean

corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration,

red blood cell distribution width and blood platelets were analyzed for both control and S. phocae

PI 80 fed group.

4.2.5. Efficacy of LAB Strain Streptococcus phocae PI 80 in the preservation of Penaeus

monodon.

Streptococcus phocae PI 80 was tested in vitro for its antagonistic activity against food borne

pathogens Vibrio parahemolyticus, Listeria monocytogenes using well diffusion method before

determining its functionality in the in vivo model. Penaeus monodon were inoculated with the S.

phocae PI 80 at about 6 Log CFU/g and were wrapped in a sterile transparent bag. Uninoculated

63

samples served as control. After inoculation, naturally occurring spoilage microbial populations

such as Vibrio parahemolyticus and Coliform counts were determined at time zero, five, ten and

fifteen days in both control and biopreserved Penaeus monodon. Spread plate technique was used

to enumerate Vibrio parahemolyticus and Coliforms using TCBS and VRBA media Lactobacillus

MRS agar was used for the enumeration of LAB. The protocol for microbial enumeration is

provided in the materials methods section (Chapter-3. Section- 3.5)

4.2.6. Co-inoculation studies in fresh Penaeus monodon

Penaeus monodon were sterilised before using it in co-inoculation experiments. Prior to

experiment, Listeria monocytogenes was cultured in Listeria selective broth at 37°C for

18-24 h and S. phocae PI 80 was cultured on MRS broth at 35°C for 18-24 h. The raw

slices of Penaeus monodon were surface inoculated with one milliliter of S. phocae PI 80

(106CFU/g) and L. monocytogenes (10

2CFU/g). After assuring good contact of inoculums

with the surface, samples were sealed in low oxygen permeability bags and stored at 4°C

for 15 days. The experimental conditions were: 1. L. monocytogenes + S. phocae PI 80. 2.

L. monocytogenes. Enumerations of bacterial populations were carried out on MRS agar

for S. phocae PI 80 and Listeria selective agar for L. monocytogenes. Bacteria populations

were enumerated at zero, five, ten and fifteen days. In addition, existence of L.

monocytogenes was also detected using PCR in both control and biopreserved Penaeus

monodon. The primer pair consisting of primer A -5‘-CATTAG TGG AAA GAT GGA

ATG -3‘ and primer B -5‘-GTA TCC TCC AGA GTG ATC GA -3‘ was used for the

amplification of a 732 bp region of the hly gene. These primers have been previously

described by Gouws & Liedemann (2005).

64

4.2.7. Determination of Total volatile base nitrogen (TVBN)

Total volatile base was measured using microdiffusion method. The complete protocol for volatile

base estimation is explained in the materials and methods section (Chapter-3. Section- 3.4)

4.2.8. Measurement of peroxide value

Peroxide value (PV) was determined using titration method. The titration was allowed to run

against standard solution of sodium thiosulfate. The complete procedure for analyzing the

oxidation rate for biopreserved and control samples are provided in the materials and methods

section (Chapter-3. Section-3.6)

5. Results

5.1. Safety assessment

Oral dose of S. phocae PI 80 produced no treatment related illness in any of the animals. Neither

weight loss nor changes in organ weight resulted with the S. phocae PI 80 fed animals compared

with the control group (Table 1). The number of positive translocation (Bacterial translocation) in

each handling group is portrayed in Table 2. There was no bacterial growth in the spleen and liver

tissue which indicates that the analyzed tissues are not contaminated by the fed protective culture

S. phocae PI 80.

5.2. Hematology

There was no significant difference in RBC, HB, HT, MCV, MCH, platelet counts, glucose,

albumin, total protein, urea, creatinine, SGPT among the wistar rats fed with S. phocae PI80

indicating that there was no induction of inflammatory response, associated with the feeding of S.

phocae PI 80 demonstrating the innocuous ability of this strain to exert a non-alteration of the

peripheral blood parameters.

65

5.3. Histology

No signs of splenomegaly or hepatomegaly were noticed in tissue samples acquired from liver

and spleen (Figure 1). Hepatocytes are regular and are orchestrated in trabecules. Trabecular

framework is clear in liver samples of control and treated. The cytoplasm of hepatocytes

comprising void vascular space is witnessed of same size in control and treated and was not found

to be expanded in treated samples. The renal tubules and glomerulli are not affected in both

control and treatment groups. No dilation of glomerulli was observed in S. phocae PI 80 supplied

groups. Renal glomeruli reflect normally a structure in both the control and treated groups.

Hypertrophy of epithelial cells or deterioration of epithelia of renal tubules by means of

infiltration of mononuclear cells and dilation of glomeruli which are hallmarks for kidney

malfunctions were not noticed. Spleen has a larger and more uniform marginal zone and a more

pronounced marginal sinus region in both the control and treatment groups. The follicle in the

spleen is better demarcated.

5.4. Bacterial translocation

It is evident from the Figure 2, that the blood smear from both treated and control in acridine

orange coated slide revealed most of green coloured cells indicative of mature erythrocytes.

Micronuclei containing fragments were not noted in both the control and S. phocae PI80 fed

animals. The proportions of mature versus immature erythrocytes were similar in both the control

and treatment groups.

5.5. Efficacy of Streptococcus phocae PI 80 in inhibition of microbial load in refrigerated sea

food

The impact of S. phocae PI 80 was bactericidal and diminished the pathogen population at the

stored temperature in Penaeus monodon (Figure 3). Treated samples exhibited reduction of

pathogen count at the end of 15 days at 4°C. In biopreserved Penaeus monodon a 0.7 and 0.84

Log reduction was observed for Coliforms and Vibrio respectively compared to the control

66

samples. More than a Log reduction of total viable count was observed in the S. phocae PI 80

supplied samples compared to control samples in the vaccum packed Penaeus monodon (Figure

8). The growth of L. monocytogenes was suppressed when co-inoculated with S. phocae PI 80, in

comparison with the samples that contained only L. monocytogenes (Figure 4). In this study, L.

monocytogenes population was inhibited by 1.2 Log in the S. phocae PI 80 inoculated Penaeus

monodon during storage in the deliberately contaminated product. The inoculated protective

culture S. phocae PI 80 antagonized the L. monocytogenes very well and became the dominant

flora in the biopreserved Penaeus monodon (Figure 5). The results showed that the inoculated

LAB strain grew rapidly during storage. Average counts of LAB exceeded above 8 Log in

Penaeus monodon. The level of LAB in biopreserved sample was comparatively 3.3 Log higher

than the uninoculated Penaeus monodon.

5.6. Peroxide value

The inaugural PV (meq peroxide/kg fish sample) value in the Penaeus monodon was 1.93±0.1

meq peroxide/kg. At the end of the storage time, lowest malonaldehyde content (3.5±0.25 meq

peroxide/kg) was observed in the biopreserved samples (Figure 6) and the highest in controls

(3.96±0.20 meq peroxide/kg).

5.7. Total volatile base

TVB values of Penaeus monodon progressively intensified with storage time for both control and

treated samples. In Penaeus monodon, TVBN values were 38±2 mg N/100 g for untreated control.

Whereas, it was 29.6±1.5 mg N/100 g for treated samples respectively (Figure 7). In vaccum

packed samples too significant difference in total volatile base evolution was noted between the

control and biopreservative treated samples. Control sample reached a value of 60.5 by the end of

storage time whereas S. phocae PI80 treated sample under vaccum condition produced only 45

mg/100gm of TVBN (Figure 9).

67

6. Discussion

A 28 day oral toxicity study in wistar rats was staged to investigate the acute toxicity of test

culture S. phocae PI 80. The consequences of such analyses furnished preliminary toxicity figures

that are handy in determining the riskiness of the culture. A translocation positive animal was

clarified as an animal that had at least one tissue sample (including blood) comprising one or

more viable bacterial cells translocated from the gut. The translocation incidence of bacteria in

our analysis was analogous between the S. phocae PI 80 fed and control group which indicates

that bacterial translocation was not correlated with the feeding of S. phocae PI 80. Translocation

of cells in the kidney epithelium, as observed in our study has been recorded for Enterococcus

faecalis in mice (Wells & Erlandsen, 1991). These findings can be further corroborated with

histological studies. In a previous study, Zhou et al. (2000) reported that four week consumption

of L. rhamnosus and Bifidobacterium sp strains to mice had no adverse effects on haematology

and biochemistry. Similarly, Tortuero et al. (1995) have also found that administration of LAB

had no effect on plasma glucose, total protein and albumin. The fact that no odd morphological

changes were contemplated in the liver, spleen and kidney of probiotics supplied animals may

manifest that the administration of S. phocae PI 80 was not toxic to the animals. Several marker

enzymes of liver and kidney are used as the biochemical markers for early acute hepatic damage.

The rise in the marker enzymes and blood parameters have been attributed to the damaged

structural integrity of the liver as they are cytoplasmic in location and are released into circulation

after cellular damage. The results of the present study demonstrated that feeding of rats with S.

phocae PI 80 had no toxic effect on blood parameters and marker enzyme profile. Formation of

micronuclei containing chromosome fragments or whole chromosomes, which are indicative of

cytogenetic damage in peripheral blood erythrocyte was not observed in any of the groups

following administration of the S. phocae PI 80 (n=8). Briefly, immature erythrocytes were

identified by their orange–red colour, mature erythrocytes by their green colour and micronuclei

by their yellowish colour as stated by Celik et al. (2005).

68

The current study focused on the surveillance of the following naturally contaminating

microflora; Coliforms, Vibrio parahemolyticus. Total viable bacteria count (TVC) is the most

common method for determination of the bacteriological quality of seafood. The results of the

present study are in concurrence with a number of studies with inoculation of different strains in

smoked salmon, crab meat, peeled shrimp and tuna (Fall et al., 2010)

In our work, we noticed that the multiplication pattern of L. monocytogenes was influenced by the

presence of protective culture S. phocae PI 80 in the co-inoculation experiments carried out in raw

Penaeus monodon samples. Nilsson et al. (1997) and Bredholt et al. (2001) verified the efficiency

of lactic acid bacteria in inhibiting L. monocytogenes in smoked salmon and meats respectively.

Synthesis of anti-listerial bacteriocin against L. monocytogenes by S. phocae PI 80 was previously

reported by Satish & Arul (2009). Though it is difficult to distinguish the inoculated LAB strain

from the natural endogenous LAB strain, differences between the control and inoculated samples

in LAB count were statically significant. Though various studies have reported the inhibitory

activity of protective culture and their secretory products against fish pathogens there are only few

reports available on the bacteria isolated from fresh aquatic product (Campos et al., 2006).

Reduction of MDA content in foods is always desirable, since MDA is known to be mutagenic

and carcinogenic. Inhibition of lipid peroxidation in foods by LAB has been observed earlier in

cheese and tuna mince (Songisepp et al., 2004). Our results approve the finding of Gelman

(2001), that starter cultures of lactic acid bacteria decrease the malonaldehyde level than the

control in fish based products. Recent reports from our research group elucidated the antioxidant

properties of this protective culture S. phocae PI 80 which have high quenching potential to

suppress free radical that cause oxidation of lipids. PV in all samples was well lesser than the

recommended acceptable level of 10–20 meq peroxide/kg fish fat (Huss, 1995). Peroxide values

reported in the present study are in accordance with those reported in sliced salmon during 15

days storage in ice at 1°C (Sallam, 2007).

69

TVB-N contents increased for all samples during the storage period with the highest values

recorded for control samples in Penaeus monodon. Both groups were rated ‗good quality‘ due to

their TVB-N values until day10 but the final values of the untreated samples exceed the upper

acceptability limit set by the EU for TVBN values of fish (35 mg N/100 g of fish flesh). In

Penaeus monodon best organoleptic results (data not shown) observed in S. phocae PI 80

preserved samples were in good correlation with the lowest TVB-N values as well as with the

lowest total bacterial counts; whereas the worst organoleptic characteristics of control samples

were accompanied by the highest values found by the same chemical and bacteriological analyses.

Lactic acid bacteria that are adapted to fishery products have a good potential to be used for the

biopreservation of fishery products. This natural way of preservation has gained special attention

for extending the shelf-life of food products. We propose that S. phocae PI 80 isolated from

Paenaeus indicus, offers opportunities as preservative culture for the prolongation of the shelf-life

of raw Penaeus monodon. Further validation studies are required to investigate the usefulness of

this protective strain towards preservation of different types of food products from sea.

70

Table 1 Effect on organ indices of wistar rats fed with protective culture S. phocae PI 80.

Means±SD with different superscripts differ significantly (ρ<0.05). (n=8)

Organs Control S. phocae PI 80

Liver 6.39±0.10

6.792±0.190

Kidney 0.65±0.01 0.706±0.024

Spleen 0.615±.02 0.642±0.020

Pancreas 0.414±0.02 0.348±0.038

Heart 0.71±0.02 0.708±0.033

Lung 1.59±0.02 1.717±0.143

Testis 0.629±0.07 0.634±0.021

Epididymis 0.260±0.05 0.310±0.031

71

Table 2 Incidence of bacterial translocation to blood, liver, kidney and spleen treated with S.

phocae PI 80. (n=8 which includes 4 animals per sex)

Item Control S. phocae PI 80

Blood

MRS 0/8 (0/4 +0/4) 0/8 (0/4 +0/4)

BHI 0/8 (0/4 +0/4) 0/8 (0/4 +0/4)

Liver

MRS 0/8 (0/4 +0/4) 0/8 (0/4 +0/4)

BHI 0/8 (0/4 +0/4) 0/8 (0/4 +0/4)

Spleen

MRS 0/8 (0/4 +0/4) 0/8 (0/4 +0/4)

BHI 0/8 (0/4 +0/4) 0/8 (0/4 +0/4)

Kidney

MRS 0/8 (0/4 +0/4) 0/8 (0/4 +0/4)

BHI 2/8 (2/4 +0/4) 1/8 (1/4+0/4)

72

Table 3 Summary of hematological and biochemical data in the oral toxicity study. Means±SD

with different superscripts differ significantly (ρ<0.05)

Parameters Streptococcus Control

phocae

RBC (10 12

/l) 6.93±0.5 6.85±0.18

Hgb (g/dl) 14.69±0.26 14.36±0.22

Hct (%) 41.88 ±1.06 38.42±0.97

PLT (10 9/l) 485.5±10.9

476.25±12.91

MCV (fL) 56.75±1.5 53.62±0.62

MCH (pg) 19.72±0.330 19.22±0.22

MCHC (g/dL) 35.95±0.9 33.87±1.75

RDW (%) 12.82±0.22 11.65±0.351

MPV (fL) 7.4±0.3 7.27±0.11

LYM (%) 83.8±1.15 76.5±2.08

MON (%) 10.17±0.79 11.25±0.793

GRA (%) 9.3±0.74 10.7±0.57

Glucose (mg/dL) 121.57±8.09 112±4.96

Albumin (g/dL) 3.13±0 .237

2.95±0.17

Urea (mol/L) 4.98±0.38 4.47±0.25

Creatinine (mmol/l) 34.59±7.0 35.25±2.75

ALKP UL-1

322.4±16.95 302±11.19

SGPT IU/L 60.24±9.27 51±5

RBC- Red Blood Corpuscles; Hgb- Haemoglobin; PLT: Platelet; GRA-Granulocytes;MON-Monocytes;

LYM-Lymphocytes; MCH-Mean Carpuscular Hemoglobin; SGPT-Serine Glutamic Pyruvic Transaminase;

ALKP- Alkaline Phosphatase.

73

Figure legends

Figure 1 Hematoxylin and Eosin stains of spleen, kidney and liver from wistar rats administered

with S. phocae PI 80. Control group received sterile skim milk.

74

Figure 2 Acridine orange stained peripheral blood erythrocytes of rat treated with S. phocae PI 80

-A, control-B.

Figure 3 Effect of S. phocae PI 80 on Vibrio and Coliform count compared with those

of control for Vibrio and Coliform respectively in Penaeus monodon. The values are

expressed as mean ± SD. * Mark indicates statistical significance at P < 0.05.

75

Figure 4 Enumeration of L.monocytogenes in control and S. phocae PI 80 treated

Penaeus monodon. The values are expressed as mean ± SD. * Mark indicates statistical

significance at P < 0.05.

Figure 5 Enumeration of Lactobacilli in control and treated Penaeus monodon. The

values are expressed as mean ± SD. * Mark indicates statistical significance at P < 0.05.

76

Figure 6 Peroxide value in control and S. phocae PI 80 treated Penaeus monodon.

The values are expressed as mean ± SD. * Mark indicates statistical significance at P < 0.05.

Figure 7 Estimation of total volatile base in control and S. phocae PI 80 treated

Penaeus monodon. * Mark indicates statistical significance at P < 0.05.

77

Figure 8 Effect of S. phocae PI 80 on Total viable count in Vaccum packed Penaeus monodon. *

Mark indicates statistical significance at P < 0.05.

Figure 9 Estimation of total volatile base in Vaccum packed Penaeus monodon. * Mark indicates

statistical significance at P < 0.05.

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5.1. Introduction

Food safety is of foremost concern in food production and processing industry currently.

A number of modern preservation techniques are being industrialised to please the

existing demands of consumer satisfaction in nutritional aspects. Increasing consumer

awareness of probable health risks allied with some of the chemical and antibiotic food

additives has led researchers to look at the likelihood of using lactic acid bacteria (LAB)

as preservative agent (Duffes et al., 1999). The handiness of LAB on fishery products are

potentially valuable because of their bio preservative properties as they grow naturally on

chilled meat, alter the environment to discourage the growth of other organisms. The

mounting curiosity in using LAB as natural preservatives is due to the secretion of

antimicrobial compounds like hydrogen peroxide, lactic acid and bacteriocins which has

led to consideration of their use as biopreservatives in aquatic food products. Varieties of

studies have reported the inhibitory activity of LAB secretory products against pathogenic

microorganisms (Apostolidis et al., 2011). However, there are merely a small number of

reports available on LAB isolated from fresh aquatic product (Campos et al., 2006).

Lactic acid bacteria are used as starter cultures for years for fermenting food products and

also for the purpose of extending the shelf life of food products. Enterococci are gram-

positive, facultative anaerobic, cocci-shaped bacteria that are gifted to produce

bacteriocin with antimicrobial activity towards a broad spectrum of pathogens.

Enterococcus spp. such as Enterococcus faecium and Enterococcus faecalis exert

admirable beneficial effects and are suggested as safe, reliable probiotic bacterium.

Among enterococci, E. faecium are well studied bacteria since they have produced most

of the bacteriocins (Enterocins). Various bacteria from Lactobacillus, Pediococcus,

79

Leuconostoc were used as starter cultures. However, genus Enterococcus gains

meticulous attention as they survive as commensals on intestine and bring out beneficial

effects. More than twenty five species belonging to Enterococcus were listed for probiotic

properties (Klien, 2003). Few members of enterococcus bacteria have been reported to

cause endocarditis and bacteraemia infections (Kayser, 2003). Other than contributing to

taste and flavour, enterococcus bacteria also act as protective cultures against food borne

pathogens. Bacteriocins secreted by these enterococcus bacteria are considered as

suitable alternatives for food preservation as they are considered as ―natural inhibitors‖

and are not found to be non-harmful to eukaryotic cells and can be digested by proteolytic

enzymes in the gut. Uses of enterococci in foods are not encouraged only for their

organoleptic properties but also for the bacteriocin they secrete called ―enterocins‖ which

inhibit food borne pathogens (Giraffa, 2003). Enterocin secreting bacterial cultures were

employed for preserving meat products previously by Callewaert et al. (2000). Enterocins

can be assisted in various hurdle technologies like MAP, antimicrobial films, Irradiation

etc. Enterocins A have limited the growth of food borne pathogens when incorporated

into alginate films and this effect was more pronounced in vaccum packed conditions

(Marcos et al., 2007). Enterococcus faecium MC13 (AY751463) strain was previously

isolated from the gut of marine fish Mughil cephalus. Optimization of temperature for the

growth and bacteriocin production of E. faecium MC13 in Mann-rogosa-sharpe broth was

determined using the methods of experimental factorial design and response surface

analysis (Kanmani et al., 2011). Defying our culture E. faecium belonging to the LAB

group, there has been controversy as to whether they have to be considered as safe for use

in food as preservatives. It is important to confirm the safety of any new strain by acute

oral toxicity tests that have been advocated as the fundamental test for probiotic safety

assessment studies (Duangjitcharoen et al., 2009). In this report, we described the safety

80

and functionality of E. faecium using a mouse model and its application in the

preservation of sea food Penaeus monodon.

5.2. Materials and methods

5.2.1. Samples and Microorganisms

Penaeus monodon was purchased from local markets. The strain E. faecium MC13 was

isolated from the gut of the Mughil cephalus (Gopalakannan, 2006; Swain et al., 2008;

Kanmani et al., 2011b). Thiosulfate citrate bile salts sucrose agar was used for

enumerating Vibrio. Listeria-selective agar was used for the enumeration of Listeria

monocytogenes (MTCC-657). All indicator strains were obtained from Microbial Type

Culture Collections (MTCC), India and the complete information regarding the microbial

cultures used in this chapter is provided in the materials methods section of Chapter-3.

5.2.2. Safety assessment of the antagonistic biopreservative Enterococcus faecium

MC-13

Safety studies using animal experiments were carried out in a recognized organization

(Reg.No. 1159 /c/07 / CPCSEA; Animal facility of the Pondicherry University, India)

according to guiding principle of the ethical committee. The central animal house is

registered with the Committee for the Purpose of Control and Supervision of Experiments

on Animals (CPCSEA). Four weeks old male wistar rats were divided into two groups

having six rats per group. The rats were maintained in a controlled environment (23±2°C)

and had free admittance to feed and water. E. faecium MC13 was inoculated in Mann-

Rogosa-Sharpe broth and incubated at 37°C for 24h (Swain et al., 2009; Kanmani et al.,

2011c). The cells were pelleted by centrifugation at 5,000 g for 10 min at 4°C. Pellets

were washed in phosphate buffered saline (PBS, pH 7.4) twice. Finally, E. faecium MC13

81

was adjusted to 109

cells in 10% sterile skim milk. The control group was fed with skim

milk under the equivalent conditions as treated by the test groups. Treatment group were

fed with 100 µL of 109

cells of E. faecium MC13. The treatment was continued for twenty

eight days during which activity, behaviour, hair lustre, water intake, feed intake and

body weight were measured.

5.2.3. Hematology

Blood samples were collected by cardiac puncture. Red blood cells, haemoglobin count,

platelet count, mean corpuscular hemoglobin, lymphocytes, monocytes, granulocytes

count, glucose, urea, creatinine, concentration of alkaline phosphatase, Serine glutamic

pyruvic transaminase enzyme concentrations were detrmined to analyse the function of

organs. Haematological parameters were determined with an automated hematology

counter (Beckman Coulter Hematology Analyser LH750).

5.2.4. Histology

At the end of experimental period, spleen, kidney and liver of the E. faecium fed group

and the control group which fed on skim milk were dissected and the samples were

analysed by histological techniques. Hematoxylin and eosin staining process was used to

analyse the sections of liver, kidney and spleen. The complete protocol is explained in the

materials and methods section (Chapter-3. Section-3.3.3).The images were processed

using Adobe Photoshop software. The sections were analysed by an experienced

hepatopathologist.

5.2.5. Bacterial translocation:

After feeding with test strains for 28 days, animals were humanely euthanazed by

isofluorane over dose. Blood was drawn via cardiac puncture of the right ventricle and

100 µl was spread-plated onto Mann Rogosa Sharpe agar (MRS) and Brain Heart

82

Infusion agar (BHI) using the methodology previously described (Chapter-3. Section-

3.3.4.). Positive growth on agar plates was defined by the presence of any micro-

organisms (even a single colony).

5.2.6. Erythrocyte staining

The micronucleus test was performed to investigate whether the supplied protective

culture E. faecium induced micronuclei in polychromatic erythrocytes in murine

peripheral blood.

Animals that are exposed to protective culture E. faecium were sacrificed at appropriate

times after treatment. Blood cells were stained and processed based on the methodology

described previously in the materials methods section (Chapter-3. Section-3.3.5.). The

proportion of mature and immature erythrocytes was determined for each animal by

analysing the erythrocytes.

5.2.7. Efficacy of Isolated LAB Strain E. faecium in preservation of sea food

Sea food samples were erratically allocated to two treatments: (1) uninoculated controls

(2) samples inoculated with 1 ml of E. faecium MC13 suspension (106 cells) on each side

of the animal. After sprawling inocula by manually rubbing down, samples were stored at

4±0.5°C. Analyses were staged on days 0, 5, 10, 15 and 20 of refrigerated storage.

5.2.8. Microbial load analysis

Penaeus monodon was inoculated with the E. faecium MC13 at about 6 Log CFU/g and

were packed in a sterile bag. Uninoculated samples were treated as control. After

treatment, spoilage microbial counts such as Vibrio and Coliform counts were observed in

both control and biopreserved samples. Spread plate technique was used to enumerate

Vibrio and Coliforms using Thiosulfate citrate bile salts sucrose agar and Violet red bile

agar media. Lactobacillus Mann Rogosa Sharpe agar was used for the enumeration of

83

LAB. Microbial counts were made on selective media and were expressed as Log CFU/g.

Microbial enumeration was carried out using the methods described in the materials and

methods section (Chapter-3. Section-3.5.).

5.2.9. Co-inoculation studies in raw Penaeus monodon

Penaeus monodon were sterilized before using it in co-inoculation experiments (Paari et

al., 2011c). The co-inoculation experiment allowed observing the effect of application of

protective culture E. faecium MC13 in limiting the population of Listeria monocytogenes.

Samples obtained from a processing plant were co-inoculated with the pathogen and

protective culture (Fall et al., 2010) with slight modifications. A 24 h culture of E.

faecium MC13 (106CFU/g) and L. monocytogenes at a final concentration of 10

4CFU/g

was added and were sealed in bags and stored at 4 ± 2ºC for 15 days. An untreated

control, with 0.25 M phosphate buffer pH 7.0, was also included. At time 0 and days 5,

10, 15 and 20, triplicate samples of each treatment were chosen for counts of L.

monocytogenes on Listeria selective agar and Mann Rogosa Sharpe agar for lactic acid

bacteria counts. For microbial analysis, tissue samples (10 gm) were transferred

aseptically to a stomacher bag containing 90 ml of 0.85 % saline, homogenized for 1 min

and was spread on the surface of specific media. Microbial counts were made on selective

media and are expressed as Log CFU/g

5.3. Determination of Total volatile base

Total volatile base was measured using microdiffusion method (Conway, 1950) explained

previously in the materials methods section of Chapter 3 (section-3.4).

84

5.3.1. Measurement of peroxide value

Peroxide value (PV) was determined using titration method described by Sallam (2007).

The complete methodology for peroxide value estimation was provided in the materials

and methods section (Chapter-3. Section-3.6.)

5.4. Results

5.4.1. Safety assessment of protective culture Enterococcus faecium MC-13

Oral toxicity test in wistar rats displayed no noticeable change in activity, behaviour all

over the study period. E. faecium MC13 demonstrated no evidence of toxicity in the

toxicity assays. Diarrhoea or any other treatment associated illness or fatality was not

recorded. No difference in organ weight was noted between the groups orally fed with E.

faecium MC13 and the control. No signs of splenomegaly (Irregular increase in spleen

size) or hepatomegaly (Irregular increase in liver size) were observed in samples acquired

from liver and spleen (Table 4). Trabecular structure is clear in liver samples of control

and treated. The cytoplasm of hepatocytes was not found to be expanded in treated

samples. The glomerulli was having normal network and structure in both control and

treatment groups. Deterioration of epithelia of renal tubules was not noticed in any of the

animal (Figure 10).

A representative fluorescence photomicrograph of peripheral blood erythrocytes of rats

exposed to protective culture is shown (Figure 11). Induction of micronuclei and no

biologically relevant increase of micronuclei were found after treatment with protective

culture. The incidence of positive translocation in each handling group is listed in Table

5. No bacterial growth in the blood and liver tissue was observed which indicates that the

analysed tissues are not translocated by the fed E. faecium MC13. In control and E.

faecium MC13 supplemented groups, bacterial penetration through the epithelium and

85

transportation to distal sites occurred to the kidney, with a translocation incidence of

25%.

There was no major variation in blood parameters amongst the control and treated group

indicating no inflammatory response, which is allied with the feeding of E. faecium

MC13. The concentration of creatinine, urea, ALKP, SGPT differed between the control

and culture fed animal, but not significantly (Table 6). Oral administration of probiotic E.

faecium MC13 had no adverse effect on food intake between the control and treatment

group throughout the experiment. Concerning tissue weights, there was no significant

difference in the weight of any tissue investigated between the control and treatment

group. Internal organs observed were normal in appearance. Absence of necrotic sections,

ulcers approves the non toxic clinical relevance of protective culture.

5.4.2. Efficacy of protective culture on preservation of sea food

Total Volatile Base contents rose for both the control and biopreserved samples during

the storage (Figure 12). Notable differences in the evolution of TVB values were

observed among the control and biopreserved group all through the study period, with the

maximum values recorded for control samples. In control, TVBN values were 60±3.5 mg

N/100 g, whereas in biopreserved; TVB-N values were 46±2 mg N/100 g. Sea foods

biopreserved using E. faecium MC13 were rated ‗good quality‘ due to their TVB-N

values until day15. In vaccum packed samples too significant difference in total volatile

base evolution was noted between the control and biopreservative treated samples.

Control sample reached a value of 60 mg/100gm of TVBN by the end of storage time

whereas E. faecium MC13 treated sample under vaccum condition produced only 38.5

mg/100gm of TVBN (Figure 18). The inaugural PV (meq peroxide/kg fish sample) value

in the Penaeus monodon was 1.3±0.3 meq peroxide/kg. At the end of the storage time,

86

lowest malonaldehyde content (6.7±0.4 meq peroxide/kg) was observed in the

biopreserved samples (Figure 13) and the highest in controls (7.7±0.4 meq peroxide/kg).

The most drastic effect i.e., reduction of Log CFU was observed in E. faecium MC13

containing samples. In control, Vibrio population increased to 6.39±0.06 Log CFU/g

whereas this was 5.1±0.07 Log CFU in biopreserved Penaeus monodon. In these

conditions, growth of coliforms was also affected by the presence of E. faecium MC13

(Figure 14). TVC was under control in the biopreserved samples compared to control

samples also in vacuum conditions. More than 2 Log reductions was observed in

biopreserved samples (Figure 17)

5.4.3. Co-inoculation experiment

Listeria monocytogenes populations increased as storage time increased for all treatments.

Listeria counts were estimated significantly low (P < 0.05) for treatment groups when

compared with the control. The result of co-inoculation experiment seems to confirm our

previous knowledge that use of protective culture limits the growth of the microbial

pathogens. In this study, during storage, L. monocytogenes population was reduced by 1.3

Log CFU in the E. faecium MC13 inoculated Penaeus monodon (Figure 15). Listeria

monocytogenes count raised to 7.7 Log from their initial 4 Log CFU in control whereas

the growth rate of listeria was limited in biopreserved samples. Their count was 6.4 Log

CFU from their initial count of 4 Log CFU. The inoculated protective culture E. faecium

MC13 antagonized the L. monocytogenes very well and became the dominant flora in the

biopreserved Penaeus monodon. The level of LAB in biopreserved sample was

comparatively 4.2 Log higher than the control Penaeus monodon respectively (Figure

16).

87

6. Discussion

A probiotic organism is a live microorganism which, when supplemented in sufficient

quantity, promotes health assistance on the host and they sustain the microbial balance of

the living. Aspiring probiotic implementations are copious, but often shortfalls proper

selection customs that ask over microbiologists to safely promote its utilitization

(Shanahan, 2003). The focus of this chapter is to present an inclusive toxicological

evaluation of the novel probiotic E. faecium MC-13 and its efficacy as a biopreservative

against foodborne pathogens

More than one hundred strains were isolated from freshly caught shrimps and fishes and

were identified as microorganisms belonging to the group LAB. Criteria crucial for

selection of strains appropriate for fish preservation under the situation of our experiment,

such as capability to grow at low temperatures, In vitro simulation of gastric transit

tolerance, and resistance to proteolytic enzymes and tolerance to bile acids, cell adhesion

assays were determined in another separate study (Satish et al., 2012). Investigations in

our laboratory have picked out two potent LAB strains, Streptococcus phocae PI-80 and

Enterococcus faecium MC-13 which exhibit strong probiotic properties (Swain et al.,

2009; Gopalakannan & Arul, 2011; Kanmani et al., 2011b). The reality that no abnormal

morphological changes were contemplated in the liver, spleen, and kidney of E. faecium

MC13 fed animals may manifest that the administration of E. faecium MC13 did not

cause any toxicity puzzles. Occurrence of micronuclei containing chromosome fragments

that are symptomatic of cytogenetic damage in peripheral blood erythrocyte was not

noted in groups fed with E. faecium. Abberations or alterations was not observed between the

responses (frequency of immature peripheral blood erythrocytes) produced by animals fed

with protective culture and the control groups. Endres et al. (2009) failed to observe any

88

significant change in the chromosomal aberrations, erythrocyte pattern in the mice models

fed with proprietary preparation of a novel probiotic Bacillus coagulans.

A bacterial translocation positive animal was defined as animal that has got translocation

had at least one tissue sample (including blood) comprising single or more viable

bacterial cells translocated through the gut when a probiotic is fed. Analogous

translocation incidence between the E. faecium MC13 fed and control group implies that

disruption of the intestinal ecological equilibrium and intestinal mucosal barrier which are

major mechanisms promoting bacterial translocation did not happen because of E.

faecium MC13

No significant difference in haemoglobin, RBC, MCH and platelet count verify the

nonexistence of anaemic condition in E. faecium fed wistar rats (Schalm et al., 1975).

Defence against microbial pathogens were mediated by monocyte (Cheesbrough, 1991).

Monocyte count was not differing significantly between the control and treated group.

The magnitude of hematological differences observed between the control and protective

culture fed rats was very low. Similar results were observed by Zhou et al. (2000) who

reported that four week consumption of L. rhamnosus and bifidobacterium strains to mice

had no adverse effects on haematology and biochemistry. Saarela et al. (2000) reviewed

that the immune modulation mediated by probiotic strains may not be related with the

inflammatory immune response that could have probable harmful effects. However

differences in total leucocytes count was noted in mice fed with probiotic Zymomonas

mobilis (De Azerêdo et al., 2010). The absence of significant difference for the organ

weight and hematological parameters added to the absence of mortality suggested that the

fed protective culture was not toxic. An increase in the weight of spleen, a decrease in

that of liver and kidney could indeed be an indication of a toxic environment (Solomon et

89

al., 1993). No macroscopic changes were observed after administration of protective

culture for 28 days.

Total volatile base may be measured as a quality catalogue of a fish which is correlated to

the activity of spoilage bacteria that causes increased ammonia and other amines

accountable for imparting off flavours to fishery products. The initial TVB were likely

similar to the values reported as 8.25 mg N/100 & 12.28 mg N/100 in cod and anchovy

fish correspondingly by Suhendon mol et al. (2007). An increase in TVB-N values was

noted with the increase with time in both the treated and control samples, indicative of a

protein degradation process. Excessive protein degradation which is connected to quality

deterioration associated with growth of spoilage bacteria are reason for higher TVB

values in control. Best organoleptic and sensory properties (not shown) noted in

biopreserved samples were in good correspondence with the lowest TVB-N values as

well as with the lowest total bacterial counts, while the worst organoleptic characteristics

of control samples were accompanied by the maximum values found in the same

chemical and bacteriological analyses. Comparable correlation between TVB, sensory

and microbial scores were reported by Oehlenschlager (1998) in ice-stored Sea cod.

Inhibition of lipid peroxidation in foods by LAB has been observed before in cheese and

tuna mince (Songisepp et al., 2004). Recent reports from our research group elucidated

the antioxidant properties of LAB, which have high potential to quench free radicals that

initiate oxidation of lipids (Kanmani et al., 2011a). Our results approve the finding of

Gelman et al. (2001), that starter cultures of lactic acid bacteria decrease the

malonaldehyde level in fish based products.

The growth pattern of L. monocytogenes was inclined by protective culture E. faecium

MC13 in the co-inoculation experiments carried out in raw Penaeus monodon. Bredholt

90

et al. (2001) demonstrated the efficiency of LAB in inhibiting L. monocytogenes in

salmon and meats respectively. Synthesis of anti-listerial bacteriocin against L.

monocytogenes by LAB was previously demonstrated in our lab (Satish & Arul, 2009).

Soni et al. (2010) reported the reductions of 0.5 and 1.2 Log CFU/g of L. monocytogenes

by Phage P100 doses of 105 and 10

6 PFU/g respectively, on raw salmon tissues. Previous

studies from our lab reported the biopreservative effect of LAB in extending the shelf life

of cheese products (Satish et al., 2010). The added protective culture showed the expected

action against the spoilage microorganisms. Similar reduction was observed when

bacteriocin-producing carnobacteria was used to preserve seafood systems (Vescovo et

al., 2006). There was a proportional decrease in L. innocua cell numbers in sea foods

during storage. In another study, Nielssen et al. (1990) reported that the extent of growth

of L. monocytogenes was reduced by 1 to 2.5 Log CFU fewer than that attained with

control. Many studies failed to prevent growth of L. monocytogenes in food products by

using preservatives (Richard et al., 2003: Yamazaki et al., 2003). Considering the fact

that the natural initial contamination levels of L. monocytogenes in shrimp are generally

very low (< 1 CFU/g) (Paranjpye et al., 2008), the protective effect of E.faeciumMC-13

demonstrated in this study could be promising to ensure the safety of sea foods towards

risk of microbial and chemical spoilage. LAB strains isolated from fish products would be

more competitive than LAB from other sources, as they would be more adapted to the

substrate to which they would potentially be added.

91

Table 4 Effect on organ indices (g) of wistar rats fed with protective culture E. faecium

MC13.

Organs Control E. faecium MC13

Liver 7.042±0.37

6.66±0.23

Kidney 0.74±0.05 0.677±0.02

Spleen 0.606±0.020 0.576±.02

Heart 0.749±0.03 0.71±0.03

Lung 1.69±0.19 1.527±0.15

Epididymis 0.33±0.05 0.312±0.04

92

Table 5 Incidence of bacterial translocation (number of positive translocation/ total

number of animal analysed) to blood, liver, kidney and spleen treated with E. faecium

MC13. (BHI- Brain Heart Infusion: MRS- Mann Rogosa Sharpe)

Item Control E. faecium MC13

Blood

MRS 0/6 0/6

BHI 0/6 0/6

Liver

MRS 0/6 0/6

BHI 0/6 0/6

Spleen

MRS 0/6 0/6

BHI 1/6 0/6

Kidney

MRS 0/6 0/6

BHI 1/6

1/6

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Table 6 Summary of hematological and biochemical data in the oral toxicity study.

Parameters E. faecium MC13 Control

RBC (10 12

/l) 7.02±0.49 6.32±0.29

Hgb (g/dl) 14.4±0.4 13.92±0.22

PLT (10 9/l) 469.5±9.46

459±8.84

MCH (pg) 22.42±0.79 20.9±0.93

LYM (%) 72.2±5.5 66.75±2.98

MON (%) 10.17±0.79 11.25±0.793

GRA (%) 11±0.53 9.25±0.73

Glucose (mg/dL) 102±11.9 88.75±8.53

Urea (mol/L) 4.37±0.33 3.62±0.33

Creatinine (mmol/l) 33.75±2.98 30±2.4

ALKP UL-1

308.75±13.7 289.5±8.54

SGPT IU/L 56±3.91 49.75±5.88

RBC- Red Blood Corpuscles; Hgb- Haemoglobin; PLT: Platelet; GRA-Granulocytes;MON-Monocytes;

LYM-Lymphocytes; MCH-Mean Carpuscular Hemoglobin; SGPT-Serine Glutamic Pyruvic Transaminase;

ALKP- Alkaline Phosphatase.

94

Figure legends

Figure 10 Hematoxylin and Eosin stains of spleen (a, b), kidney (c, d) and liver (e, f)

from wistar rats administered with E. faecium MC13. Control group received sterile skim

milk.

95

Figure 11 Acridine orange stained peripheral blood erythrocytes of wistar rats fed with E.

faecium MC13.

Figure 12 Estimation of total volatile base in control and treated Penaeus

monodon . The values are expressed as mean ± SD. Asterisk indicates statistical

significance at p< 0.05

96

Figure 13 Peroxide value in control and treated Penaeus monodon. The values are

expressed as mean ± SD. Asterisk indicates statistical significance at p< 0.05.

Figure 14 Effect of E. faecium MC13 on Vibrio and Coliform count compared

with those of control for Vibrio and Coliform respectively in Penaeus

monodon. The values are expressed as mean ± SD. Asterisk indicates statistical

significance at p< 0.05.

97

Figure 15 Enumeration of L.monocytogenes in control and treated Penaeus

monodon. The values are expressed as mean ± SD. Asterisk indicates statistical

significance at p< 0.05.

Figure 16 Enumeration of Lactobacilli in control and treated Penaeus

monodon. The values are expressed as mean ± SD. Asterisk indicates statistical

significance at p< 0.05.

98

Figure 17 Effect of E. faecium MC13 on Total viable count in Vaccum packed Penaeus

monodon. Asterisk indicates statistical significance at p< 0.05.

Figure 18 Estimation of total volatile base in Vaccum packed Penaeus monodon. Asterisk

indicates statistical significance at p< 0.05.

99

6.1. Introduction

A number of potent preservation techniques for extending the shelf life of food products

are being developed to satisfy the existing challenges of food preservation. Consumer

fulfilment with regards to nutritional and sensory aspects of food was given preference

during preservation of foods. Chemicals like ethylene dibromide, most of benzoates like

sodium benzoate and ethylene oxide were used to preserve food against insects and

microorganisms in various countries (Theron & Rykers Lues, 2011). Various researches

have identified the hazardous nature of chemicals in food to human health. Induction of

tumour like symptoms have been reported by commonly used food preservatives like

Butylated hydroxy anisole (Witschi, 1986). This has made people to reject chemically

preserved foods and consumers look for foods, free of chemical preservatives being

aware of chemical free food stuffs. Identification and application of appropriate

processing techniques that promise food security and safety is the immediate requirement

of time for sea food industries. Of the various techniques used for preservation of food,

gamma radiation at low dose which limits or eliminates microbial contamination by

electron beams appears to be the easily applicable source for preserving food products

(Thayer & Rajkowski, 1999). Radiation pasteurisation can be used as a tool to extend the

shelf life of sea foods (Kwon & Byun, 1995; Manisha & Warrier, 2002; Venugopal et al.,

1999; Ashie et al., 1996). Nearly 50 products in sixty countries are sterilised using

gamma irradiation. Various reports say that food irradiated up to a dose of 10 kGy is safe

to consume without causing toxicity related symptoms (WHO, 1999). The Codex

Alimentarius Commission jointly organised by the Food and Agricultural organisation

and World Health Organisation which sets the standards for irradiated foods and the

regulations for irradiation technology has limited the irradiation dose up to 10 kGy

100

(Codex general standards, 1984). No toxicological evidences were reported from food

stuffs irradiated at higher doses (WHO, 1999). Consumers demand for additive free,

considerably natural and fresh products with microbial safety that can be provided by

employing the technique Gamma irradiation (Gould, 1996). Patterson & Loaharanu

(2000) reported minimal alterations in the sensory quality of food products when

sterilised using gamma irradiation. Radiation threshold or tolerance differs from one food

product to other. As all food products respond in a different way to ionising radiations,

radiation dose threshold has to be identified for each product. Low doses of irradiation

were preferred than higher doses as flavour changes were noticed in samples irradiated at

high dose (Nickerson et al., 1983). Two to three fold increase in shelf life was observed

for sardines in a study conducted by Kasimoglu (2003). A trial study was carried out to

examine the potential of irradiation technique for foods transported between Korea and

India and found the technique was versatile in preserving the chemical and microbial

indices of spoilage (Kwon et al., 2004). Irradiation may restrain the augmentation of

pathogenic microorganisms on sea foods but reduction of quality by chemical basis can

still take place (Ahn et al., 1998). Selman et al. (1982) reported that the nutritional and

sensory aspects of foods will not be affected during irradiation. However, changes in

odour and flavour due to oxidation of lipids and alterations in colour due to reddening

effect were considered as factors that interrupt consumer acceptance for irradiated foods.

(Egan & Wills, 1985; Byun et al., 2002). The NaNO2 formed in the irradiated food are

reported to have cancer inducing properties (Gray, 1976). The adverse changes happening

during the process of food irradiation has to be controlled and standardised for each

foods. The emergence of oxidation products produced due to irradiation resulted in poor

organoleptic property which limits the storage and processing quality of fishery products

(Gardner, 1979). Fish from fresh water and marine source has got significant level of

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polyunsaturated fatty acids like eicosapentaenoic acid. Rancidity is an intolerable

shortcoming in food quality which causes degradation in lipid constituents. The extent of

rancidity is not only based on the amount of lipid present in the fish but also the

composition of lipid and also based on the location of lipid in tissue. High content of

unsaturated fatty acid in fishery products is also a reason for rancidity. Tocopherol and

carotenoid like materials in fish matrixes also co-oxidize with lipids during storage (Allen

& Hamilton, 1994). The autoxidation of lipids leading to rancidity in fishery products is

initiated by free radicals generated during high energy irradiations. The reaction between

generated free radicals and lipids in the fish is considered as a vital feature of cellular

injury. Free radical mediated oxidation of lipids particularly affects the PUFA due to their

high degree of unsaturation. Various diseases such as atherosclerosis, cancer and

myopathy were reported for consumption of food containing oxidation products (Hunter

& Mohammad, 1986). Antioxidants that inhibit the process of oxidation by transferring

electrons to oxidizing agent act as reducing agent. This aptitude of antioxidants to

respond with the generated free radicals during irradiation makes them molecule of

special interest. In this study, we evaluated the effect of gamma irradiation on the quality

of Penaeus monodon and also the potential of antioxidants in preserving the quality of

irradiated fishery products were determined.

6.2. Materials and methods

6.2.1. Irradiation treatment

Penaeus monodon (15 gm) were purchased from landing centres near Pondicherry

University. Gut and head from Penaeus monodon was removed and were grouped into

following batches: Samples irradiated with antioxidant: Samples irradiated without

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antioxidant. Samples from the above mentioned batch were analysed for microbiological,

biochemical analyses and electron spin paramagnetic resonance investigations.

6.2.2. Experimental design

Shrimp collection

Degutting and beheading

Antioxidant treatment ( 0.1% Curcumin, BHA)

Gamma irradiation (1-10kGy)

Evaluation of radical emergence (EPR)

Antioxidant assays

TVB TMA TBARS Microbial count EPR Spectral

assessment

6.2.3. Chemical analysis

Total volatile base was estimated using the Conway microdiffusion method with few

modifications. Twenty five gm of fish sample was homogenised with seventy five ml of

distilled water and filtered using a whatman filter paper. 2 ml of this filterate was added

to the central ring. Following this, 2 ml of 0.025N Hcl was added to the outer ring along

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with 1 ml of saturated potassium carbonate and the system was incubated in dark

condition for 24 h. After incubation, the content of the inner ring was titrated with 0.025

N NaOH. Tashiros reagent was used as indicator to denote the end point. Results were

noted as milligrams TVB-N/100g. A blank was conducted simultaneously using 2%

trichloroacetic acid solution instead of sample extract. Similar process was followed to

estimate the concentration of TMA, except the addition of 1% formaldehyde to the outer

chamber of the conway unit. Trimethyl amine concentrations were expressed as mg

N/100 gm fish.

Lipid oxidation was determined by the TBA method. 1 gm of fish muscle was mixed with

9 ml of 0.25 M sucrose and homogenized. Homogenized substances were filtered using

Whatman filter paper. To 0.2 ml of this filtrate, 0.2 ml of SDS, 1.5 ml of TBA was mixed

and heated at 70oC for 60 min and absorbance was read at 532 nm. TBARS value was

expressed as mg of malonaldehyde (MDA) per kg.

6.2.4. Microbial load analysis

10 gm of tissue from control and irradiated samples (1- 10kGy) were homogenized in 90

ml of 0.1% saline in a stomacher and plated onto selective media. Bacterial count was

expressed as log CFU/g. the complete protocol for microbial load analysis was provided

in the materials and methods section (Chapter-3. Section-3.5.)

6.2.5. Electron paramagnetic resonance analysis

EPR studies were done using an EPR spectrometer (JEOL) at room temperature and the

spectra were recorded at the X-band (9.3GHz). 100 mg sample was placed in quartz EPR

tubes (3 mm of internal diameter with a wall thickness about 0.1 mm). EPR Spectrum

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was recorded using the following parameters: microwave frequency, 9.5 GHz; microwave

power, 0.63-31.73 mW; centre field, 250 mT; Sweep width, 500 mT.

7. Results

Total viable count was 3.8± 0.12 log CFU/g at day 0. Microbial growth was not observed

till day-5 in 6, 8 and 10 kGy irradiated samples respectively. During final days of storage,

higher microbial count (7.1±0.15 log CFU/g) was noticed for control whereas it was only

2.6±0.12 log CFU/g for 10 kGy irradiated samples (Figure 19).

7.1. Evolution of TMA, TVB and T-BARS content in irradiated Penaeus monodon

During storage, Trimethylamine content was 17mg/100gm for control; whereas it was

only 5 mg/100gm in 10 kGy irradiated samples. Trimethylamine values for 2, 4, 6, 8 kGy

irradiated samples were 14.5, 13.15, 9.3, and 6.3 mg/100gm respectively (Figure 20).

TMA value was under the admissible level throughout the study period for samples

irradiated beyond 4 kGy.

Total volatile base value was high (74±4.2 mg per 100 gm) for control, whereas lowest

total volatile base (58±4.24 mg per 100 gm) was noted in 1 kGy irradiated sample.

Lowest TVB-N value (26±2.8 mg) was noted in 10 kGy irradiated samples (Figure 21).

TBARS value was determined for both control and irradiated samples. Lower lipid

oxidation rate was noted in samples irradiated at lower doses (1-3 kGy) whereas

increased oxidation rate was observed in samples irradiated at higher irradiation doses (4-

10 kGy). Final TBARS values were 0.71± 0.03, 0.5± 0.04, 0.49± 0.04, 0.76± 0.07, 0.8±

0.05, 1.02± 0.01 mg malanoaldehyde/Kg for non-irradiated, 2, 4, 6, 8 and 10 kGy

irradiated samples correspondingly (Figure 22).

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7.2. Detection of irradiated Penaeus monodon

Irradiated samples exhibited radical peak in spectral resonance. Samples irradiated at

higher doses showed peaks of greater intensity whereas samples irradiated at lower doses

showed EPR peaks of lower intensity. The non-irradiated samples were EPR silent

without any radical peaks. Radiation induced free radical generation was strong at higher

doses where the strongest intensity in radical peak was noted in 10 kGy irradiated

samples (Figure 28). Reduction in signal strength for free radical was noted for irradiated

samples upon storage. The decay kinetics observed after 30 days and 45 days of storage

showed reduction in their intensity (Figure 29 & 30). Although absolute decay was not

observed, considerable decline in the radical induced signal was noted.

7.3. Effect of antioxidants on t-bars evolution in irradiated Penaeus monodon

The potential role of antioxidants in controlling the radiation induced oxidation of lipid

was studied at two doses (2.5 kGy and 5 kGy). The potential of antioxidant was noted to

be 79.95 % and 71.65 % for BHA and curcumin correspondingly in DPPH radical assay

and was 79.6 % and 70.85 % in Beta carotene bleaching antioxidant assay for BHA and

curcumin respectively. The antioxidants curcumin and BHA showed excellent radical

suppressing effect. Samples irradiated not including antioxidant exhibited higher lipid

oxidation rate compared to samples treated with antioxidants BHA and curcumin.

Analogous tendency was observed for peroxide value in the non-irradiated control.

Addition of BHA and curcumin postponed the formation of peroxides. Remarkable

differences (P>0.05) were observed between the control (7±0.5) and curcumin (5.6±0.28)

and BHA (3.1±0.4) treated samples in 5 kGy irradiated samples (Figure 23). Parallel

trend was seen in 2.5 kGy irradiated samples (Figure 24), where lower peroxide value

was obtained in samples infused with antioxidants compared to control.

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During final days of storage, TBARS value for irradiated control devoid of antioxidant

was 1.31±0.12 and 0.91±0.04 for 5 kGy and 2.5 kGy respectively. However, final day

TBARS value were 1±0.07 (curcumin), 0.88±0.02 (BHA) for 5 kGy irradiated sample

(Figure 25) and it was 0.72±0.02 and 0.6±0.01 for curcumin and BHA treated 2.5 kGy

irradiated samples respectively (Figure 26). Results of TBARS assay was supported with

the results of Electron Paramagnetic Resonance Assay, where the antioxidant supplied

samples showed peaks of lower intensity (Figure 27). Radical quenching potential of

curcumin and BHA was clearly visible from the EPR spectra.

8. Discussion

High microbial and chemical spoilage reports were noted in Penaeus monodon because of

the poor production and distribution modes that are followed. Also the moisture content

and water activity (Aw) of the shrimp make it a perishable food where enzymatic and

chemical spoilage will occur easily (Labuza, 1970). Development of a processing

procedure to preserve the quality of shrimp product is of high need. Though irradiation at

lower dose can offer microbial protection to foods, few chemical and sensory damages

occur at higher doses. Hence a processing system was developed to preserve the quality

of irradiated foods by supplying antioxidants prior to irradiation treatment. DPPH radical

quenching assay, β-carotene assay, t-bars assay and Electron spin paramagnetic resonance

spectral readings were carried out to analyse the antioxidant nature of extracts used for

preserving irradiated foods. This study suggest that shared mutual effect of gamma

irradiation and antioxidant packaging as an effectual processing method for preservation

the quality of tiger prawn Penaeus monodon.

Total viable count method is generally used to access the microbial quality of sea food

products. Microbial and biochemical deterioration in Penaeus monodon during

refrigerated storage are controlled to a noteworthy level at various doses of irradiation.

107

The effect of gamma irradiation was exactly noticeable in all doses. Microbial load was

lower in irradiated samples this was visible through the drop of log CFU in irradiated

samples whereas the Total viable count was high for non-irradiated control samples. The

reduction of Log CFU was dose dependent. The outcome of this trial assures the

association between the increase of radiation dosage and reduction of microbial count.

Reduction of microbial load by gamma irradiation has been reported previously in

anchovy (Lakshmanan et al., 1999), tilapia (Cozzo-Siqueira et al., 2003), spanish

mackerel (Abu-Tarboush et al., 1996).

The shelf life and the quality of Penaeus monodon was also determined by estimating the

chemical indices of spoilage. Trimethylamine evolution in the control and irradiated

samples gave a clear picture regarding the control of chemical spoilage by irradiation

process. Trimethyl amine oxide is an osmoregulator present in fish which plays the role

of a protein stabilizer. The fishy odour is due to the conversion of trimethylamine oxide to

trimethyl amine (Shakila et al., 2003). The threshold level or tolerability of 10-15 mg/100

gm set for TMA was reached in control by ninth day of storage, whereas in irradiated

samples, delayed evolution of trimethylamine was noted.

The amount of dimethyl amine, ammonia and trimethyl amine produced during storage of

sea food Penaeus monodon can be analysed by measuring the concentration of total

volatile base. TVB estimation is also measured as an indicator for chemical spoilage in

sea foods. In fishery products, 15–20 mg N/100 gm of TVB value denotes a good quality

and if the value crosses 50 mg N/100 gm it indicates poor grade due to deterioration

caused to tissue protein. In control and irradiated samples the evolution of TVB was

found to be increasing. Al-Kahtani et al. (1996) reported the reduction pattern of TVB in

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1.5–10 kGy irradiated tilapia. Reduction of TVB values in irradiated samples may be due

to the decrease in microbial population which is accountable for formation of amines

Oxidative degradation is the major problem associated with food irradiation. The lipids in

the food are oxidised by the electron trapping ability of free radicals generated by the

process of irradiation. As they have high concentration of lipids and polyunsaturated fatty

acids, oxidative degradation of fatty substances at higher doses becomes the reason for

increased TBARS value at higher irradiation doses. The emergence of free radicals due to

irradiation at higher doses has been detected by electron paramagnetic resonance

(Arvanitoyannis, 2010; Gordy et al., 1955). In our research, the intensity of EPR radical

signal extended linearly with the raise in the irradiation dose. Raise in EPR signal with

the higher irradiation dose with respect to free radical generation was reported by

Stachowicz et al. (1992). Raffi et al. (2000) noticed a reduction of 50%-80% radiation

induced signal in irradiated fruits after 210 days of storage.

Oxidation of lipids happens due to the addition of oxygen molecule to fatty acid

substances resulting in the formation of hydroperoxides. These hydroperoxides form

malanoldehyde which cause rancid odour. High fat content in fish could be the reason for

increased oxidation rate in the stored Penaeus monodon. TBARS value which measures

the quantity of malondialdehyde in sample was found to be low in the antioxidant treated

samples. With the increase in the storage time, the TBA value or the oxidation rate also

increases which could be due to the decomposition of lipid hydroperoxides. Quenching

the initial free radical reactions or by breaking of radicals chain reaction by donating

electron is the process carried out by antioxidants to prevent lipid oxidation. Irradiation

mediated lipid oxidation were studied in a dose dependent mode by Thayer et al. (1993).

Saeed & Howell (2002) reported the decline of TBARS value in the existence of

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antioxidant in Atlantic mackerel. Sensory analysis carried out by Ozogul et al. (2004)

suggested that antioxidant packaging will reduce the damages caused by irradiation. The

outcome of the study confirmed that BHA and curcumin improved oxidative stability for

irradiated Penaeus monodon.

110

Figure

Figure 19 Changes in total viable count in refrigerated Penaeus monodon as affected by

irradiation. The values are expressed as mean ± SD for three samples.

Figure 20 Changes in TMA -N values of Penaeus monodon with or without irradiation,

stored under refrigeration.The values are expressed as mean ± SD. Asterisk indicates

statistical significance at p< 0.05.

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Figure 21 Changes in TVB N values of Penaeus monodon with or without irradiation,

stored under refrigeration. The values are expressed as mean ± SD for three samples.

Asterisk indicates statistical significance at p< 0.05.

Figure 22 Changes in TBA values of Penaeus monodon with or without irradiation. The

values are expressed as mean ± SD. Asterisk indicates statistical significance at p< 0.05.

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Figure 23 Effect of antioxidants - control, - curcumin, - BHA on the

retardation of Peroxide value by irradiation at 5 kGy. The values are expressed as mean ±

SD. Asterisk indicates statistical significance at p< 0.05.

Figure 24 Effect of antioxidants - BHA - curcumin - control on the

retardation of Peroxide value affected by irradiation at 2.5 kGy. The values are expressed

as mean ± SD. Asterisk indicates statistical significance at p< 0.05.

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Figure 25 Effect of antioxidants - control, - curcumin, - BHA on the

retardation of TBARS affected by irradiation at 5 kGy. The values are expressed as mean

± SD. Asterisk indicates statistical significance at p< 0.05.

Figure 26 Effect of antioxidants - control, - curcumin, - BHA on the

retardation of TBARS affected by irradiation at 2.5 kGy. The values are expressed as

mean ± SD. Asterisk indicates statistical significance at p< 0.05.

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Figure 27 X-band EPR spectra of tiger prawn treated with antioxidant. A) Control- 2.5

kGy B) 2.5 kGy + 1% curcumin C) 2.5 kGy +1% BHA D) control-5 kGy E) 5 kGy + 1%

curcumin F) 5 kGy + 1% BHA

Figure 28 X-band EPR spectra of tiger prawn irradiated at different doses at day -0. (A-

2; B-4kGy;C-6 kGy;D-8kGy;E-10kGy)

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Figure 29 X-band EPR spectra of tiger prawn irradiated at different doses after 30 days

of storage. (A- control; B-2kGy;C-4 kGy;D-6kGy;E-8kGy,F-10kGy)

Figure 30 X-band EPR spectra of tiger prawn irradiated at different doses after 45 days

of storage. (A- Control; B-2kGy;C-4 kGy;D-6kGy;E-8kGy,F-10kGy)

A

F

A A A A A A

C

A

B

A

E

D

A

A

F

A A A A A A

E

D

A

B

A

C

A

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7.1. Introduction

The worldwide requirement for sea food products is mounting and so there is an emerging

need for using efficient preservative approaches. Food spoilage caused due to pathogenic

microorganisms was a serious problem in food industries (Niemira, 2010). Gamma

irradiation has been employed for sterilization of animal foods (Mahrour et al., 2003;

Chwla et al., 2003). Despite the fact that gamma irradiation and refrigerated storage may

effectively control the growth of pathogenic microorganisms, damages caused due to

chemical and physical origins can happen even at low temperature (Ahn et al., 1998).

Fishery products are extremely sensitive to oxidation as they comprise fatty acids which

are prone to rancidification. The oxidative products formed due to irradiation counteract

with muscle components leading to alterations in sensory quality as well as limit the

storage stability (Gardner, 1979). Antioxidants can suppress free radicals and extend the

shelf life of fishery products by decelerating the process of lipid peroxidation, which is

one of the main reasons for deterioration of food products during storage (Fwell, 1997).

Usage of antioxidant combinations displayed marked reduction in reducing the negative

shades of irradiation and operate as admirable lipid stabilizers (Pazos et al., 2005). As a

result, adding up of either synthetic or natural scavengers to quench radiolytic products is

required for the preservation of sea food. In the past few years, the suspected toxicity of

some synthetic antioxidants used in food has raised curiosity in natural products as they

are safer and more acceptable to the human body (Stone et al., 2003). Hence a call for

identifying natural sources, particularly of plant source has notably increased in recent

years (Skerget et al., 2005). Despite major interest in naturally occurring antioxidants,

there is still research needed on their spectrum of action, explicitly in food products.

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Addition of natural compounds obtained from plants has been employed in an attempt to

improve the oxidative stability in fish muscle. Tea catechins (Tang et al., 2001) rosemary

(Vareltzis et al., 1997), grapes (Yerlikayav & Gokoglu, 2010), pomegranate (Gokoglu et

al., 2009), citrus peel (Kang et al., 2006) and ginger extracts (Fagbenro & Jauncey, 1994)

have successfully inhibited rancidity of different seafood. Grape oligomeric catechins are

immensely efficacious in delaying lipid oxidation in fish muscle during frozen storage

(Pazos et al., 2005) and their compounds are aspiring candidates as food supplements for

their antiradical properties (Matito et al., 2003). Antioxidant compounds appear in bound

form with insoluble polymer matrix (Niwa & Miyachi 1986). Processing technologies are

now developed to enhance the antioxidant potential from natural sources (Avila-Sosa et

al., 2010). Gamma irradiation suggested for sterilisation of food has been reported to have

some enhancing effects on the antioxidant aspects of plant extracts (Choi et al., 2006).

This technology was used to enhance the antioxidant properties of selected fruit extracts.

Distinctive methodologies were reviewed in order to measure the multifunctional

antioxidant activity of fruit peel extracts as well as to compare the results produced by

different heat treatments on enhancement of antioxidant activity. The aim of this work

was to assess the radio protecting capability of four Indian fruit extracts subjected to

various doses of gamma irradiation and heat treatments and to demonstrate their practical

utility in a sea food Penaeus monodon.

7.2. Materials and methods

7.2.1. Sample collection

Grape (Vitus vinifera), Lemon (Citrus limon), Pomegranate (Punica granatum), Orange

(Citrus sinensis) were obtained from a local market. In this study, heating and/or

irradiation effects on the radical scavenging activity of fruit peels, under the experimental

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conditions of heating at 50, 100 and 150°C for 60 min and/or irradiating at 2.5, 5 and 10

kGy was determined. Fruit peels were heated in a heat furnace and irradiated using

Cobalt-60 gamma chamber before extraction. Extracts from Punica granatum was

prepared using the method described by Singh et al. (2002). The peels were manually

separated and shade dried and was extracted with 100 ml of methanol at room

temperature for 24 h. The extracts were filtered and was re-extracted with same solvent

and concentrated under reduced pressure through rotator evaporator. Method of Li et al.

(2006) was employed for extraction from Citrus limon and Citrus sinensis. Briefly, frozen

peels were dipped in liquid nitrogen and ground to fine powder. The powdered peels were

extracted using 100 ml of ethanol for 24 h for complete extraction. Vitus vinifera seeds

were powdered and extracted in a soxhlet extractor with hexane for 6 h for the removal of

fatty matter. The defatted powder was extracted in a soxhlet extractor for 24 h with 100

ml methanol at 60-70°C as described by Jayaprakasha et al. (2001).

7.2.2. DPPH radical Scavenging Activity

The capacity to scavenge the ‗‗stable‘‘ free radical DPPH was monitored following the

method of Blois (1958). The radical-scavenging activity was calculated as a percentage of

DPPH discoloration. The complete protocol is explained previously in the materials and

methods section (Chapter-3. Section-3.8.1).

7.2.3. Reducing Power

The reducing power of extracts was calculated based on the method of Oyaizu (1986).

Higher absorbance of the mixture indicates stronger reducing power. The complete

protocol is explained previously in the materials and methods section (Chapter-3. Section-

3.8.2).

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7.2.4. β-carotene bleaching assay

β-carotene bleaching assay was done according to the method of Matthaus (2002). The

complete protocol is explained previously in the materials and methods section (Chapter-

3. Section- 3.8.3).

7.2.5. Nitrite scavenging activity

This assay was carried out as described by Saha et al. (2004) with some modifications.

The complete protocol is explained previously in the materials and methods section

(Chapter-3. Section- 3.8.4).

7.2.6. Hydroxyl radical scavenging activity

Hydroxyl radical scavenging activity was determined following the method of Smirnoff

& Cumbes (1989) with a few modifications. The complete protocol is explained

previously in the materials and methods section (Chapter-3. Section-3.8.5).

.

7.2.7. Electron Paramagnetic Resonance spectroscopy

EPR investigations on RSA of extracts against a stable radical were measured using the

method described by Nanjo et al. (1996). A methanol solution of 60 µl each sample was

added to 60 µl of DPPH (60 µmol l-1

). After mixing vigorously, the solutions were

transferred into an aqueous cell quartz tube and fitted into the cavity of the EPR

spectrometer. The spin adduct was measured on EPR spectrometer exactly 2 min later in

an X-band EPR spectrometer at room temperature using standard rectangular cavity

operating at 9.4 GHz with a 100 kHZ modulation frequency. The microwave power and

modulation amplitude were 4 mW and 1 G, respectively.

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7.2.8. Gas Chromatography-Mass Spectrometry analysis of natural Extracts

Dry powder of selected material showing maximum antioxidant properties (10 kGy

irradiated sample) was subjected to column chromatography employing silica gel (60-120

mesh size) and eluted stepwise using a linear gradient of chloroform: methanol. The

active fraction which showed maximum DPPH radical scavenging activity in each

extracts was subjected for GC-MS analysis using the methods described in materials and

methods section (Chapter-3, section-3.8.7).

7.2.9. Evaluation of antioxidant potential in Penaeus monodon after irradiation

treatment

Antioxidant activity in the meat system was determined using the method of Moller et al.

(1999) with modifications. Penaeus monodon were separated into sections in sterile

plastic bags and were treated with selected natural extracts as well as with BHA, a

synthetic antioxidant. The Percentage of extract that was used is 1% for all cases prior to

irradiation. The irradiated samples were stored at 4°C to examine their efficacy of natural

extracts in irradiated samples.

7.2.9.1. Peroxide value

Peroxide value (PV) was determined using the method described by the American Oil

Chemists Society (1990). 5 gm of sample was dissolved in 30 ml glacial acetic acid-

chloroform solution (3/2 v/v) and 1 ml KI solution (14 g/ 10 ml) was added. Distilled

water was added after 1 min and the mixture was titrated with 0.01N sodium thiosulfate

until the blue colour disappeared. PV was calculated using the following formula

100W

NBVKg/mEqPV

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Where, V is the volume of sodium thiosulfate consumed, B is the volume of normal

sodium thiosulfate consumed during a blank titration, W is the weight of the sample and

N is the normality of sodium thiosulfate multiplied by a factor.

7.2.9.2. TBA assay

Oxidation of lipids is assessed by the TBA assay which is based on the reaction between

thiobarbituric acid and malondialdehyde as described by Ruberto & Baratta (1999).

Briefly, 0.05 g of each sample was mixed with distilled water (1 ml), 1.5 ml of 20 %

acidic acid and 1.5 ml 0.8% of TBA in 1.1% SDS and heated to 100°C for 60 min in a

water bath. After cooling, 5 ml butan-1-ol was added. Samples were then centrifuged at

10000 rpm for 10 min. The upper layer absorbance was read at 532 nm.

7.2.9.3. DPPH assay

Measurements with the DPPH assay were taken using a method used by Tepe et al.

(2005). 0.05 g of the fish samples and 5 ml of 0.004 % DPPH in methanol were mixed.

The samples were incubated at room temperature for 30 min. Absorbance was measured

at 517 nm, using methanol as a blank. Measurements were expressed as absorbance.

Decreasing absorbance levels indicated increasing antioxidant activity of the extracts.

7.3. Results

7.3.1. Scavenging activity of natural extracts

Effect of fruit extracts on hydroxyl radicals was moderate for Citrus sinensis

(47.41±4.54%) and Punica granatum peel (57.6±4.05%) as compared to BHA, which

showed 68.26±2.95% inhibition (Figure 31A). The antioxidant activity has been reported

to be concomitant with the reducing power. The high reducing power (0.554 (Citrus

limon)–0.874 (Vitus vinifera seed) ASE/ml) indicated their potential as electron donors to

scavenge free radicals efficiently. Reducing powers obtained for extracts were in the

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following order: Vitus vinifera seed > Punica granatum peel> Citrus sinensis peel>

Citrus limon peel. Nitrite scavenging effect of naturally occurring fruit peel extract

proved it to be a potential nitrite scavenger. As shown in Figure 31B, the scavenging

capacities of Citrus limon (53.5±5.15%) and Citrus sinensis (59±5.90%) towards nitrite

were relatively feeble whereas, the scavenging activities became stronger with Vitus

vinifera seed (78.53±6.55%) and Punica granatum peel (76.70±5.41%). Figure 31C

shows the antioxidant activity of the fruit peel and seed extracts as measured by the

bleaching of β-carotene. Antioxidant activities were 42±4.71%, 47.7±4.40%,

70.1±2.13%, 67.4±1.73% for Citrus limon, Citrus sinensis, Vitus vinifera seed and Punica

granatum extracts, respectively, but antioxidant activity of BHA reached 79.5±3.9%.

DPPH Proton radical scavenging potential was higher in Vitus vinifera seed extract

(64.9±2.65%) followed by Punica granatum peel (61.6±1.1%) and Citrus sinensis peel

extract (39.7±1.0%). Minimal DPPH scavenging activity was observed in Citrus limon

extract (23.3±3.41%) whereas, positive control BHA exhibited a RSA of 78.61±3.0%.

7.3.2. Effect of heating conditions on the antioxidant activity of natural fruit extracts

Effect of heat and irradiation treatment on the antioxidant capacities of extracts were

evaluated by two assays, DPPH radical scavenging activity (Figure 32A) and beta

carotene bleaching assay (Figure 32B). RSA is observed to increase notably after the heat

treatment at higher temperatures (150°C and 100°C) compared to lower temperature

(50°C). Thermal treatment at 150°C increased the RSA of fruit peel extract from

30.19±3.46, 40.98±1.98, 68.26±1.13, 69.02±1.38 to 38.96±1.33, 49.46±3.80, 79.2±2.64,

80.17±2.37 for Citrus limon peel extract, Citrus sinensis peel extract, Vitus vinifera seed

extract and Punica granatum peel extract, correspondingly. Similar to the results of

DPPH assay, the scavenging potential increased in beta carotene assay as the heating

123

temperature is increased. Maximum activity was observed in Punica granatum peel

(84.53±1.12) and Vitus vinifera seed extract (86.3±4.20) when heated to 150°C

suggesting the possibility of using it as a natural antioxidant for applications at higher

temperatures.

7.3.3. Effect of Irradiation conditions on the antioxidant activity of extracts

Radical scavengers were assessed after exposing it to three different doses 2.5, 5 and 10

kGy. The percentage antioxidant activity increased significantly upon irradiation

treatment from 69±4.09, 69.51±1.59 for 2.5 kGy irradiated samples to 84.15±3.60,

81.8±4.01 by increasing the dose to 10 kGy for Vitus vinifera seed and Punica granatum

peel extracts respectively in DPPH assay. Similar to the results of DPPH assay, the

scavenging potential increased in beta carotene assay with higher doses. The antioxidant

potential elevated from 75.82±2.73 to 84.29±2.06 for Punica granatum peel and from

72.33±1.90 to 81.5±3.23 for Vitus vinifera seed extract with increasing dosage of gamma

irradiation from 2.5 kGy to 10 kGy. Maximum activity was contemplated at 150°C and

10 kGy treated samples which elucidates the affinity between the temperature and

radiation dose with the radical scavenging activity.

7.3.4. Electron paramagnetic resonance studies on radical scavenging activity

EPR spectrum of the DPPH radical is recognised by its five lines of relative intensities. It

is important to note that the higher the antioxidant potential of fruit peel extracts, the

smaller the signal of DPPH. The EPR signal intensity in the system with fruit peel and

seed extracts is smaller (Figure 33B-E) than that observed without extracts (Figure 33A).

7.3.5. Gas chromatography mass spectrometry of partially purified extracts

124

GC–MS analysis of active fraction of fruit peel extracts revealed the presence of

significant quantities of n-alkanes namely tridicane, hexadecane, pentadecane in the Vitus

vinifera seed extracts (Table 1). Abundance of carboxylic acids like propanoic acid and

butanoic acid was seen in Citrus limon samples. Hexadecoinic acid, an antioxidant from

tea leaves (Jang et al. 2010) was also found in Citrus limon extract (Table 2). Benzoic

acid, tetradecane, dodecane, Phenol, 2, 4-bis (1, 1-dimethylethyl), tetradeconic acid, and

nonadecane comprise the antioxidant properties for Citrus sinensis extract (Table 3).

7.3.6. Effect of natural extracts on t-bars and peroxide value evolution in irradiated

samples

Addition of fruit peels extracts delayed the generation of peroxides (Figure 35) and

TBARS values (Figure 36). The initial peroxide value was 0.75±.21 and 0.9±.14 in 2.5

and 5 kGy irradiated samples, respectively. By the end of storage time, significant

differences (P>0.05) were observed between the control (3.4±.28) and each of Vitus

vinifera, Punica granatum, Citrus sinensis and Citrus limon extract treated samples,

which exhibited values of 1.5±.21, 1.65±.2, 2.4±.4, 2.5±.42, respectively (Figure 35A).

Analogous trend was observed in 5 kGy irradiated samples, where the Vitus vinifera seed

extract and Punica granatum extract exhibited better PV compared with the control and

other counterparts (Figure 35B).

Initial TBA values were low in all treated samples compared to the control as in the PV.

No difference in TBARS was noticed amid the dipping treatments during initial days of

storage. However, final day value was 3±.28, 1.95±.07, 2.35±.07,1.55±.007, 1.35±.21 for

control, Citrus limon peel, Citrus sinensis peel, Punica granatum peel and Vitus vinifera

seed extracts respectively (Figure 36A). It was prominent that formation of TBARS in

Penaeus monodon increased when the irradiation dose was increased from 2.5 kGy to 5

125

kGy. Final day TBARS value for 5 kGy irradiated control sample was 4.65±.035 whereas

it was 3.1±.28, 3.2±.2, 2.35±.3, 2.45±.007 for Citrus limon peel, Citrus sinensis peel,

Vitus vinifera seed, Punica granatum peels respectively (Figure 36B). BHA supplied

samples showed better tbars value than the natural extracts treated group. However, the

difference was not significant for Punica granatum and Vitus vinifera seed extracts.

7.3.7. Demonstration of antioxidant activity in irradiated fish samples by DPPH

assay

Data generated using the DPPH assay Penaeus monodon irradiated at 2.5 and 5 kGy are

presented in Figure 34A and Figure 34B respectively. Throughout the study period,

antioxidant supplied samples exhibited lower DPPH absorbance values indicative of

higher antioxidant activity than the control samples which are irradiated without

antioxidant. Overall, fish samples infused with Punica granatum and Vitus vinifera seed

extract displayed higher antioxidant activity (i.e., lower absorbance) than did the Citrus

limon, Citrus sinensis and control samples. A final DPPH absorbance value of 0.19±.05

was seen for control, while it was 0.178±.01, 0.182±.005, 0.172±.007, 0.17±.004 for

Citrus limon, Citrus sinensis, Punica granatum, Vitus vinifera seed respectively.

Analogous reduction was observed in 5 kGy irradiated samples. A final DPPH value of

0.68±.006 was observed for control samples, while DPPH absorbance value for samples

supplied with Citrus limon, Citrus sinensis, Punica granatum and Vitus vinifera seed

extracts were 0.53±.01, 0.58±.007, 0.5±.007 and 0.47±.011 respectively.

7.4. Discussion

126

Hydroxyl radical is the most effective radical that react with lipid, proteins and with

oxygen resulting in the production of peroxy radical. Thus removing hydroxyl radical is

important for antioxidant defence in food systems (Aruoma, 1998). The reducing

properties are generally associated with the presence of reductones (Singh et al., 2002)

which break down the free radical chain by donating a hydrogen atom. The polyphenols

of the antioxidant extracts are believed to act in a similar fashion. Exposure to excess

nitrite from the diet is implicated as a potential etiological factor in the development of

colorectal cancers (Kato & Puck, 1971). In β-carotene assay the absorbance decreased

rapidly in samples without antioxidant whereas, in the presence of an antioxidant, they

retained their colour for a longer time indicating the antioxidant potential of extracts. It is

apparent that the components in the fruit extracts reduce the extent of β-carotene

destruction by neutralizing the linoleate free radical formed in the system. DPPH assay is

a standard test in antioxidant studies and offers a rapid technique for screening the RSA

of specific extracts. The reduction capability of the DPPH radical is determined by the

decrease in its absorbance at 517 nm induced by antioxidants (Bidchol et al., 2009). The

better free radical scavenging by BHA is credited to their methoxy group, which increases

the accessibility of the radical centre of DPPH to BHA (Gow- Chin & Pin-Der, 1994).

One can notice that the RSA significantly increased after the heat treatment at higher

temperatures compared to lower temperature. We infer that the increase is primarily due

to the degradation and release of bound antioxidant compounds from the matrix. The

enhancing effect of simple heat treatment on the antioxidant activity of fruit peel extracts

represented in this study was consistent with that of previous reports (Lin et al., 2008; Xu

et al., 2007). However this result contradicts the findings of Bchir et al. (2010) who

127

reported that an increase in temperature caused a decrease of the antioxidant activity in

pomegranate.

The percentage antioxidant activity increased significantly upon irradiation treatment.

Release of phenolic compounds from unextractable insoluble polymers to an extractable

form was considered a reasonable mechanism to be responsible for the elevated

antioxidant activity. Radiation induced enhancement of antioxidant activity was shown by

Variyar et al. (2004) in soybean.

The decline in the EPR signal after incubation with extract preparations may be due to the

pairing of odd electron of DPPH, reflecting the protective role of these extracts in

restricting the generated radicals. It is also very apparent from the spectra that Punica

granatum peel and Vitus vinifera seed extract minimized the intensity of the radical peak

to a greater extent in comparison to other extracts. Similar decrease in the amount of EPR

spectra was observed after the addition of grape skin extracts by Stavikova et al. (2008).

Liolios et al. (2009) also reported the presence of those compounds in fruits of date palm.

Hexadecoinic acid, an antioxidant from tea leaves (Jang et al., 2010) was also found in

Citrus limon extract. The main component Benzopyran 2 one in Citrus limon extract has

been reported for its biological activity in lemon peel oil by Su and Horvat et al. (1987).

Presence of benzoic acid, 2-Furancarboxaldehyde in orange juices and their antioxidant

nature has already been proved in many studies (Jerković & Marijanović 2010).

Tetradecanoic acid as one among the fatty acid compositions has been studied for its

radical scavenging properties by Kırmızıgül et al. (2007). Benzene di carboxylic acid, the

major compound in Punica granatum extract in our study was also detected in heat

treated citrus peels by Lee et al. (2003). Some compounds which have been reported as

major constituents in our study were absent or expressed in low quantity from other

128

authors reports. This may be due to different chemotype of the plant other than

geographical divergence and ecological condition.

The intense susceptibility of Penaeus monodon to suffer oxidation during irradiation and

storage is mainly due to the synchronous presence of high amounts of unsaturated lipids.

An increase in the peroxide value is most useful as an index of the earlier stages of

oxidation. The decline of the PV at the end of storage may occur owing to decomposition

of hydroperoxides into secondary oxidation products. As peroxide value measurement

are not reliable in assessing oxidation of highly unsaturated fish samples because of their

nature to form secondary oxidation products, PV was used in conjunction with TBA and

DPPH assay.

A significant increase in TBARS was observed at 12th

day of storage, perhaps due to the

fact that TBARS are secondary products of lipid oxidation formed from decomposed lipid

hydroperoxides. Inhibition of lipid oxidation in irradiated samples could be due to either

suppression of free radicals during initiation step or interruption of free radicals chain

reaction by acting as electron donors. A similar result was observed by Kang et al. (2006)

in fish homogenate by the addition of citrus peel extract.

The purple coloured DPPH (a stable free radical) is reduced to the yellow-coloured 1,1,-

diphenyl-2-picrylhydrazine reacting with the free radicals of the sample (Kirby &

Schmidt 1997). The lower gathering of free radicals in antioxidant supplied samples

might be the reason for having lower absorbance (i.e., higher anti-oxidant reactivity).

Overall, the antioxidant activity of all samples lessened with storage time. Fasseas et al.

(2007) demonstrated similar lower DPPH absorbance in meat samples preserved with

oregano and sage essential oils. Oxidative damage caused by free radicals and other

reactive oxygen species is believed to be associated with the development of a range of

129

diseases. This research has demonstrated that the extract from fruits, which is commonly

used as a natural food, is an excellent free radical scavenger. Furthermore, the outcomes

of the present study suggest that antioxidant activity can be enhanced by external stimuli.

Supply of food samples with natural antioxidants prior to gamma irradiation treatment

had reduced the occurrence of lipid oxidation to a larger extent during preservation. In the

near future, it is possible that the application of synthetic antioxidants will decrease still

further and consumers will be more willing to accept safe natural antioxidants for

preservation of sea foods.

130

Table 7. Detected compounds in gas chromatography from Vitus vinifera seed extracts

S. no RT Compound Area

1. 10.17 Benzoic acid, 4-ethoxy-, ethyl ester 7.71

2. 10.17 Benzoic acid, 4-ethoxy-, ethyl ester 7.71

3. 23.47 Benzenamine, 3-methoxy-2,4,6-trimethyl 3.71

4. 23.58 Benzenamine, 3-methoxy-2,4,6-trimethyl 1.70

5. 31.6 2-Butyn-1-ol, 4-methoxy- 1.74

6. 27.0 4H-1-Benzopyran-4-one 1.96

7. 27.6 1,2-Benzenedicarboxylic acid 2.46

8. 33.35 10,11-(3'-6'-Dihydrobenzo)[3.2]par 2.20

acyclophane-4'-carboxylic acid

9. 32.57 2-Ethoxycarbonyl-3-methyl-7-nitro 13.66

10. 9.349 Heneicosane 2.49

11. 8.0 Hexadecane 8.06

12 1.2 Hexadecane 3.87

13 33.53 1-Heptatriacotanol 5.07

14 10.0 1-Iodo-2-methylundecane 1.1

15 12.9 Nonadecane, 2-methyl- 2.57

16 14.9 Octadecane 2.44

17 28.69 Octadecane, 1-chloro- 3.60

18 31.1 7- Oxabicyclo[4.1.0]heptane, 1-meth 10.82

yl-4-(2-methyloxiranyl)-

19 9.98 Phenol, 2,4-bis(1,1-dimethylethyl) 4.12

20 29.3 Stigmasta-5,22-dien-3-ol 7

21 8.0 Tetradecane 2.63

22 6.34 Tridecane 1. 02

131

Table 8. Detected compounds in gas chromatography from Citrus limon peel extracts

S. no RT Compound Area

1 8.6 Bicyclo [3.1.1]hept-2-ene, 2,6-dime 1.75

thyl-6-(4-methyl-3-pentenyl)-

2 10.1 Benzoic acid, 4-ethoxy-, ethyl ester 1.31

3 32.14 beta.-D-Fructopyranose, 3.35

4 32.36 4H-1-Benzopyran-4-one 3.43

5 33.3 4H-1-Benzopyran-4-one 7.09

6 6.95 Benzocycloheptatriene 1.16

7 33.87 Benzene, 1,2,4-trimethyl-5-(1-methylethyl)- 1.53

8 31.8 Butanoic acid, 4.02

9 18.82 2H-1-Benzopyran-2-one, 5,7-dimethoxy 12.82

10 18.5 Hexadecanoic acid, ethyl ester 1.09

11 33.15 5-Hydroxymethyl tricyclazole 9.34

12 12.6 1H-Pyrrole, 2-ethyl-4-methyl- 1.02

13 23.07 Pimpinellin 2.92

7H-Furo[3,2-g][1]benzopyran-7-one 4,9-dimethoxy-

14 31.9 Propanoic acid 1.67

15 29.07 Pyridine, 3-methyl-2,6-diphenyl 1.23

Dicyclooctanopyridazine

16 29.83 1-Naphthalenamine, N-phenyl- 1.02

132

Table 9. Detected compounds in gas chromatography from Citrus sinensis peel

extracts

S. no RT Compound Area

1. 6.95 Benzocycloheptatriene 1.16

2. 10.17 Benzoic acid, 4-ethoxy-, ethyl ester 3.82

3. 6.34 Dodecane, Eicosane 2.08

4. 20.74 Eicosane, 2-methyl- 1.64

5. 6.1 2-Furancarboxaldehyde, 5-(hydroxymethyl 2.36

6. 11.2 Hexadecane 2.35

7. 28.07 Hentriacontane 1.22

8. 9.3 1-Iodo-2-methylundecane 1.71

9. 12.9 Nonadecane, 2-methyl- 2.28

10. 14.9 Octadecane 1.44

11. 9.9 Phenol, 2,4-bis(1,1-dimethylethyl) 3.14

12. 8.0 Tetradecane 2.91

13. 14.6 Tetradecanoic acid 3.33

133

Table 10. Detected compounds in gas chromatography from Punica granatum peel

extracts

S. no RT Compound Area

1. 32.3 4-azafluorenone, phenylimine 11.6

2. 32.5 4H-1-Benzopyran-4-one, 2-(3,4-dime 2.98

thoxyphenyl)-5,6,7-trimethoxy

3. 27.6 1,2-Benzenedicarboxylic acid 44.77

4. 26.1 2H,8H-Benzo[1,2-b:3,4-b']dipyran-6 1.01

-propanol, 5-methoxy-2,2,8,8-tetra

methyl-, acetate

5. 10.1 Benzoic acid, 4-ethoxy-, ethyl ester 2.88

6. 31.4 4-[p-Chlorostyryl]-6-methoxy-8-[2, 2.16

5-dimethylpyrryl]quinoline

7. 31.78 Disulfide, di-tert-dodecyl 1.55

8 32.4 1,3-Dioxo-2-[4-(3,4-xylyloxy)pheny 5.79

l]-5-isoindolinecarboxylic acid.

9 6.1 2-Furancarboxaldehyde, 5-(hydroxymethyl 5.74

10. 11.2 Hexadecane 1.60

11. 12.9 Heptacosane 1.00

12. 9.9 Phenol, 2, 4-bis (1, 1-dimethylethyl) 1.53

13. 8.064 Tetradecane 1.123

14. 27.9 Tetratetracontane 1.86

15. 14.5 Tetradecanoic acid 14.5

134

Figure

Figure 31 Antioxidant potential of fruit peel extracts: A- Hydroxyl radical scavenging

activity, B- Nitrite scavenging activity, C-β carotene bleaching, D-DPPH, E- Reducing

power. Results are shown as means±SD of three independent experiments.

Figure. 32A Effect of heat treatments (A- 50°C; B-100°C; 150°C) and irradiation dose

(D- 2.5kGy; E-5 kGy; F-10 kGy) on DPPH radical scavenging activity.

Figure. 32B Effect of heat treatments (A- 50°C; B-100°C; 150°C) and irradiation dose

(D- 2.5kGy; E-5 kGy; F-10 kGy) on antioxidant potential of fruit peel extracts in β-

carotene model system. Asterisk indicates statistical significance at p< 0.05

135

Figure 33 The typical EPR spectra of DPPH methanolic solution (60 µmol l-1

) recorded

in the presence of fruit extracts illustrating their antioxidant action (A- Control, B- Citrus

limon, C- Citrus sinensis D- Vitus vinifera, E- Punica granatum). Spectra were recorded

2 min after the extracts addition. EPR spectra were measured at 298 K using microwave

power 5 mW. X-axis refers to magnetic field from 315 mT to 335 mT. Y- axis refers to

the intensity of DPPH radical.

136

Figure 34A Profiles of DPPH antioxidant activity level in 2.5-kGy irradiated Penaeus

monodon infused with natural fruit extracts during refrigerated storage. Results are shown

as means ± SD of three independent experiments. Asterisk indicates statistical

significance at p< 0.05

Figure 34B Profiles of DPPH antioxidant activity level in 5-kGy irradiated Penaeus

monodon infused with natural fruit extracts during refrigerated storage. Results are shown

as means ± SD of three independent experiments. Asterisk indicates statistical

significance at p< 0.05

137

Figure 35A Formation of peroxides (mEq/kg) in 2.5-kGy irradiated Penaeus monodon

without and with exogenous antioxidative treatment during refrigerated storage. Results

are shown as means ± SD of three independent experiments. Asterisk indicates statistical

significance at p< 0.05

Figure 35B Formation of peroxides (mEq/kg) in 5-kGy irradiated Penaeus monodon

without and with exogenous antioxidative treatment during refrigerated storage. Results

are shown as means ± SD of three independent experiments. Asterisk indicates statistical

significance at p< 0.05

138

Figure 36A TBARS values (mg MDA kg−1 of fish) of 2.5-kGy irradiated Penaeus

monodon added with natural fruit extracts during refrigerated storage. Results are shown

as means ± SD of three independent experiments.

Figure 36B TBARS values (mg MDA kg−1 of fish) of 5-kGy irradiated Penaeus

monodon added with natural fruit extracts during refrigerated storage. Results are shown

as means ± SD of three independent experiments

139

8.1. Introduction

Fish are perishable food products which spoil generally sooner than other muscle foods.

Mounting awareness about the safety of sea food commodities have led to copious

developments in the field of fish preservation. Storage and processing of seafood products

are restricted due to the progress of lipid oxidation related problems and off-flavors

associated rancidity (Petillo et al., 1998). In order to evade the usage of synthetic

chemical preservatives, numerous studies are focused on employing natural substances

that carry a ‗green‘ image for preservation of sea food. Chitosan, a natural polysaccharide

comprising copolymers of glucosamine and N-acetylglucosamine, has been far and wide

used in food processing, medicine and biotechnology fields (Majeti & Ravi, 2000; Harish

Prashanth & Tharanathan, 2007). Chitosan becomes an appealing molecule that has

engrossed a enormous deal of attention as a food additive due to its antibacterial, film

forming property, antioxidative and biodegradable ability (Fan et al., 2009; Shahidi et al.,

1999; Kim & Thomas, 2007; Rhoades & Roller, 2000). Inherent antimicrobial

characteristics of chitosan could be the prime dynamic force in the improvement of novel

applications for this underused biopolymer. Wide spectrum of antimicrobial activity for

chitosan was reported against human pathogenic microorganisms (Chen et al., 2010;

Raafat et al., 2009). Although quite a lot of studies have been published, the precise

mechanism of the antimicrobial activity of chitosan remains vague. A recent study states

that the antibacterial effect of chitosan is due to the distraction of the cell membrane of

pathogens (Fang et al., 2010). Shelf life of fishery products was extended by inhibiting

the growth of Pseudomonas and Shewanella by the addition of chitosan (Cao et al.,

2009). Radiation is a convenient tool for modification of chitosan that has been

investigated for enhancement of their biological activity (Feng et al., 2008). Irradiation

140

was effective in increasing the antimicrobial activity of chitosan against Escherichia coli

(Kume et al., 2002). However, a research carried out by Lopez-Caballero et al. (2005)

demonstrated that the addition of powdered chitosan to fish patties had no consequence

on bacterial growth. Investigations on the radiation effects of chitosan proved that

irradiation could increase its antimicrobial activity and antioxidant properties

(Czechowska-Biskup et al., 2005; Park et al., 2004). Enhancement of antioxidant activity

of chitosan by gamma irradiation at three different doses were reported by Feng et al.

(2008). Suitability of irradiated chitosan in inhibiting oxidative rancidity in meat sample

has been reported (Kanatt et al., 2004). Among the diverse modifications that are

achievable, grafting of synthetic polymer is also a handy mode. The properties of chitosan

are personalized and a novel flexible material could be developed by chitosan blending

(Suyatma et al., 2004; Pourjavadi et al., 2003; Mahdavinia et al., 2004; Don et al., 2002).

PMMA was blended with chitosan for usage in various biomedical applications and the

non cytotoxic nature of MMA grafted chitosan has been proved (Radhakumary et al.,

2005). El-Tahlawy et al. (2006) investigated the ultimate changes in the chitosan

properties grafted by methyl acrylate and found that the grafted cross linked chitosan

graft copolymer was having enhanced antiviral property compared to the unmodified

chitosan. Development of modified chitosan enables us to achieve efficient chitosan

based materials with special feature and with minimum loss of the native properties.

Synergistic effects of chitosan covalently bonded with antimicrobials have been reported

(Song et al., 2002; Chen et al., 1996). In this study, the antioxidant and antimicrobial

activity of chitosan was enhanced using irradiation. The effect of modified chitosan

derivatives were analysed during the preservation of Penaeus monodon.

141

8.2. Materials and methods

Chitosan was irradiated at doses of 5 and 10 kGy in the gamma chamber facility of the

Central Instrumentation Facility (CIF), Pondicherry University. Cobalt-60 was the

irradiation source. Irradiated chitosan was prepared according to the method of Kannat et

al. (2004). Treatment groups for Penaeus monodon preservation were divided as: control

(without chitosan treatment), 5 kGy irradiated chitosan, 10 kGy irradiated chitosan,

Blended chitosan. Penaeus monodon was purchased from the local retail market. Instantly

after purchase, they were taken back to lab under sterile conditions.

8.2.1. Synthesis of poly (methyl acrylate) polymer grafted chitosan (Chitosan-g-

PMMA)

A 2g of chitosan was prepared in 250 ml of 1 % aqueous acetic acid (2% w/v solution).

The reaction was done with a nitrogen atmosphere at 70° C. 0.1M Ceric Ammonium

Nitrate in 1N nitric acid (10 ml) was added. 4g MMA was added drop by drop with

uninterrupted stirring. After 4h, the reaction was stopped and the product was precipitated

using sodium hydroxide solution with vigorous stirring. The obtained precipitate was

washed thoroughly by distilled water and filtered. The homopolymer was obtained from

the grafted product in a soxhelet extractor using acetone as solvent. The grafting

percentage was also calculated from Thermogravimetric analysis.

8.2.3. Scheme of the graft

142

O

O

OOO

HO

OH

NH2

HO

OHNH2

OH

HO

NH2

O

OCAN

O

O

OOO

HO

OH

NH2

HO

ONH

HO

NH

O

O

OO

OO

O

O

O

O

O

O

OO

OO

O

PMMA

Structure of Chitosan

Scheme-1 Structure of Chitosan-g-PMMA

8.2.4. Characterization

FT-IR spectra of chitosan and its graft copolymer was recorded in the range of wave

numbers 4000–500 cm-1

using Nicolet Nexus 470 spectrometer (Nicolet Co., Ltd.,USA).

The X-ray diffraction (XRD) patterns and Scanning electron micrograph (SEM) images

of chitosan and its graft copolymer was obtained. The degradation process and thermal

stability of chitosan and its graft copolymer were investigated using a thermogravimetric

analyzer

8.2.5. Antioxidant assay

8.2.5.1. DPPH radical Scavenging Activity

The capacity to supress the free radical DPPH was monitored according to the method of

Blois (1958). Chitosan (0.1 ml) was mixed with 0.9 ml of DPPH (0.041 mM). After 60

min dark incubation, the reduction of the radical intensity was determined by measuring

the absorbance at 517 nm. The radical scavenging activity was calculated using the

equation:

100

A

AARSA%

DPPH

sDPPH

143

8.2.5.2. Reducing Power

The reducing power of extracts was determined by the method of Oyaizu (1986).

Chitosan (1 ml) was mixed with 0.2 M phosphate buffer (1 ml) and potassium

ferricyanide (10 mg/ml) and incubated at 50°C. Following incubation, 1 ml of

trichloroacetic acid (100 mg/ml) was added and centrifuged for 10 min. To the upper

layer, 1 ml of water and 0.1 ml of ferric chloride (1.0 mg/ml) was added and the

absorbance was measured at 700 nm. Higher absorbance of the reaction mixture denotes

stronger reducing power.

8.2.5.3. β-carotene bleaching assay

β-carotene bleaching assay was carried out based on the method of Matthaus (2002). β-

carotene was prepared by dissolving in 1 ml of chloroform. After removal of chloroform

under vacuum, 40 mg of linoleic acid, 100 ml of distilled water and Tween 80 emulsifier

were added. 0.2 ml of prepared chitosan extracts were added to this mixture and

incubated at 50°C. The zero time absorbance was measured at 470 nm. Absorbance

readings were then recorded at 30 min intervals until the control sample had changed

colour. Antioxidant activity was calculated using the following equation

100contentcaroteneInitial

assayofmin120aftercontentcaroteneactivitytAntioxidan

8.2.5.4. TBA assay

Oxidation of lipids was measured by the TBA assay as ascribed by Ruberto & Baratta

(1999). 0.05 g of sample (Penaeus monodon) was mixed with distilled water, 1.5 ml of

20 % acidic acid and 1.5 ml 0.8% of TBA in 1.1% SDS and heated to 100°C for 60 min.

After cooling, 5 ml butanol was added. Samples were then centrifuged at 10000 rpm for

15 min. The absorbance of the upper layer was determined spectrophotometrically at 532

nm.

144

8.2.5.5. DPPH assay

DPPH assay was carried out using a method followed by Tepe et al. (2005). 0.05 g of the

sample (Penaeus monodon) and 5 ml of 0.004 % DPPH in methanol were mixed. The

samples were incubated at room temperature for 30 min. Absorbance was measured at

517 nm. DPPH measurements were expressed by means of absorbance. Decreasing

absorbance levels indicated increasing antioxidant activity of the extracts.

8.2.6. Determination of Total volatile base nitrogen (TVBN)

Microdiffusion method was employed to analyse total volatile base in Penaeus monodon.

Briefly, 1 ml of H2S04 was dropped into the inner chamber of the conway unit together

with Tashiro‘s indicator. In the outer unit of conway apparatus, 1 ml of 20%

trichloroacetic acid extract and potassium carbonate was added. Following absorption, the

contents of the inner chamber of conway unit was titrated against sodium hydroxide (0.1

N) until a colour change was observed. A blank was conducted at the same time with 2%

TCA solution in its place of sample.

8.2.7. Microbial count in raw Penaeus monodon

Total viable count was determined in the stored samples from control and all treatment

groups. At time 0 and days 5, 10, 15, triplicate samples of each treatment were chosen for

total plate count using the protocol explained in the materials and methods section

(Chapter-3. Section-3.5). Microbial counts were expressed as log CFU/g

8.3. Results

8.3.1. Characterisation

In the IR spectrum of chitosan-g-Poly methyl methacrylate, the characteristic absorption

bands around 3420 can be observed (Figure. 37A). The broad absorption band at 3420cm-

145

1 indicate that -OH and -NH2 groups are hydrogen bonded and absorption at 1658 cm

-1

indicate the amide linkage of chitosan backbone with external grafting polymer.

The change of chitosan structure after graft polymerization was investigated by means of

the powder X-ray diffraction. Four peaks of pure chitosan showing the maximum

intensity in the XRD was obtained at 20 0, 44

0 52

0 and 72

0. The powder XRD results

showed successful grafting on the chitosan surface. Figure 37B (a, b) shows the powder

X-ray diffractograms obtained for pure chitosan and chitosan-g-PMMA.

As seen in scanning electron microscopic pictures, chitosan is in the form of

homogeneous particles. Scanning electron image of native chitosan was used as

reference. The exterior surfaces of grafts were accumulated in the form of globules,

which may be due to the porosity of the graft that are the implications of successful graft

copolymer. Surface evidence of SEM image supports the grafting. The SEM image of

chitosan showed hefty clustered configuration, indicating the interactions between

chitosan molecules (Figure 38).

DSC shows the difference in Glass transition temperature (TG) between the pure chitosan

and the grafted chitosan (Figure. 39A). Increase in molecular weight due to random

polymerization and decrease in glacious crystalline nature of grafted chitosan resulted in

the decrease of glass transition temperature. The blue shift in glass transition temperature

of grafts is due to external polymerization. Exothermic peak is found to be decreased in

grafts due to increased molecular weight caused due to external polymerization.

The degradation process and thermal stability of chitosan and chitosan grafts were

evaluated through thermo gravimetric analysis (TGA). Weight loss in pure chitosan is

54.16% and starts at 245°C whereas weight loss starts at 208°C for PMMA grafted

chitosan and the total weight loss for PMMA grafted chitosan was 49.63%. Figure 39B,

146

shows thermograms of (a) pure chitosan and (b) chitosan-g-PMMA respectively. Pure

chitosan shows an endothermic peak at temperature around 91.84 0C, which ascribes to

the water holding capacity of chitosan by the -OH and unsubstituted free –NH2 groups.

Additionally, chitosan exhibits an exothermic peak at 306.60 0C informing decomposition

of polysaccharide unit. In the case of figure 39B(b), endotherms peak or glass transition

temperature appear at 102.92 0C and exothermic peak or decomposing temperatures

appear at 286.05 0C, which explores the decreased decomposing peak of chitosan grafted

polymer, indicating successful polymerization.

8.3.2. Scavenging activity of modified chitosan

Antioxidant activity of the chitosan measured by the bleaching of β-carotene was

presented in figure. 40. Antioxidant activities were 62.33±3.39%, 67.3±5%, 81.2±2.34%,

87.9±2.2%, for unmodified chitosan, blended chitosan grafts, 5 kGy irradiated chitosan

derivative, 10 kGy irradiated chitosan derivatives respectively.

The antioxidant activity has been reported to be concomitant with the reducing power of

chitosan samples. The highest reducing power was observed for irradiated chitosan

derivatives compared to the unmodified and grafted chitosan. Difference in reducing

power was also observed between the groups subjected to irradiation. A higher irradiation

dose (10 kGy) exhibited higher radical scavenging activity and higher reducing power

compared to chitosan irradiated at 5 kGy. Reducing powers obtained were in the

following order: unmodified chitosan > blended chitosan> 5kGy irradiated chitosan >

10kGy irradiated chitosan derivative.

DPPH assay offers a rapid method for screening the radical scavenging activity of

compounds. Proton radical scavenging potential was higher in 10 kGy irradiated chitosan

147

derivative (84±2.5%) followed by 5 kGy irradiated chitosan derivative (77.35±2.57%),

blended chitosan (65±1). Minimal DPPH scavenging activity was observed in unmodified

natural chitosan (63.11±1.1%).

8.3.3. Effect of chitosan on t-bars evolution

This study demonstrated the effectiveness of irradiated chitosan as a natural anti-oxidant,

when it was supplemented to the Penaeus monodon. Data generated using the TBARS

assay in Penaeus monodon during refrigerated storage are presented in figure. 41.

Throughout the study period, Penaeus monodon treated with modified chitosan exhibited

lower TBARS values indicative of higher antioxidant activity than the control (no

treatment). Though Penaeus monodon treated with unmodified chitosan exhibited

antioxidant activity better than control (without chitosan treatment), it was lower than the

activity executed by the modified counterparts. By the end of storage time, significant

differences (P>0.05) were observed between the control (2.03±.15) and each of native

chitosan, blended chitosan, 5 kGy , 10 kGy irradiated chitosan infused Penaeus monodon,

which exhibited values of 1.43±.15, 1.3±.05, 1.03±.15, 1±.1 respectively (Figure. 41).

8.3.4. Total volatile base

TVB-N contents augmented for all samples during the storage period with the utmost

values recorded for control samples followed by chitosan, blended chitosan, 5 kGy

irradiated, 10 kGy irradiated chitosan (Figure. 42). TVB values also intensified for

chitosan treated samples during storage period. At the end of storage period, TVB value

of control P. monodon was found to be 36±3.5which was higher than chitosan (26±2),

blended chitosan treated (25±2), 5 kGy irradiated chitosan treated (24.3±2.5) 10 kGy

chitosan treated (21±2). No lag phase was detected for control samples for total volatile

base generation. A rapid increase in TVB was evident from day-5. Control samples

148

surpassed the acceptability margin (35 mg TVBN/100 g of fish) set by the European

Union for total volatile base values of fish. Though limitation of TVB was not significant

between the treatment groups, all chitosan treated samples, limited the generation of TVB

throughout the storage period. A TVB value of 30mg which is considered to be spoilage

level for human consumption (Harpaz et al., 2003) was attained in control samples by

day-10 whereas, none of the chitosan treated groups reached this boundary. Log reduction

was high in irradiated chitosan treated samples (1.5 Log) compared to the unmodified

chitosan (1.3 Log) treated samples. Though Log reduction was not significant during

early days of storage, marked reduction was noted during later days of storage between

the control and treatment groups (Figure. 43).

8.4. Discussion

The full potential of chitosan can be explored using various modification techniques.

Modification methods like nitration, phosphorylation, sulphation, acylation, alkylation are

generally used for preparing modified chitosan (Van Luyen and Huong, 1996; De smedt.

et al., 2000; Roberts, 1992; Avadi et al., 2004). Hybrid materials made of bio and

synthetic polymers are developed for lot of applications by a technique called graft

copolymerisation (Jenkins, 2001). Introducing side chains during the process of chitosan

grafting pave way for the synthesis of novel materials for application in various fields like

water treatment, medicine, food processing etc (Kurita, 2001). Vinyl monomers like

methaacrylate, methyl methacrylate, acrylonitrile were polymerised with chitosan using

graft polymerisation for better functioning (Kurita, 2001). Water solubility was enhanced

by grafting chitosan with phenolic compounds (Kumar et al., 1999). Radical

polymerisation is the method used to prepare copolymers. Free radicals are generated in

the back bone of main polymer and the generated radicals function as initiator for grafting

149

vinyl/acrylic monomers (Mourya, 1999). Though the radicals can be generated using

chemical and irradiation method, in this study chemical method for radical generation

was employed. The amine group of the chitosan is believed to form polymerised graft due

to nucleophilic attack.The grafting confirmation was obtained from the FT-IR spectra of

pure chitosan and chitosan-g-PMMA. In figure 1A (b) the bending frequency of -NH2

(1457cm-1

) and -OH (1590cm-1

) peaks are shifted around 15cm-1

compared to native

chitosan which confirms the successful polymerization. The covalent link between

chitosan and chitosan grafted PMMA polymer were confirmed by FT-IR spectroscopy. In

the spectrum of chitosan-g-PMMA, characteristic stretching and bending frequencies was

observed at 1590 cm-1

(Harish Prashanth et al., 2005). These broad peaks are attained due

to formation of hydrogen bond between –OH and NH2 groups of core chitosan polymer.

In the case of PMMA co-polymer grafted chitosan, new peaks appear at 1658 cm-1

which

indicates the presence of -C=O of the amide and ester groups of the side chain

copolymers (Mohamed et al., 2007). The bending frequency of -NH2 and -OH, peaks

were found to be shifed around 15cm-1

compared to native chitosan confirming the

grafting of external copolymer. Four peaks of pure chitosan showing the maximum

intensity in the XRD was obtained at 2θ = 20 0, 44

0. 52

0 and 72

0, matches the values

reported (Joshi & Sinha, 2006), indicating the highly crystalline nature of chitosan. X-ray

diffraction was carried out in order to compare the crystalline nature of the chitosan and

chitosan grafted PMMA network. The molecular and crystal structure of the chitosan was

determined by the X-ray diffraction method. The four peaks showing the maximum

intensity corresponds to h00, 0k0, 00l, lattice plane of the chitosan orthorhombic crystals

indicating chitosan is highly crystalline in nature (Okuyama et al., 1997). After carrying

out polymerisation on the chitosan surface, three peaks completely disappeared indicating

the loss of crystalline nature. A broad peak around 20 0 which indicates the crystalline

150

structure of chitosan was converted in to amorphous nature due to the external

polymeraisation (Liu et al., 2004). In the case of cross-linked chitosan there was

significant difference in the intensity of characteristic peaks of chitosan. The distinct

differences in the diffraction patterns of chitosan and cross-linked chitosan could be

attributed to modification in the arrangement of molecules in the crystal lattice. Following

graft copolymerization, irregular rod like and globular shapes were clearly observed in

the scanning electron microscope images. It may be ascribed to the polar difference and

the destruction of the intermolecular hydrogen bonds. Decreased weight loss in case of

grafted chitosan compared to native chitosan might be due to the reduction of saccharide

units and increased polymerization. Similarly, the glass transition temperature of the

chitosan-g-PMMA gradually increased which confirms the decreased crystalline nature of

chitosan due to the grafting. The thermal stability and degradation behaviour of chitosan

and PMMA grafted chitosan were evaluated by TGA under nitrogen atmosphere. The

grafted content of the PMMA side chain co-polymer were determined by measuring the

weight loss between 200 °C and 800 °C (Liu et al., 2006; Wang et al., 1994). The TGA

results demonstrate that pure chitosan obtained 11% weight loss of water molecule

between 40-100 0C temperature ranges. In the case of Chitosan-g-PMMA weight loss

obtained for side chain polymer was 40.8% within the temperature range of 240-790 0C.

Pure chitosan shows the endothermic peak at around 91.84 0C, ascribable to the water

holding capacity, which is due to the –OH and unsubstituted free –NH2 groups of

chitosan. Furthermore, chitosan exhibits an exothermic peak at 306.60 0C that informs the

decomposition of polysaccride unit. In the case of grafted chitosan, endotherms peak or

glass transition temperature appear at 102.92 0C and exothermic peak or decomposing

temperatures appear at 296.05 0C. The results explores that the grafted chitosan has

attained decreased decomposing peak due to successful polymerisation on the chitosan

151

(Radhakumary et al., 2005). Similarly the glass transition temperature of the chitosan-g-

PMMA gradually increased indicating the decline of crystalline nature of chitosan due to

the polymerisation on the chitosan surface.

In the presence of customized chitosan, samples retained their colour for a longer time

indicating the antioxidant potential of modified chitosan. The extent or potential of

radical neutralisation varied with different modified chitosans. Modified chitosan reduced

the extent of β-carotene destruction by neutralizing the linoleate free radical formed in the

system. The high radical quenching property of chitosan may be due to reaction between

the generated free radicals and the residual free amino groups (Xie et al., 2001). The

modified chitosan showed higher potential as electron donors to quench free radicals

capably compared to the control (unmodified chitosan). The radical reducing properties

are in general coupled with the existence of reductones, which split down the free radical

chain by donating a hydrogen atom (Singh et al., 2002).

It is evident that irradiation of chitosan enhance the antioxidant activity of chitosan. Also

the enhancement or increase in antioxidant potential is dose dependent in case of

irradiation.

A similar study carried out by Kannat et al. (2004) revealed that irradiated chitosan

exhibited enhanced antioxidant activity than the unirradiated chitosan. In another study

carried out by Feng et al (2008). Chitosan irradiated at 20 kGy exhibited higher reductive

capacity and better radical scavenging potential. Irradiation treatment has been reported to

depolymerise chitosan, thus revealing the free amino group which increases the DPPH

radical scavenging activity (Kannat et al., 2004).

The intense susceptibility of fishery products to endure rancidity mediated disorders

during storage is chiefly due to the elevated amounts of unsaturated lipids. Throughout

152

the study period, difference in TBARS value was not significant between the unmodified

chitosan and blended chitosan but was significant with the samples treated with irradiated

chitosan. Similarly, irradiated chitosan exhibited a better antioxidant activity in lamb

meat than autoclaved chitosan (Kannat et al., 2004). Darmadji & Izumimoto (1994)

proposed that the antioxidative properties of chitosan are accountable for minimising the

TBA values in minced beef.

Reduction in TVB values by chitosan treatment was reported in fishery products (Jeon et

al., 2002). Reduction of microbial load was evident in chitosan treated samples (Figure.

7). Chitosan has been reported for its antimicrobial nature due to its polycationic

character in various studies (Chen et al., 1998; Shin et al., 2001; Xie et al., 2002).

Efficiency of irradiated chitosan on inhibiting the growth of E. coli was studied by

Matsuhashi & Kume (1997).

153

Figure

Figure 37. (37A) -FT-IR spectrumof chitosan (a) and chitosan-g-PMMA (37B). (1B)-

XRD spectrum of pure chitosan (a) and chitosan-g-PMMA (b).

154

Figure 38 SEM image of (a,b) pure chitosan (c,d) chitosan-g-PMMA.

155

Figure 39 (39A)- DSC of (a) pure chitosan and (b) chitosan-g-PMMA. (39B)- Thermal

gravimetric analysis (TGA) of (a) pure chitosan (b) chitosan-g-PMMA.

156

Figure 40 Antioxidant potential of chitosan extracts: A-chitosan. B-Blended chitosan C-

5 kGy irradiated chitosan. D- 10 kGy irradiated chitosan. Results are shown as means±SD

of three independent experiments.

Figure 41 Evolution of TBARS values in Penaeus monodon (mg MDA Kg-1

of fish)

during refrigerated storage. Results are shown as means±SD of three independent

experiments. Asterisk indicates statistical significance at p< 0.05

157

Figure 42 Evolution of TVB values in Penaeus monodon (TVB-N mg/100g) during

refrigerated storage. Results are shown as means±SD of three independent experiments.

Asterisk indicates statistical significance at p< 0.05

Figure 43 Profiles of antimicrobial activity in Penaeus monodon treated with chitosan

during refrigerated storage. Results are shown as means±SD of three independent

experiments. Asterisk indicates statistical significance at p< 0.05

158

SUMMARY

Gone are the golden days where fishery processing was as easy. Offlate, Plenty of new techniques

arebeing developed to meet the demands of customers replacing the function of chemical and

antibiotic residues. Natural way of preserving foods using biological, physical and chemical tools

help for extending the shelf life of sea food and for enhancing the trade of sea food commodities.

The influence of Streptococcus phocae PI80 on the shelf life of Penaeus monodon was

investigated by measurement of microbial and chemical analysis after appraising the safety of the

protective probiotic culture S. phocae PI80 in wistar rat‘s model. The results of this safety

assessment indicate that oral administration of S. phocae PI80 does not demonstrate any

toxicological effects. Treatment of S. phocae PI80 had no adverse effects on animal‘s general

health status, haematology, blood biochemistry, histology parameters, or on the incidence of

bacterial translocation. The effect of S. phocae PI80 is very evident with the reduction of Listeria

monocytogenes, Vibrio parahemolyticus & Coliforms. During storage, a marked decline in total

volatile base and peroxide value was observed in protective culture treated samples than the

control. The strain S. phocae PI80 looks promising as a protective culture for the preservation of

fish products.

The effects of another protective culture Enterococcus faecium MC-13 on the shelf life of

beheaded, scaled and gutted tiger shrimp (Penaeus monodon) during refrigerated storage was also

examined. The control and the treated samples were analysed periodically for chemical (PV,

TVB-N), microbiological (total viable count) and sensory characteristics. The in vivo safety

following oral administration to wistar rats was investigated before its usage as a bio preservative.

By the end of the experiment, the lowest total volatile base, microbial load was observed in E.

faecium MC-13 inoculated sea food samples and the highest in controls. Addition of protective

culture to the artificially contaminated food sample in the assayed storage condition inhibited

Listeria monocytogenes by 1.3 log CFU/g in Penaeus monodon. Our results validate the

159

feasibility of using E. faecium MC-13 and S. phocae PI80 as a possible candidate for use as a

starter culture in increasing the quality, and nutritive value in foods from the sea.

The efficiency of gamma radiation in combination with antioxidant wrapping for extending the

quality and shelf-life of fishery products was also evaluated. Quality assessment was studied by

monitoring the chemical (TVN, TMA, TBARS), microbiological (TBC) and Electro paramagnetic

resonance spectral analysis (EPR). A dose dependent reduction of TVB, TMA was contemplated

with irradiated samples. Lowest TVB and TMA were seen in 10 kGy irradiated samples

compared to control TVB and TMA values. Antioxidant supplied samples exhibited lower t-bars

values indicative of higher antioxidant activity than the control. Infusion of fish samples with

antioxidants diminished the intensity of EPR radical peak in irradiated samples. Outcome of the

study propose irradiation plus antioxidant wrapping as an efficient tool in preservation of foods

from sea. With the need to find safe natural antioxidants as an alternative to chemical and

antibiotic food substitutes, a chapter was contributed to find potent antioxidants from fruit peels.

The quenching capacities of Vitus vinifera seed extract, Citrus limon peel extract, Punica

granatum peel extract, Citrus sinensis peel extract were studied together with their antioxidant

activity in Penaeus monodon. The functionality of the extracts was evaluated using β-carotene-

linoleic acid model system, reducing power assay, DPPH, hydroxyl, nitrite radical scavenging

assay. Vitus vinifera and Punica granatum extract demonstrated best radical scavenging potential

in all multifunctional antioxidant assays. Radical scavenging activity measured by electron

paramagnetic resonance against a stable radical 1,1,-diphenyl-2-picrylhydrazil, revealed radical

peaks of lower intensity in antioxidant infused samples. Compounds possessing antioxidant

properties were identified from purified fruit extracts by GC-MS analysis. Treatments with these

extracts increased the stability of irradiated samples against lipid oxidation. TBARS values for

irradiated control was higher than the values reported for the samples treated with BHA, Punica

granatum peel, Vitus vinifera seed, Citrus sinensis peel and Citrus limon peel extracts treated

samples, respectively. This study also elucidated the relationship between heating temperature and

irradiation dose on the antioxidant activity of extracts. Maximum antioxidant activity was

160

observed at 150°C heated and 10 kGy irradiated extracts. These results suggest that fruit peels

will be a potential material for extracting antioxidants. Chitosan, a natural polymer with green

image which has wide application in various food industries was also employed for preserving

foods. Native chitosan, irradiated chitosan (5kGy and 10 kGy) and grafted chitosan was

characterized and employed for the preservation of sea food Penaeus monodon. The grafting of

metha acrylate onto natural native polymer chitosan was executed and the configuration of

covalent bonds in the grafted chitosan was demonstrated by performing, SEM, XRD, FTIR, TG

and DSC analyses. The antioxidant properties of modified chitosan conjugates were compared

with that of a native chitosan, treated in the alike conditions. The modified chitosan conferred

antioxidant and antibacterial potential equivalent to or better than that of the unmodified chitosan

in Penaeus monodon. Modified chitosan treated Penaeus monodon produced less TBARS and

TVB values than the control group.

161

REFERENCE

Abu-Tarboush, H.M., Al-Khatami, H.A., Atia, M., Abou-Arab, A.A., Bajaber, A.S., El-

Mojaddidi, M.A., 1996. Irradiation and post irradiation storage at 2°C of tilapia

(Tilapia nilotica X T. aurea) and Spanish mackerel (Scomeromorus commerson):

sensory and microbial assessment. J. Food Prot. 59, 1041–1048.

AFSSA., 2002. Recommendations on the presentation of data for assessing the safety of

micro-organisms used in the feed/food sector — new or modified strains — a

different application of strains already in use. AFSSA recommendation of 2002.

11–22.

Ahmad, J.I., 1996. Free radicals and health: Is vitamin E the answer. J. Food Sci.

Technol. 10, 147–152.

Ahmed, I.O., Alur, M.D., Kamat, A.S., Bandekar, J.R., Thomas, P., 1997. Influence of

processing on the extension of shelf-life of Nagli-fish (Sillago sihamo) by gamma

radiation. Int. J. Food Sci. Technol. 32, 325–332.

Ahn, D.U., Sell, C., Jo, C., Chen, X., Wu, C., Lee, J.I., 1998. Effects of dietary Vitamin E

supplementation on lipid oxidation and volatiles content of irradiated cooked turkey

meat patties with different packaging. Poultry Sci. 77, 912–920.

Ahn, D.U., Nam, K.C., 2003. Use of antioxidants to reduce lipid oxidation and off-odour

volatiles of irradiated pork homogenates and patties. Meat Sci. 63, 1–8.

Ahn, D.U., Love, J., Lee, J., 2006. Effect of Antioxidants on Consumer Acceptance of

Irradiated Turkey Meat. J. Food Sci. 68, 1659–1663.

162

Akhtar, P., Gray, I.J., 1988. Effect of dietary components and surface application of

oleoresin rosemary on lipid stability of rainbow trout (Oncorhynchus mykiss)

muscle during refrigerated and frozen storage. J. Food Lipids. 5, 43–58.

Al Bulushi, I., Poole, S., Deeth, H.C., Dykes, G.A., 2009. Biogenic Amines in Fish: Roles

in Intoxication, Spoilage, and Nitrosamine Formation–A Review, Critic. Rev. Food

Sci. Nutri. 49, 369–377.

Al-Kahtani, H.A., Abu-Tarboush, M.H., Bajaber, A.S., Atia, M., Abou-Arab, A.A., El-

Mojaddidi, M.A., 1996. Chemical changes after irradiation and post-irradiation

storage in Tilapia and Spanish mackerel. J. Food Sci. 61849, 729–733.

Allen, J.C., Hamilton, R.J., 1994. Rancidity in foods. Third edition. Aspen publishing

groups. New York. Chapman and hall. 256–269.

Alur, M.D., Doke, S.N., Warrier, S.B., Nair, P.M., 1995. Biochemical methods for

determination of spoilage of foods of animal origin: a critical evaluation. J. Food

Sci. Technol. 32, 181–188.

Alur, M.D., Venugopal, V., Nerkar, D.P., 1989. Spoilage potential of some contaminant

bacteria isolated from Indian mackerel. J. Food Sci. 54, 1111–1114.

American oil chemist‘s society. 1990. Official methods and recommended practices of

the American oil chemists society, 5th

edn. American oil chemist society, IL, USA.

Ananou, S., Garriga, M., Hugas, M., Maqueda, M., Martínez-Bueno, M., Gálvez, A.,

Valdivia, E., 2005. Control of Listeria monocytogenes in model sausages by

enterocin AS-48. Int. J. Food Microbiol. 103, 179–190.

Andrews, L.S., Grodner, R.M., 2004. Ionizing radiation of seafood. In: Irradiation of food

and Packaging: recent developments. ACS Symposium Series. Amer Chemical Soc,

Washington, DC, USA. 875,151–164.

163

Aoki, T., 1975. Effects of chemotherapeutics on bacterial ecology in the water of ponds

and the intestinal tracts of cultured fish, ayu (Plecoglossus altivelis). Jpn. J.

Microbiol. 19, 7–12.

Apostolidis, E., Kwon, Y.I., Rahul Shinde., Reza Ghaedian., Shetty, K., 2011. Inhibition

of Helicobacter pylori by fermented milk and soy milk using selected lactic acid

bacteria and link to extent lactic acid content and phenolic content. Food

Biotechnol. 25, 58–76.

Ashie, I.N.A., Smith, J.P., Simpson, B.K., 1996. Spoilage and shelf life extension of fresh

fish and shellfish. Crit. Rev. Food Sci. Nutri. 36, 87–121.

Attouchi, M., Sadok, S., 2010. The effect of powdered thyme sprinkling on quality

changes of wild and farmed gilthead sea bream fillets stored in ice. Food Chem.

119, 1527–1534.

Avadi, M.R., Sadeghi, A.M.M., Tahzibi, A., Bayati, K.H., Pouladzadeh, M., Zohuriaan-

Mehr M.J., Rafiee-Tehrani, M., 2004. Diethylmethyl chitosan as an antimicrobial

agent: Synthesis, characterization and antibacterial effects. Eur. Polym. J. 40, 1355–

1361.

Avila-Sosa, R., Gastélum-Franco, M.G., Camacho-Dávila, A., Torres-Muñoz, J.V.,

Nevárez Moorillón, G.V., 2010. Extracts of Mexican Oregano (Lippia berlandieri

Schauer) with Antioxidant and Antimicrobial Activity. Food Bioprocess Technol.

3, 434–440.

Aruoma, O.I., 1998. Free radicals, oxidative stress and antioxidants in human health and

disease. J. Am. Oil Chem.Soc. 75, 199–212.

Bao, H.N.D., Osako, K., Ohshima, T., 2010. Value-added use of mushroom ergothioneine

as a colour stabilizer in processed fish meats. J. Sci. Food Agric. 90, 1634–1641.

164

Bari, M.L., Ukuku, D.O., Kawasaki, T., Inatsu, Y., Isshiki, K., Kawamoto, S., 2005.

Combined efficacy of nisin and pediocin with sodium lactate, citric acid, phytic

acid and potassium sorbate and EDTA in reducing the Listeria monocytogenes

population of inoculated fresh cut produce. J. Food Prot. 68, 1381–1387.

Bchir, B., Besbes, S., Karoui, R., Attia, H., Paquot, M., Blecker, C., 2010. Effect of Air-

Drying Conditions on Physico-chemical Properties of Osmotically Pre-treated

Pomegranate Seeds. Food and Bioprocess Technol. Doi: 10.1007/s11947-010-0469-

3.

Bidchol, A.M., Wilfred, A., Abhijna, P., Harish, R., 2009. Free Radical Scavenging

Activity of Aqueous and Ethanolic Extract of Brassica oleracea L. var. Italic. Food

Bioprocess Technol. Doi: 10.1007/s11947-009-0196-9.

Blois, M.S., 1958. Antioxidant determination by the use of a stable free radical. Nature.

181, 1999–1200.

Bonev, B.B., Chan, W.C., Bycroft, B.W., Roberts, G.C.K., Watts, A., 2000. Interaction of

the l-antibiotic Nisin with mixed lipid bilayers: AP-31 and H-2 NMR study.

Biochem. 39, 11425–11433.

Borriello, S.P., Hammes, W.P., Holzapfel, W., 2003. Safety of probiotics that contain

lactobacilli or bifidobacteria. Clin Infect. Dis. 36, 775– 80.

Boulanger, J.M., Lee, E., 1991. Enterococcal bacteremia in a pediatric institution: a four-

year review. Rev. Infect. Dis. 13, 847–856.

Bowe, W.P., Logan, A.C., 2011. Acne vulgaris, probiotics and the gut-brain-skin axis -

back to the future ? Gut pathog. 3, 1–11.

165

Boyle, R.J., Robins-Browne, R.M., Tang, M.L.K., 2006. Probiotic use in clinical practice:

what are the risks? Am. J. Clin. Nutr. 83, 1256–1264.

Bredholt, S., Nesbakken, T., Holck, A., 2001. Industrial application of an anti listerial

strain of Lactobacillus sakei as a protective culture and its effect on the sensory

acceptability of cooked, sliced, vacuum-packaged meats. Int. J. Food Microbiol. 66,

191–196.

Bremner, H.A., Olley, J., Statham, J.A., Vail, A.M.A., 1988. Nucleotide catabolism:

influence of storage life of tropical species of fish from the North West Shelf of

Australia, J. Food Sci. 53, 6–11.

Breukink, E., De Kruijff, B., 2006. Lipid II as a target for antibiotics. Nat. Rev. Drug

Discov. 5, 321–332.

Byun, M.W., Lee, J.W., Yook, H.S., Lee, K.H., Kim, H.Y., 2002. Improvement of shelf

stability and processing properties of meat products by gamma irradiation. Radiat.

Phys. Chem. 63, 361–364.

Cabello, F.C., 2006. Heavy use of prophylactic antibiotics in aquaculture: a growing

problem for human and animal health and for the environment. Env. Microbiol. 8,

1137–1144.

Calderón-Miranda, M.L., Barbosa-Canovas, G.V., Swanson, B.G., 1999. Inactivation of

Listeria innocua in liquid whole egg by pulsed electric fields and nisin. Int. J. Food

Microbiol. 51, 7–17.

Callewaert, R., Hugas, M., De Vuyst, L., 2000. Competitiveness and bacteriocin

production of enterococci in the production of Spanish-style dry fermented

sausages. Int. J. Food Microbiol. 57, 33–42.

166

Campos, C., Rodriguez, O., Calo-Mata, P., Prado, M., Barros-Velazquez, J., 2006.

Preliminary characterization of bacteriocins from Lactococcus lactis, Enterococcus

faecium and Enterococcus mundtii strains isolated from turbot (Psetta maxima).

Food Res. Int. 39, 356–364.

Cao, R., Xue, C.H., Liu, Q., 2009. Changes in microbial flora of Pacific oysters

(Crassostrea gigas) during refrigerated storage and its shelf-life extension by

chitosan. Int. J. Food Microbiol.131, 272–276.

Casaus, P., Nilsen, T., Cintas, L.M., Nes, I.F., Hernandez, P.E., Holo, H., 1997. Enterocin

B, a new bacteriocin from Enterococcus faecium T136 which can act synergistically

with enterocin A. Microbiol. 143, 2287–2294.

Casey, P.G., Casey, G.D., Gardiner, G.E., Tangney, M., Stanton, C., Ross, R.P., Hill, C.,

Fitzgerald, G.F., 2004. Isolation and characterization of anti-salmonella Lactic acid

bacteria from the porcine gastrointestinal tract. Lett. Appl. Microbiol. 39, 431–438.

Celik, A., Ogenler, O., Comelekoglu, U., 2005. The evaluation of micronucleus

frequency by acridine orange fluorescent staining in peripheral blood of rats treated

with lead acetate. Mutagenesis. 20, 411–415.

Cheesborough, M., 1991. Medical laboratory manual for tropical countries. 2nd

edition

Tropical Health Technology and Butterworth Scientific Ltd. 1, 494–526.

Chen, L.C., Kung, S.K., Chen, H.H., Lin, S.B., 2010. Evaluation of zeta potential

difference as an indicator for antibacterial strength of low molecular weight

chitosan. Carbohydr. Polym. 82, 913–919.

Chen, C., Lian, W., Isai, G., 1998. Antibacterial effects of N-sulfonated and N-

sulfobenzoyl chitosan and application to oyster preservation. J. Food Prot. 61,

1124–1128.

167

Chen, M., Yeh, G.H., Chiang, B., 1996. Antimicrobial and physicochemical properties of

methylcellulose and chitosan films containing a preservative. J. Food Process.

Preserv. 20, 379–90.

Chikindas, M.L., Montville, T.J. (2002). Perspectives for application of bacteriocins as

food preservatives‘ in Juneja, VK and Sofos, JN, control of foodborne

microorganisms, Marcel, Dekker, Newyork.

Cho, G.S., Hanak, A., Huch, M., Holzapfel,W.H., Franz, C.M.A.P., 2010. Investigation in

to the potential of bacteriocinogenic Lactobacillus plantarum BFE 5092 for

biopreservation of Raw Turkey meat. Probiotics & Antimicro. Prot. 2, 77–89.

Choi, Y., Lee, S.M., Chun, J., Lee, H.B., Lee, J., 2006. Influence of heat treatment on

antioxidant activities and polyphenolic compounds of Shiitake (Lentinus edodes)

mushroom. Food Chem. 2, 381–387.

Chou, C.H., Silva, J.L., Wang, C., 2006. Prevalence and typing of Listeria

monocytogenes in raw cat fish fillets. J. Food Prot. 69, 815–819.

Chouliara, I., Sawaidis, L.N., Riganakos, K., Kontaminas, M.G., 2004. Preservation of

salted, vacuum-packaged, refrigerated sea bream (Sparus aurata) fillets by

irradiation: microbiological chemical and sensory attributes. Food Microbiol. 21,

351–359.

Chwla, S.P., Kim, D.H., Jo, C., Lee, J.W., Song, H.P., Byun, M.W., 2003. Effect of

gamma irradiation on survival of pathogens in Kwamegi, a Korean traditional semi-

dried seafood product. J. Food Prot. 66, 2093–2096.

Cintas, L.M., Casaus, P., Havarstein, L.S., Hernandez, P.E., Nes, I.F., 1997. Biochemical

and genetic characterization of enterocin P, a novel sec-dependent bacteriocin

from Enterococcus faecium P13 with a broad antimicrobial spectrum. Appl.

Environ. Microbiol. 63, 4321–4330.

168

Cleveland, J., Montville, T.J., Nes, I.F., Chikindas, M.L., 2001. Bacteriocins: safe, natural

antimicrobials for food preservation. Int. J. Food Microbiol. 71, 1–20.

Codex general standards- Codex General Standard for Irradiated Foods and

Recommended International Code of Practice for the Operation of Radiation

Facilities Used for the Treatment of Food. 1984. Rome, Food and Agriculture

Organisation of the United Nations, (CAV/Vol. XV-Ed. 1).

Codex., 2003. General standard for irradiated foods. Codex Stan 106-1983, Rev. 1-2003.

Rome: Codex Alimentarius Commission.

Commission of the European Communities., 1996. Commission directive on processed

cereal-based foods and baby foods for infants and young children. Luxemburg City,

Luxembourg: European Commission. L49, 17–96.

Conway, E.J., 1950. Microdiffusion Analysis and Volumetric Error. Third edition.

Crosby Lockwood and Son Ltd, London.

Cozzo-Siqueira, A., Oetterer, M., Gallo, C. R., 2003. Effects of irradiation and

refrigeration on the nutrients and shelf-life of tilapia (Oreochromis niloticus). J.

Aquat. Food Prod. Technol. 12, 85–101.

Crandall, A.D., Montville, T.J., 1998. Nisin Resistance in Listeria monocytogenes ATCC

700302 Is a Complex Phenotype. Appl. Environ. Microbiol. 64, 231–237.

Crawford, L.M., Ruff, E.H., 1996. A review of the safety of cold pasteurization through

irradiation. Food Cont. 7, 87–97.

Czechowska-Biskup, R., Rokita, B., Ulanski, P., Rosiak, J.M., 2005. Radiation-induced

and sono chemical degradation of chitosan as a way to increase its fat binding

capacity. Nuclr. Intr. Methd. Phys. Res. 236, 383–390.

169

Daeschel, M.A.Z., 1989. Antimicrobial substances from lactic acid bacteria for use as

food preservatives. Food Technol. 43, 164–169.

Darmadji, P., Izumimoto, M., 1994. Effect of chitosan in meat preservation. Meat Sci. 38,

243–254.

Davies, E.A., Bevis, H.E., Delves-Broughton, J., 1997. The use of the bacteriocin, nisin,

as a preservative in ricotta-type cheeses to control the food-borne pathogen

Listeria monocytogenes. Lett. Appl. Microbiol. 24, 343–346.

Dawson, P.L., Harmon, L., Sotthibandhu, A., Han, I.Y., 2005. Antimicrobial activity of

nisin-adsorbed silica and corn starch powders. Food Microbiol. 22, 93–99.

De azerêdo, G.A., Montenegro stamford, T.L., De souza, E.L., Fonseca veras, F.,

Rodrigues de almeida, E., De araújo, J.M., 2010. In vivo assessment of possible

probiotic properties of Zymomonas mobilis in a wistar rat model. Electr. J.

Biotechnol. 13, 1–7.

De Martinis, E.C.P., Públio, M.R.P., Santarosa, P.R., Freitas, F.Z., 2001. Antilisterial

activity of lactic acid bacteria isolated from vacuum-packaged Brazilian meat and

meat products. Braz. J. Microbiol. 32, 32–37.

De Smedt S.C., Demeester, J., Hennink, W.E., 2000.Cationic polymer based gene

delivery systems, Pharma Res. 17, 113–126.

Deegan, L.H., Cotter, P.D., Hill, C., Ross, P., 2006. Bacteriocins: biological tools for bio-

preservation and shelf-life extension. Int. Dairy J. 16, 1058–1071.

Deeley, C.M., 2006. The development of food irradiation to-date in Asia Pacific, the

Americas, Europe and Africa. In: Tutorial presented to the International Meeting on

Radiation Processing (IMRP), Kuala Lumpur.

170

Degnan, A.J., Luchansky, J.B., 1992. Influence of beef tallow and muscle on the

antilisterial activity of pediocin AcH and liposome-encapsulated pediocin AcH. J.

Food Prot. 55, 552–554.

Delves-Broughton, J., 1990. Nisin and its uses as a food preservative. Food Technol. 44,

100–117.

Delves-Broughton, J., 2005. Nisin as a food preservative. Food Aust. 57, 525–527.

Diehl, J.F., Hasselmann, C., Kilcast, D., 1991. ‗Regulation of food irradiation in the

European Community: Is nutrition an issue?. Food Cont. 2, 212–219.

Diehl, J.F., 1993. Will irradiation enhance or reduce food safety? Food policy. 18, 143–

151.

Diehl, J.F., 2002. Food irradiation. A past, present and future. Radiat. Phys. Chem. 63,

211–215.

Dillon, R., Patel, T.R., 1992. Listeria in seafoods: a review, J. Food Prot. 55, 1005–1015.

Don, T.M., King, C.F., Chiu, W.Y., 2002. Synthesis and properties of chitosan-modified

poly (vinyl acetate). J. Appl. Polym. Sci. 86, 3075–3063.

Donohue, D.C., Salminen, S., Marteau, P., 1998. Safety of probiotic bacteria. In:

Salminen, S., von Wright, A. (Eds.), Lactic Acid Bacteria, 2nd Edition, Marcel

Dekker Inc, New York, pp. 369–383.

Dorrington, T., Gomez-Chiarri, M., 2008. Antimicrobial peptides for use in oyster

aquaculture: effect on pathogens, commensals and eukaryotic expression systems. J.

Shellfish Res. 27, 365–374.

Doyle, M.P., 1989. Ed., Foodborne Bacterial Pathogens, Marcel Dekker, New York.

Duangjitcharoen, Y., Kantachote, D., Ongsakul, M., Poosaran, N., Chaiyasut, C., 2009.

Potential use of probiotic Lactobacillus plantarum SS2 isolated from a fermented

171

plant beverage: safety assessment and persistence in the murine gastrointestinal

tract. World J. Microbiol. Biotechnol. 25, 315–321.

Duffes, F., Leroi, F., Boyaval, P., Dousset, X., 1999. Inhibition of Listeria

monocytogenes by Carnobacterium spp. strains in a simulated cold smoked fish

system stored at 4ºC. Int. J. Food Microbiol. 47, 33–42.

Egan, A.F., Wills, P.A., 1985. The preservation of meats using irradiation. CSIRO Food

Res. Q 45–49.

Ehlermann, D.A.E., 2005. Four decades in food irradiation. Radiat. Phys. Chem. 73, 346–

347.

El-Tahlawy, K.F., El-Rafie, S.M., Aly Sayed Aly., 2006. Preparation and application of

chitosan/poly (methacrylic acid) graft copolymer. Carbohydr. Polym. 66, 176–183.

Endres, J.R., Clewell, A., Jade, K.A., Farber, T., Hauswirth, J., Schauss, A.G., 2009.

Safety assessment of a proprietary preparation of a novel probiotic, Bacillus

coagulans, as a food ingredient. Food Chem. Toxicol. 47, 1231–1238.

Ercolini, D., Storia, A., Villani, F., Mauriello, G., 2006. Effect of a bacteriocin activated

polythene film on Listeria monocytogenes as evaluated by viable staining and epi

fluorescence microscopy. J. Appl. Microbiol. 100, 765–772.

Escribano-Bailo´n, M.T., Guerra, M.T., Rivas-Gonzalo, J.C., Santos-Buelga, C., 1995.

Proanthocyanidins in skins from different grape varieties. Zeitschrift für

Lebensmittel Untersuchung und Forschung. 200, 201–224.

Fagbenro, O., Jauncey, K., 1994. Chemical and nutritional quality of fermented fish

silage containing potato extracts, formalin or ginger extracts. Food Chem. 50, 383–

388.

172

Fall, P.A., Leroi, F., Chevalier, F., Guérin, C., Pilet, M.F., 2010. Protective Effect of a

Non-Bacteriocinogenic Lactococcus piscium CNCMI-4031 Strain Against Listeria

monocytogenes in Sterilized Tropical Cooked Peeled Shrimp. J. Aquat. Food Prod.

Technol. 19, 84–92.

Fall, P.A., Leroi, F., Cardinal, M., Chevalier, F., Pilet, M.F., 2010. Inhibition of

Brochothrix thermosphacta and sensory improvement of tropical peeled cooked

shrimp by Lactococcus piscium CNCM I-4031. Lett. Appl. Microbiol. 50, 357–361.

Fan, W., Sun, J., Chen, Y., Qiu, J., Zhang, Y., Chi, Y., 2009. Effects of chitosan coatings

on quality and shelf life of silver carp during frozen storage. Food Chem. 115, 66–

70.

Fang, T.J., Tsai, H.C., 2003. Growth patterns of Escherichia coli O157:H7 in ground beef

treated with nisin, chelators, organic acids and their combinations immobilized in

calcium alginate gels. Food Microbiol. 20, 243–253.

Fang, Y., Lou, M.M., Li, B., Xie, G.L., Wang, F., Zhang, L.X., 2010. Characterisation of

Burkholderia cepacia complex from cystic fibriosis patients in China and their

chitosan susceptibility. World J. Microbiol. Biotechnol. 26, 443–450.

FAO., 2005.World inventory of fisheries. Reducing post-harvest losses. Issues Fact

Sheets. Text by Lahsen Ababouch. In: FAO Fisheries and Aquaculture Department.

FAO/WHO., 2001. Regulatory and clinical aspects of dairy probiotics. Cordoba,

Argentina.

Farkas, J., 1985. Five years experience of the international facility for food irradiation

technology. Radiat. Phys. Chem. 25, 227–231.

Farkas, J., 1998. Irradiation as a method for decontaminating food: a review. J. Food

Microbiol. 44, 189–204.

173

Fasseas, M.K., Mountzouris, K.C., Tarantilis, P.A., Polissiou, M., Zervas, G., 2007.

Antioxidant activity in meat treated with oregano and sage essential oils. Food

Chem. 106, 1188–1194.

Feng, T., Du, Y., LI, J., Hu, Y., Kennedy, J.F., 2008. Enhancement of antioxidant activity

of chitosan by irradiation. Carbohydr. Polym. 73, 126–132.

Fleury, Y., Dayem, M.A., 1996. Covalent Structure, Synthesis, and Structure-Function

Studies of Mesentericin Y 105, a Defensive Peptide from Gram-positive

Bacteria Leuconostoc mesenteroides. J. Biological Chem. 271, 14421–14429.

Fuller, R., 1989. Probiotics in man and animals. A review. J. Appl. Bacteriol. 66, 365–

378.

Fregeau Gallagher, N.L., Sailer, M., Niemczura, W.P., Nakashima, T.T., Stiles,

M.E., Vederas J.C.1997. Three-Dimensional Structure of Leucocin A in

Trifluoroethanol and Dodecylphosphocholine Micelles:  Spatial Location of

Residues Critical for Biological Activity in Type IIa Bacteriocins from Lactic Acid

Bacteria. Biochem. 36, 15062–15072.

Gálvez, A., Abriouel, H., López, R.L., Ben Omar, N., 2007. Bacteriocin-based strategies

for food biopreservation. Int. J. Food Microbiol. 120, 51–70.

Gardner, H.W., 1979. Lipid hydroperoxide reactivity with proteins and amino acids: A

review. J. Agric. Food Chem. 27, 220–229.

Gelman, A., Drabkin, V., Glatman, L., 2001. Evaluation of lactic acid bacteria, isolated

from lightly preserved fish products, as starter cultures for new fish-based food

products. Innov. Food Sci. Emerg. Technol. 1, 219–226.

174

Georgalaki, M.D., Berghe, E.V.D., Kritikos, D., Devreese, B., Beeumen, J.V.,

Kalantzopoulos, G., Vuyst, L.D., Tsakalidou, E., 2002. Macedocin, a food-grade

lantibiotic produced by Streptococcus macedonicus ACA-DC 198. Appl. Environ.

Microbiol. 68, 5891–5903.

Ghadi, S.V., Venugopal, V., 1991. Influence of γ-irradiation and ice storage on fat

oxidation in three Indian fish. Int. J. Food Sci. Technol. 26, 397–401.

Giraffa, G., 2003. Functionality of enterococci in dairy products. Int. J. Food Microbiol.

88, 215–222.

Gillor, O., Etzion, A., Riley, M.A., 2008. The dual role of bacteriocins as anti and

probiotics. Appl. Microbiol. Biotechnol. 81, 591–606.

Gokoglu, N., Yerlikaya, P., 2008. Inhibition effects of grape seed extracts on melanosis

formation in shrimp (Parapenaeus longirostris). Int. J. Food Sci. Technol. 43,

1004–1008.

Gokoglu, N., Topuz, O.K., Yerlikaya, P., 2009. Effects of pomegranate sauce on quality

of marinated anchovy during refrigerated storage. LWT Food Sci. Technol. 42,

113–118.

Gomez Guillen, M.C., Montero, M.P., 2007. Polyphenol uses in sea food conservation.

Am. J. Food Technol. 2593–601.

Gomez-Gil, B., Tron-Mayen, L., Roque, A., Turnbull, J.F., Inglis, V., Guerra-Flores,

A.L., 1998. Species of Vibrio isolated from hepatopancreas, haemolymph and

digestive tract of a population of healthy juvenile Penaeus vannamei. Aquacult.

163, 1–9.

175

Gonza´lez-Parama´s, A.M., Estebam-Ruano, S., Santos-Buelga, C., Pascual-Teresa, S.,

Rivas-Gonzalo, J. C., 2004. Flavanol content and antioxidant activity in winery

byproducts. J. Agric. Food Chem. 52, 234–238.

Gopalakannan, A., 2006. Studies on the control of Aeromonas hydrophila infection and

Cyprinus carpio and Tilapia mossambicus by Immunostimulants and probiotics,

Ph.D Thesis, Pondicherry University, Pondicherry.

Gopalakannan, A., Arul, V., 2011. Inhibitory activity of probiotic Enterococcus faecium

MC13 against Aeromonas hydrophila confers protection against hemorrhagic

septicemia in common carp Cyprinus carpio. Aquacult. Int. 19, 973–985.

Gopalakannan, A., Venkatesan Arul., 2006. Immunomodulatory effects of dietary intake

of chitin, chitosan and levamisole on the immune system of Cyprinus carpio and

control of Aeromonas hydrophila infection in ponds. Aquacult. 255, 179–187.

Gordon, S., 2008. Elie Metchnikoff: father of natural immunity. Eur. J. Immunol. 38,

3257–3264.

Gouws, P.I., Liedemann, I., 2005. Evaluation of Diagnostic PCR for the Detection of

Listeria monocytogenes in Food Products. Food Technol. Biotechnol. 43, 201–205.

Gordy, W., Ard, W.B., Shields, H., 1955. Microwave spectroscopy of biological

substances. I. Paramagnetic resonance in x-irradiated amino acids and proteins.

Proc. Natl. Acad. Sci. 41, 983–996.

Gow- Chin, Y., Pin-Der, D., 1994. Scavenging effect of methanolic extracts of pea nut

hulls on free radicals and active oxygen species. J. Agric. Food Chem. 42, 629–632.

Gould, G.W., 1996. Methods for preservation and extension of shelf life Int. J. Food

Microbiol. 33, 51–64.

176

Gram, L., Huss, H.H., 1996. Microbiological spoilage of fish and fish products, Int. J.

Food Microbiol. 33, 121–137.

Gray, J.I., 1976. N-nitrosamines and their precursors in bacon: a review. J. Milk Food

Technol. 39, 686–690.

Guerra, N.P., Macias,C.L., Agrasar, A.J.M., Castro, L.P., 2005. Development of a

bioactive packaging cellophane using Nisaplin as biopreservative agent. Lett. Appl.

Microbiol. 40, 106–110.

Gomez-Guillen, M.C., Montero, M.P., 2007. Polyphenol Uses in Seafood

Conservation. Am. J. Food Technol. 2, 593–601.

González-Martínez, B.E., Gómez-Treviño, M., Jiménez-Salas, Z., 2003. Bacteriocinas de

probióticos. Rev. Salud Pública y Nutrición, 4.

Halliwell, B., 1997. Antioxidants in human health and disease. Annu. Rev. Nutr.16, 33–

50.

Hanlin, M.B., Kalchayanand, N., Ray, P., Ray, B., 1993. Bacteriocins of lactic acid

bacteria in combination have greater antibacterial activity. J. Food Prot. 56, 252–

255.

Harish Prashanth, K.V., Tharanathan, R.N., 2007. Chitin/chitosan: modifications and their

unlimited application potential—an overview. Trends Food Sci. Technol. 18, 117–

131.

Harish Prashanth, K.V., Kshama Lakshman, T.R., Shamala, R.N., Tharanathan., 2005.

Biodegradation of chitosan-graft-polymethylmethacrylate films. Int. Biodet.

Biodeg. 56, 115–120.

177

Harpaz, S., Glatman, L., Drabkin, V., Gelman, A., 2003. Effects of herbal essential oils

used to extend the shelf-life of fresh water reared Asian sea bass fish (Lates

calcarifer). J. Food Prot. 66, 410–417.

Harty, D.W., Oakey, H.J., Patrikakis, M., Hume, E.B., Knox, K.W., 1994. Pathogenic

potential of Lactobacilli. Int. J. Food Microbiol. 24, 179–189.

Hassan, Z.H., Zwartkruis-Nahuis, J.T.M., De Boer, E., 2012. Occurrence of Vibrio

parahaemolyticus in retailed seafood in The Netherlands. Int. Food Res. J. 19, 39–

43.

Havenaar, R., Huis in 't Veld, J.H.J., 1992. Probiotics: a general view. In: Wood, B.J.B.,

Eds., The Lactic Acid Bacteria. The Lactic Acid Bacteria in Health and Disease.

Elsevier Appl Sci, London.151–170.

He, Y., Shahidi, F., 1997. Antioxidant activity of green tea and its catechins in a fish meat

model system. J. Agric. Food. Chem. 45, 4262–4266.

Helander, I.M., Nurmiaho-Lassila, E.L., Ahvenainen, R., Rhoades, J., Roller, S., 2001.

Chitosan disrupts the barrier properties of the outer membrane of gram negative

bacteria. Int. J. Food Microbiol. 71, 235–244.

Helander, I.M., Von Wright, A., Mattila-Sandholm, T.M., 1997. Potential of lactic acid

bacteria and novel antimicrobials against Gram-negative bacteria. Trends Food Sci.

Technol. 8, 146–150.

Huey-Shilye, L., Chiu-Yin, K., Joo-Ann, E., Wai-Yee, F., Min-Tze, L., 2009. The

Improvement of Hypertension by Probiotics: Effects on Cholesterol, Diabetes,

Renin, and Phytoestrogens. Int. J. Mol. Sci. 10, 3755–3775.

Humasong, L., 1979. Animal tissue techniques, 4th

ed. W. H. Freeman and Co., San

Francisco, 661.

178

Hunter, M.I.S., Mohamed, J.B., 1986. Plasma antioxidants and lipid oxidation products in

Duchenne muscular dystrophy. Clin. Chim. Acta.155, 123–132.

Hurst, A., Hoover, D.G., 1993. Nisin. In: Davidson P.M, Branen A.L, editors.

Antimicrobials in foods. New York: Marcel Dekker. 369–394.

Huss, H.H., Jeppesen, V.F., Johansen, C., Gram, L., 1995. Biopreservation of fish

products - a review of recent approaches and results. J. Aquat. Food Prod Technol.

4, 5–25.

Hyink, O., Balakrishnan, M., Tagg, J.R., 2005. Streptococcus rattus strain BHT produces

both a class I two-component lantibiotic and a class II bacteriocin. FEMS

Microbiol. Lett. 252, 235–241.

Jack, R.W., Tagg, J.R., Ray, B., 1995. Bacteriocins of Gram-positive bacteria. Microbiol.

Rev., 59, 171–200.

Jacobsen, T., Budde, B.B., Koch, A.G., 2003. Application of Leuconostoc carnosum for

biopreservation of cooked meat products. J. Appl. Microbiol. 95, 242–249.

Jamet, E., Chamba, C.F., 2008. Contribution to the safety assessment of technological

microflora found in fermented dairy products. Int. J. Food Microbiol. 126, 263–266.

Jang, J., Yang, Y.C., Zhang, G.H., Chen, H., Lu, J.L., Du, Y.Y., Ye, J.H., Ye, D.,

Borthakur, D., Zheng, X.Q., Liang, Y.R., 2010. Effect of ultra- violet B on release

of volatiles in tea leaf. Int. J. Food Prop. 13, 608–617.

Je, J.Y., Kim, S.K., 2006. Chitosan derivatives killed bacteria by disrupting the outer and

inner membrane. J. Agric. Food Chem. 54, 6629–6633.

Jeevanandam, K., Kakatkar, A., Doke, S.N., Bongiwar, V., Venugopal, V., 2001.

Influence of salting and gamma irradiation on the shelf-life extension of threadfin

bream in ice. Food Res. Int. 34, 739–746.

179

Jenkins, D.W., Hudson, S.M., 2001. Review of vinyl graft copolymerization featuring

recent advances toward controlled radical-based reactions and illustrated with

chitin/ chitosan trunk polymers. Chem. Rev. 101, 3245–3273.

Jeon, Y.J., Kamil, J.Y.V.A., Shahidi, F., 2002. Chitosan as an edible invisible film for

quality preservation of herring and Atlantic cod. J. Agric. Food Chem. 50, 5167–

78.

Jeon, Y.J., Park, P.J., Kim, S.K., 2001. Antimicrobial effect of chitooligosaccharides

produced by bioreactor. Carbohydr. Polym. 44, 71–76.

Jeppesen, V.F., Huss, H.H., 1993. Antagonistic activity of two strains of lactic acid

bacteria against Listeria monocytogenes and Yersinia enterocolitica in a model fish

product at 5°C. Int. J. Food Microbiol. 19, 179–186.

Jerković, I., Marijanović, Z., 2010. Oak (Quercus frainetto Ten.) Honeydew Honey-

Approach to Screening of Volatile Organic Composition and Antioxidant Capacity

(DPPH and FRAP Assay). Molecules. 15, 3744–3756.

Jo, C., Lee, W.D., Kim, D.H., Kim, J.H., Ahn, H.J., Byun, M.W., 2004. Quality attributes

of low salt Changran Jeotkal (aged and seasoned intestine of Alaska Pollock,

Theragra chalcogramma) developed using gamma irradiation. Food Cont. 15, 435–

440.

Joerger, R.D., 2007. Antimicrobial films for food applications: a quantitative analysis of

their effectiveness. Packag. Tech. Sci. 20, 231–273.

Joerger, R.D., 2003. Alternatives to antibiotics: bacteriocins, antimicrobial peptides and

bacteriophages. Poult. Sci. 82, 640–647.

180

Joshi, J.M., Sinha V.K., 2006. Synthesis and Characterization of Carboxymethyl Chitosan

Grafted Methacrylic Acid Initiated by Ceric Ammonium Nitrate. J. Polym. Res. 13,

387–395.

Kamil, J.Y.V.A., Jeon, Y.J., Shahidi, F., 2002. Antioxidative activity of chitosans of

different viscosity in cooked comminuted flesh of herring (Clupea harengus). Food

Chem. 79, 69–77.

Kamat, A.,Warke, R., Kamat, M., Thomas, P., 2000. Low-dose irradiation as a measure to

improve microbial quality of ice cream. Int. J. Food Microbiol. 62, 27–35.

Kanatt, S.R., Chander, R., Sharma, A., 2007. Antioxidant potential of mint (Mentha

spicata L.) in radiation-processed lamb meat. Food Chem. 100, 451–458.

Kanatt, S.R., Chander, R., Sharma, A., 2004. Effect of irradiated chitosan on the

rancidity of radiationprocessed lamb meat. Int. J. Food Sci. Technol. 39, 997–1003.

Kang, H.J., Chawla, S.P., Jo, C., Kwon, J.H., Byun, M.W., 2006. Studies on development

of functional powder from citrus peel. Biores. Technol. 97, 614–620.

Kanmani, P., 2011a. 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.

Kanmani, P., Satish ramraj., Yuvaraj, N., Paari, K.A., Pattukumar, V., Arul, V. 2011b.

Design and optimization of fermentation medium for enhanced bacteriocin

production by probiotic Enterococcus faecium MC-13. Prep. Biochem. Biotech. 41,

40–52.

Kanmani, P., Satish, R., Yuvaraj, N., Paari, K.A., Pattukumar, V., Arul, V., 2011c.

Production and purification of a novel exopolysaccharide from lactic acid bacterium

181

Streptococuus phocae PI80 and its functional characteristics activity in vitro.

Biores. Technol. 102, 4827–4833.

Kanmani, P., Satish, R., Yuvaraj, N., Paari, K.A., Pattukumar, V., Arul, V., 2011c. The

role of environmental factors and medium composition on bacteriocin production

by an aquaculture probiotic Enterococcus faecium MC-13 isolated from fish

intestine. Kor. J. Chem. Eng. 28, 860–866.

Kanmani, P., Satish, R., Yuvaraj, N., Paari, K.A., Pattukumar, V., Arul, V., 2011d.

Optimization of media components for enhanced production of Streptococcus

phocae PI80 and its bacteriocin using response surface methodology. Braz. J.

Microbiol. 42, 716–720.

Kasimoglu, A., Denli, E., Iç, E., 2003. The extension of the shelf-life of sardine which

were packaged in a vacuum stored under refrigeration and treated by δ-irradiation.

Int. J. Food Sci. Technol. 38, 529–535.

Kato, F.T., Puck, T.T., 1971. Mutagenesis by carcinogenic nitroso compounds. J. Cell

Physiol. 78, 139–144.

Kayser, F.H., 2003. Safety aspects of enterococci from the medical point of view, Int. J

Food Microbiol. 88, 255–262.

Kesarcodi-Watson, A., Kaspar, H., Lategan, M.J., Gibson, L., 2008. Probiotics in

aquaculture: The need, principles and mechanisms of action and screening

processes. Aquacult. 274, 1–14.

Kim, K.W., Thomas, R.L., 2007. Antioxidative activity of chitosans with varying

molecular weights. Food Chem. 101, 308–313.

Kirby, A.J., Schmith, R.J., 1997. The antioxidant activity of Chinese herbs for eczema

and of placebo herbs-I. J. Ethnopharmacol. 56, 103–108.

182

Kırmızıgül, S., Böke, N., Sümbül, H., Göktürk, R. S., Arda, N., 2007. Essential fatty acid

components and antioxidant activities of eight Cephalaria species from south

western Anatolia. Pure Appl. Chem. 79, 2297–2304.

Kiss, I.F., Beczner, I.J., Zachariev, G.Y., Kovacs, S., 1990. Irradiation of meat products,

chicken and use of irradiated spices for sausages. Radiat. Phys. Chem. 36, 295–299.

Klein, G., 2003. Taxonomy, ecology and antibiotic resistance of enterococci from food

and the gastro-intestinal tract. Int. J. Food Microbiol. 88, 123–131.

Krull, R.E., Chen, P., Novak, J., Kirk, M., Barnes, S., Baker, J., Krishna, N.R., Caufield,

P.W., 2000. Biochemical structural analysis of the lantibiotic mutacin II. J. Biol.

Chem. 275, 15845–15850.

Kumar, G., Smith, P.J., Payne, G.F., 1999. Enzymatic grafting of a natural product onto

chitosan to confer water solubility under basic conditions. Biotechnol. Bioeng. 63,

154–165.

Kume, T., 1985. Economic viability and commercial experience with Shihoro potato

irradiator. In: Proceedings of the ASEAN Workshop on Food Irradiation. ASEAN

Food Handling Bureau, 78–89.

Kume, T., Furuta, M., Todoriki, S., Uenoyama, N., Kobayashi, Y., 2009. Status of food

irradiation in the world. Radiat. Phys. Chem. 78, 222–226.

Kume, T., Nagasawa, N., Yoshii, F., 2002. Utilization of carbohydrates by radiation

processing. Radiat. Phys. Chem. 63, 625–627.

Kurath, G., 2008. Biotechnology and DNA vaccines for aquatic animals. Rev. Sci. Tech.

Off. Int. Epiz. 27, 175–196.

183

Kurita, K., 2001.Controlled functionalization of polysaccharide chitin. Prog. Polym. Sci.

26, 1921–1971.

Kwon, J.H., Kausar, T., Noh, J.E., Warrier, S.B., Venugopal, V., Karani, M., Artik, A.,

Bhushan, B., Byun, M.W., Kim, S.J., Kim, K.H., Kim, K.S., 2004. Inter-country

transportation of irradiated dried Korean fish to prove its quality and identity.

Radiat. Phy. Chem. 71, 81–85.

Kwon, J.H., Byun, M.W., 1995. Gamma irradiation combined with improved packaging

for preserving and improving the quality of dried fish (Engraulis encrasicholus).

Radiat. Phy. Chem. 46, 725–729.

Kyrana, V.R., Lougovois, V.P., 2002. Sensory, chemical and microbiological assessment

of farm-raised European sea bass (Dicentrarchus labrax) stored in melting ice. Int.

J. Food Sci. Technol. 37, 319–328.

Labuza, T.P., 1970. Water content and stability of low moisture content and intermediate

moisture foods. Food Tech. 24, 543–550.

Lakshmanan, R., Venugopal, V., Venketashvaran, K., Bongirwar, D.R., 1999. Bulk

preservation of small pelagic fish by gamma irradiation: studies on a model storage

system using anchovies. Food Res. Int. 32, 707–713.

Laukova, A., Strompfova, V., Skrivanova, V., Volek, Z., Jindrichova, E., Marounek, M.,

2006. Bacteriocin- producing strain of Enterococcus faecium EK13 with probiotic

character and its application in the digestive tract of rabbits. Biologia Bratislava. 61,

779–782.

Lee, J.W., Cho, K.H., Yook, H.S., Jo, S., Kim, D.H., Byun, M.W. 2002. The effect of

gamma irradiation on the stability and hygienic quality of semi-dried Pacific saury

(Cololabis seira) flesh. Radiat. Phys. Chem. 64, 309–315.

184

Lee, S.C., Kim, J.H., Jeong, S.M., Kim, D.R., Ha, J.U., Nam, K.C., Ahn, D.U., 2003.

Effect of far-infrared radiation on the antioxidant activity of rice hulls. J. Agric.

Food Chem. 51, 4400–4403.

Leistner, L., Gorris, L.G.M., 1995. Food preservation by hurdle technology. Trends Food

Sci. Technol. 6, 41–46.

Leroi, F., 2010. Occurrence and role of lactic acid bacteria in seafood products. Food

Microbiol. 27, 698–709.

Li, B.B., Smith, B., Hossain, M., 2006. Extraction of phenolics from citrus peels: solvent

extraction method. Sep. Purific. Technol. 48, 182–188.

Lin, C.C., Hung, P.F., Ho, S.C., 2008. Heat treatment enhances the NO-suppressing and

peroxynitrite intercepting activities of kumquats (Fortunells margarita swingle)

peel. Food chem. 109, 95–103.

Lin, Y.T., Labbe, R.G., Kalidas Shetty., 2005. Inhibition of Vibrio parahaemolyticus in

seafood systems using oregano and cranberry phytochemical synergies and lactic

acid. Innov. Food Sci. Emer. Technol. 6, 453–458.

Liolios, C.C., Sotiroudis, G.T., Chinou, I., 2009. Fatty Acids, Sterols, Phenols and

Antioxidant Activity of Phoenix theophrasti Fruits Growing in Crete, Greece. Plant

Foods Hum. Nutri. 64, 52–61.

Liu, Y., Zhang, R., Zhang, J., Zhou, W., Li .S., 2006. Graft Copolymerization of Sodium

Acrylate onto Chitosan via Redox Polymerization. Iran. Polym. J. 15, 935–942.

Liu, L., Li, Y., Zhang, W., Zhang, G., Fang, Y., 2004. Homogeneous graft

copolymerization of chitosan with methyl methacrylate by γ -irradiation via a

phthaloylchitosan intermediate. Polym. Int. 53, 1491–1494.

185

Löliger, J., 1989.Natural antioxidants for the stabilization of foods, In Flavor Chemistry

(D. B. Min and T. Smouse, Eds.), AOCS, Champaign, IL.302.

Lopez-Caballero, M.E., Gomez-Guillen, M.C., Perez-Mateos, M., Montero, P., 2005. A

chitosan-gelating blend as a coating for fish patties. Food Hydrocoll. 19, 303–311.

Luchansky, J.B., 1999. Overview on applications for bacteriocin-producing lactic acid

bacteria and their bacteriocins. Antonie van leeuwenhoek. 76, 335.

Lutgendorff, F., Nijmeijer, R.M., Sandstro¨m, P.A., Trulsson, L.M., Magnusson, K.E.,

2009. Probiotics prevent intestinal barrier dysfunction in acute pancreatitis in rats

via induction of ileal mucosal glutathione biosynthesis. PLoS ONE. 4, e4512, 1–13.

Lyon, W.J., Glatz, B.A., 1993. Isolation and purification of propionicin PLG-1, a

bacteriocin produced by a strain of Propionibacterium thoenii. Appl. Environ.

Microbiol. 59, 83–88.

Mazza, G., 1995. Anthocyanins in grapes and grapes products. Critic. Rev. Food Sci.

Nutri. 35, 341–371.

Mahdavinia, G.R., Zohuriaan-Mehr, M.J., Pourjavadi, A., 2004. Modified chitosan. III.

Super absorbency, salt and pH sensitivity of smart ampholytic hydrogels from

chitosan g-polyacrylonitrile. Polym. Adv. Technol. 15, 173–180.

Mahrour, A., Caillet, S., Nketsa-Tabiri, J., Lacroix, M., 2003. Microbial and sensory

quality of marinated and irradiated chicken. J. Food Prot. 66, 2156–2159.

Majeti, N., Ravi, K., 2000. A review of chitin and chitosan applications. React. Funct.

Polym. 46, 1–27.

Manisha, N.K., Warrier, S.B., 2002. Quality improvement of sun dried Acetes indicus

(Jawala prawns) by radiation processing. Fish. Chimes. 22, 25–27.

186

Mantovani, H.C., Hu, H., Worobo, R.W., Russell, J.B., 2002. Bovicin HC5, a bacteriocin

from Streptococcus bovis HC5. Microbiol. 148, 3347–3352.

Marciset, O., Stratingh, J.M.C., Mollet, B., Poolman, B., 1997. Thermophilin 13, a

nontypical antilisterial poration complex bacteriocin that functions without a

receptor. J. Biol. Chem. 272, 14277–14284.

Marcos, B., Aymerich, T., Monfort, J.M., Garriga, M., 2007. Use of antimicrobial

biodegradable packaging to control Listeria monocytogenes during storage of

cooked ham. Int. J. Food Microbiol. 120, 152–158.

Matito, C., Mastorakou, F., Centelles, J.J., Torres, J.L., Cascante, M., 2003.

Antiproliferative effect of antioxidant polyphenols from grape in murine Hepa-

1c1c7. Eur. J. Nutr. 42, 43–49.

Matsuhashi, S., Kume, T., 1997. Enhancement of antimicrobial activity of chitosan

by irradiation. J. Sci. Food Agri. 73, 237–241.

Matthaus, B., 2002. Antioxidant activity of extracts obtained from residues of different oil

seeds. J. Agric. Food Chem. 50, 3444–3452.

Mauriello, G., De Luca, E., La storia, A., Villani, E., Ercolini, D., 2005. Antimicrobial

activity of a nisin activated plastic film for food packaging. Lett. Appl. Microbiol.

41, 464–469.

Mäyrä-Mäkien, A., Bigret, M., 1993. Industrial use and production of lactic acid bacteria.

In: Salminen S, von Wright A, eds. Lactic acid bacteria. New York: Marcel Dekker,

65–95.

187

McAuliffe, O., Ryan, M.P., Ross, R.P., Hill, C., Breeuwer, P., Abee, T., 1998. Lacticin

3147, a broad-spectrum bacteriocin which selectively dissipates the membrane

potential. Appl. Environ. Microbiol. 64, 439–445.

Mendes, R., Silva, H.A., Nunes, M.L., Abecassis, J.M., 2005. Effect of low dose

irradiation and refrigeration on the microflora, sensory characteristics and biogenic

amines of Atlantic horse mackerel (Trachurus trachurus). Eur. Food Res. Technol.

221, 329–335.

Mohamed, K.R., El-Bassyouni, G.T., Beherei, H.H., 2007. Chitosan Graft Copolymers-

HA/DBM Biocomposites: Preparation, Characterization, and In Vitro Evaluation. J.

Appl. Polym. Sci. 105, 2553–2563.

Moharem, A.S., Charithraj, A.P., Janardhana, G.R., 2007. Incidence of listeria species in

seafood products of mysore, India. J. Food Safety. 27, 362–372.

Moll, G.N., Konings, W.N., Driessen, A.J.M., 1999. Bacteriocins: mechanism of

membrane insertion and pore formation. Antonie Van Leeuwnhoek. 76, 185–197.

Moller, J.K.S., Madsen, H.L., Aaltonen, T., Skibsted, L.H., 1999. Dittany (Origanum

dictamnus) as source of water extractable antioxidant. Food Chem. 64, 215–219.

Mottet, C., Michetti, P., 2005. Probiotics: wanted dead or alive. Dig. Liver Dis. 37, 3–6.

Mourya, V.K., Inamdar, N.N., 2008. Chitosan modifications and applications:

Opportunities galore. React. Funct. Polym. 68, 1013–1051.

Nanjo, F., Goto, K., Seto, R., Suzuki, M., Sakai, M., Hara, Y., 1996. Scavenging effects

of tea catechins and their derivatives on 1,1,-diphenyl-2-picrylydrazyl radical. Free

Rad. Biol. Med. 21, 895–902.

Neetoo, H., Ye, M., Chen, H., 2007. Effectiveness and stability of plastic films coated

with Nisin for inhibition of Listeria monocytogenes. J. Food Prot. 70, 1267–1271.

188

Nes, I.F., Diep, B.D., Holo, H., 2007. Bacteriocin Diversity in Streptococcus and

Enterococcus. J. Bacteriol. 189, 1189–1198.

Nickerson, J.T.R., Licciardello, J.J., Ronsivalli, J., 1983. Radurization and rancidation:

fish and shellfish, Preservation of Food by Ionizing Radiation, Vol. III (E.S.

Josephson, and M.S. Peterson, eds.), CRC Press, Boca Raton, FL.

Nielsen, J.W., Dickson, J.S., Crouse, J.D., 1990. Use of a Bacteriocin Produced by

Pediococcus acidilactici To Inhibit Listeria monocytogenes Associated with Fresh

Meat. Appl. Environ. Microbiol. 56, 2142–2145.

Niemira, B.A., 2010. Irradiation Sensitivity of Planktonic and Biofilm-associated Listeria

monocytogenes and L. innocua as Influenced by Temperature of Biofilm Formation.

Food Bioprocess Technol. 3, 257–264.

Nilsson, L., Huss, H.H., Gram, L., 1997. Inhibition of Listeria monocytogenes on cold-

smoked salmon by nisin and carbon dioxide atmosphere. Int. J. Food Microbiol. 38,

217–227.

Niwa, Y., Miyachi, Y., 1986. Antioxidant action of natural health products and Chinese

herbs. Inflammation, 10, 79–97.

No, H.K., Meyers, S.P., Prinyawiwatkul, W., Xu, Z., 2007. Applications of chitosan for

improvement of quality and shelf life of foods: A Review. J. Food Sci. 72, 87–100

No, H.K., Park, N.Y., Lee, S.H., Meyers, S.P., 2002. Antibacterial activity of chitosans

and chitosan oligomers with different molecular weights. Int. J. Food Microbiol. 74,

65–72.

O‘ Sullivan, L., Ross, R.P., Hill, C., 2002. Potential of bacteriocin producing lactic acid

bacteria for improvements in food safety and quality. Biochem. 84, 593–604.

189

Oehlenschlager, J., 1998. Detailed Experimental Iced-Storage Characteristics of Barents

Sea Cod. Inf. Fischwirtsch. 45, 35–42.

Okuyama, K., Noguchi, K., Miyazawa, T., 1997. Molecular and Crystal Structure of

Hydrated Chitosan. Macromolecules. 30, 5849–5855.

Ordonez, J.A., Hierro, E.M., Bruna, J.M., de la Hoz, L., 1999. Changes in the components

of dry-fermented sausages during ripening. Critic. Rev. Food Sci. Nutri. 39, 329–

367.

Ouwehand, A.C., Vesterlund, S., 2003. Health aspects of probiotics, I Drugs. 6, 573–580.

Oyaizu, M., 1986. Studies on products of browning reaction: antioxidative activities of

products of browning reaction prepared from glucosamine. Jap. J. Nutri. 44, 307–

315.

Ozden, O., Inugur, M., Erkan, N., 2007. Effect of different dose gamma radiation and

refrigeration on the chemical and sensory properties and microbiological status of

aquacultured sea bass (Dicentrarchus labrax). Radiat. Phys. Chem. 76, 1169–1178.

Özogul, F., Özogul, Y., 2007. The ability of biogenic amines and ammonia production by

single bacterial cultures. Eur. Food Res. Technol. 225, 385–394.

Ozogul, F., Polat, A., Ozogul, Y., 2004. The effect of modified atmosphere packaging

and vacuum packaging on chemical, sensory and microbiological changes of

sardines (Sardina pilchardus). Food Chem. 85, 49–57.

190

Paari, K.A., Hari krishnam naidu., Kanmani, P., Satish, R , Yuvaraj, N., Pattukumar, V.,

Arul, V., 2011a. Evaluation of Irradiation and Heat Treatment on Antioxidant

Properties of Fruit Peel Extracts and Its Potential Application During Preservation

of Goat Fish Parupenaeus indicus. Food Bioprocess Technol. 5, 1860–1870.

Paari, K.A., Kanmani, P., Satish ramraj., Yuvaraj, N., Pattukumar, V., Mona agrawal.,

Arul, V., 2011b. The combined effect of irradiation and antioxidant packaging on

shelf life extension of goat fish (Prupenaeus indicus): Microbial, chemical and EPR

spectral assessment. J. Food Process. Preserv. 36, 152–160.

Paari, K.A., Kanmani, P., Satish ramraj., Yuvaraj, N., Pattukumar, V., Ponni, S., Arul,

V., 2011c. Biopreservation of Sardinella longiceps and Penaeus monodon Using

Protective Culture Streptococcus phocae PI80 Isolated from Marine Shrimp

Penaeus indicus. Probiotics & Antimicro. Prot. 3, 103-111.

Paari, K.A., Kanmani, P., Satish Kumar, R., Yuvaraj, N., Pattukumar, V., Arul, V., 2012.

Potential function of a novel protective culture Enterococcus faecium MC13

isolated from the gut of Mugil cephalus: Safety assessment and its custom as

biopreservative. Food Biotechnol. 26, 180–197.

Parada, J.L., Caron, C.R., Medeiros, ABP., Soccol, C.R., 2007. Bacteriocins from Lactic

Acid Bacteria: Purification, Properties and use as Biopreservatives. Braz. Arch.

Biol. Technol. 50, 521–542.

Paranjpye, R.N., Peterson, M.E., Poysky, F.T., Eklund, M.W., 2008. Incidence, growth,

and inactivation of Listeria monocytogenes in cooked and peeled cold-water

shrimp. J. Aquat. Food Prod. Technol. 17, 266–284.

191

Parente, E., Giglio, M.A., Riccardi, A., Clementi, F., 1998. The combined effect of nisin,

leucocin F10, pH, NaCl and EDTA on the survival of Listeria monocytogenes in

broth. Int. J. Food Microbiol. 40, 65–75.

Park, P.J., Je, J.Y., Kim, S.W., 2004. Free radical scavenging activities of different

deacetylated chitosans using an ESR Spectrometer. Carbohydr. Polym. 55, 17–22.

Park, S.H., Itoh, K., Fujisawa, T., 2003. Characteristics and identification of enterocins

produced by Enterococcus faecium JCM 5804T. J. Appl. Microbiol. 95, 294–300.

Parker, R.B., 1974. Probiotics, the other half of the antimicrobial story. Anim. Nutr.

Health. 29, 4–8.

Patel, A.K., Deshattiwar, M.K., Chaudhari, B.L., Chincholkar, S.B., 2009. Production,

purification and chemical characterization of the catecholate siderophore from

potent probiotic strains of Bacillus spp. Bioresour. Technol. 100, 368–373.

Patterson, M.F., Loaharanu, P., 2000. Food Irradiation. In: The Microbiological Safety

and Quality of foods. B. Lund, Gould, G.W. and Baird-Parker, T. Aspen, USA, 65–

88.

Pattukumar, V., Maloy, K. S., Murugan, M., Vijayabasakara, G.S., Sivakumar, K., Arul,

V., 2010. Population of Vibrio Parahaemolyticus (Pathogen) and Bacillus

(Beneficial Bacteria) in Penaeus Monodon (Fabricus, 1798) Culture. Online J. Biol.

Sci. 10, 142–150.

Pawar, D.D., Malik, S.V.S., Bhilegaonkar, K.N., Barbuddhe, S.B., 2000. Effect of nisin

and its combination with sodium chloride on the survival of Listeria monocytogenes

added to raw buffalo meat mince. Meat Sci. 56, 215–219.

192

Pazos, M., Gallardo, J.M., Torres, J.L., Medina, I., 2005a. Activity of grape polyphenols

as inhibitors of the oxidation of fish lipids and frozen fish muscle. Food Chem. 92,

547–557.

Pazos, M., Gonza´lez, M.J., Gallardo, J.M., Torres, J.L., 2005b. Preservation of the

endogenous antioxidant system of fish muscle by grape polyphenols during frozen

storage. Eur. Food Res. Technol. 220, 514–519.

Peláez, C., Requena, T., 2005. Exploiting the potential of bacteria in the cheese

ecosystem. Int. Dairy J. 15, 831–844.

Pellegrini, N., Simonetti, P., Gardana, C., Brenna, O., Brighenti, F., Pietta, P., 2000.

Polyphenol content and total antioxidant activity of Vini Novelli (Young red wines).

J.Agric. Food Chem. 48, 732–735.

Peschel, W., Sánchez-Rabaneda, F., Dn, W., Plescher, A., Gartzia, I., Jimenez, D.,

Lamuela-Raventos, R., Buxaderas, S., Condina, C., 2006. An industrial approach in

the search of natural antioxidants from vegetable and fruit wastes. Food Chem. 97,

137–150.

Petillo, D., Hultin, H.O., Krzynowek, J., Autio, W.R., 1998. Kinetics of antioxidant loss

in mackerel light and dark muscle. J. Agric. Food Chem. 46, 4128–4137.

Poli, B.M., Messini, A., Parisi, G., Scappini, F., Figiani, V., Giorni, G., Vincenzini, M.,

2006. Sensory, physical, chemical and microbiological changes in European sea

bass (Dicentrarchus labrax) fillets packaged under modified atmosphere/air or

prepared from whole fish stored in ice. Int. J. Food Sci. Technol. 41, 444–454.

Poole, S.E., Mitchell, G.E., Mayze, J.L., 1994. Lowdose irradiation affects

microbiological and sensory quality of sub-tropical seafood. J. Food Sci. 59, 85–87.

193

Pourjavadi, A., Mahdavinia, G.R., Zohuriaan-Mehr, M.J., 2003. Modified chitosan. II. H-

Chito PAN, a novel pH-responsive superbasorbent hydrogel. J. Appl. Polym. Sci.

90, 3115–3121.

Proctor, B.E., Bhatia, B.S., 1950. Effect of electron radiation on haddock fillets, Food

Technol. 4, 357–359.

Przybylski, L.A., Finerty, M.W., Grodner, R.M., Gerdes, D.L., 1989. Extension of shelf-

life of iced fresh channel catfish fillets using modified atmospheric packaging and

low-dose irradiation, J. Food Sci. 54, 269–273.

Pyrgotou, N., Giatrakou, V., Ntzimani, A., Savvaidis, I.N., 2010. Quality assessment of

salted, modified atmosphere packaged rainbow trout under treatment with oregano

essential oil. J. Food Sci. 75, M406–M411.

Raafat, D., Sahl, H.G., 2009. Chitosan, and its antimicrobial potential—a critical

literature survey. Microbial Biotechnol. 2, 186–201.

Radhakumary, C., Nair, P.D., Suresh Mathew., Reghunadhan Nair, C.P., 2005.

Biopolymer Composite of Chitosan and Methyl Methacrylate for Medical

Applications. Trends Biomater. Artif. Organs. 18, 117–124.

Ramiah, K., Ten Doeschate, K., Smith, R., Dicks, L.M.T., 2009. Safety Assessment of

Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA Determined in

Trials with Wistar Rats. Probiotics & Antimicro. Prot.1, 15–23.

Reid, G., 2005 "From Bench to Bedside to Consumer: Where are the Regulatory

Problems for Probiotics?," J. Complement Int. Med. 2, 4.

Reid, G., Zalai, C., Gardiner, G., 2001. Urogenital lactobacilli probiotics, reliability and

regulatory issues. J. Dairy Sci. 84, 164–169.

194

Resta-Lenert, S., Barrett, K.E., 2003. Live probiotics protect intestinal epithelial cells

from the effects of infection with enteroinvasive Escherichia coli (EIEC). Gut. 52,

988– 997.

Resta-Lenert, S., Barrett, K.E., 2009. Modulation of intestinal barrier properties by

probiotics: role in reversing colitis. Ann. NY. Acad. Sci. 1165, 175–182.

Rhoades, J., Roller, S., 2000. Antimicrobial Actions of Degraded and Native Chitosan

against Spoilage Organisms in Laboratory Media and Foods. Appl. Environ.

Microbiol. 6, 80–86.

Richard, C., Brillet, A., Pilet, M.F., Prévost, H., Drider, D., 2003. Evidence on inhibition

of Listeria monocytogenes by divercin V41 action. Lett. Appl. Microbiol. 36, 288–

292.

Ring, E., Gatesoupe, F.J., 1998. Lactic acid bacteria in Fish: a review. Aquacult. 100,

177–203.

Roberts, G.A.F., 1992. Structure of chitin and chitosan. In: Roberts GAF, ed. Chitin

Chemistry. MacMillan, Houndmills.1–53.

Ronsivalli, L.J., Ampolla, V.G., King, F.J., Holston, J.A., 1968. A study of irradiated

pasteurized fishery products. Report to USAEC on Contract No.AT (49-11)-1889,

Bur. Com. Fish Tech. Lab, Gloucester, MA

Ross, K.F., Ronson, C.W., Tagg, J.R., 1993. Isolation and characterization of the

lantibiotic salivaricin A and its structural gene salA from Streptococcus salivarius

20P3. Appl. Environ. Microbiol. 59, 2014–2021.

Ross, R.P., Morgan, S., Hill, C., 2002. Preservation and fermentation: past, present and

future. Int. J. Food Microbiol. 79, 3–16.

195

Ross, R.P., Stanton, C., Hill, C., Fitzgerald, G.F., Coffey, A., 2000. Novel cultures for

cheese improvement. Trends Food Sci. Technol. 11, 96–104.

Ruberto, G., Baratta, M.T., 1999. Antioxidant activity of selected essential oil

components in two lipid model systems. Food Chem. 69, 167–174.

Rudrapatnam, N.T., Farooqahmed, S.K., 2003. Chitin the undisputed biomolecule of

great potential. Critic. Rev. Food Sci. Nutri. 43, 61–87.

Saeed, S., Howell, N.K., 2002. Effect of lipid oxidation and frozen storage on muscle

proteins of Atlantic mackerel (Scomber scombrus). J. Sci. Food Agric. 82, 579–

586.

Sa´nchez-Alonso, I., Jime´nez-Escrig, A., Saura-Calixto, F., Borderı´as, A.J., 2007. Effect

of grape antioxidant dietary fibre on the prevention of lipid oxidation in minced

fish: Evaluation by different methodologies. Food Chem. 101, 372–378.

Saarela, M., Mogensen, G., Fonden, R., Matto, J., Mattila-Sandholm, T., 2000. Probiotic

bacteria: safety, functional and technological properties. J. Biotechnol. 84, 197–215.

Sachetti, G., Maietti, S., Muzzoli, M., Scaglianti, M., Manfredini, S., Radice, M., Renato-

Bruni, R., 2005. Comparative evaluation of 11 essential oils of different origin as

functional antioxidants, antiradicals and antimicrobials in foods. Food Chem. 91,

621–632.

Saha, K., Lajis, N.H., Israf, D.A., 2004. Evaluation of antioxidant and nitric oxide

inhibitory activities of selected Malaysian medicinal plants. J. Ethnopharmacol. 92,

263–267.

Selmi, S., Sadok, S., 2008. The effect of natural antioxidant (Thymus vulgaris Linnaeus)

on flesh quality of tuna (Thunnus thynnus (Linnaeus) during chilled storage. Pan.

Am. J. Aquat. Sci. 3, 36–45.

196

Sallam, K.I., 2007. Antimicrobial and antioxidant effects of sodium acetate, sodium

lactate, and sodium citrate in refrigerated sliced salmon. Food Cont. 18, 566–575.

Samir, A., Margarita, G., Marta, H., Mercedes, M., Manual, M.B., Antonio, G., Eva, V.,

2005. Control of Listeria monocytogenes in model sausages by enterocin AS-48.

Int. J. Food Microbiol. 103, 179–190.

Satish Kumar, R., Arul, V., 2009. Purification and characterization of Phocaecin PI80: An

anti-listerial bacteriocin produced by Streptococcus phocae PI80 isolated from the

gut of Peneaus indicus (Indian White Shrimp). J. Microbiol. Biotechnol. 19, 1393–

1400.

Satish Kumar, R., Varman, D.R., Kanmani, P., Yuvaraj, N., Paari, K.A., Pattukumar,

V., Arul, V., 2010. Isolation, Characterization and Identification of a Potential

Probiont from South Indian Fermented Foods (Kallappam, Koozh and Mor

Kuzhambu) and Its Use as Biopreservative. Probiotics & Antimicro prot. 2, 145–

151.

Satish Kumar, R., Kanmani, P., Yuvaraj, N., Paari, K.A., Pattukumar, V., Arul, V., 2011.

Lactobacillus plantarum AS1 binds to cultured human intestinal cell line HT-29

and inhibits cell attachment by enterovirulent bacterium Vibrio parahaemolyticus.

Lett. Appl. Microbiol. 53, 481–487.

Savvaidis, I.N., Skandamis, P., Riganakos, K.A., Panagiotakis, N., Kontominas, M.G.,

2002. Control of Natural Microbial Flora and Listeria monocytogenes in Vacuum-

Packaged Trout at 4 and 10◦C Using Irradiation. J. Food Prot. 65, 515–522.

Schalm, O.W., Jain, N.C., Carrol, E.J., 1975. Veterinary Haematology 3rd

Edition. Lea

and Febiger, Philadephia, pp, 421–538.

197

Schillinger, U., Geisen, R., Holzapfel, W.H., 1996. Potential of antagonistic

microorganisms and bacteriocins for the biological preservation of foods. Trends

Food Sci. Technol. 71, 58–64.

Schnurar, J., Magnusson, J., 2005. Antifungal lactic acid bacteria as biopreservatives.

Trends Food. Sci. Technol. 16, 1–9.

Scientific Committee for Food., 1987. Report on /radiated Foods, EUR Series Food

Science and Techniques, No 10840, Brussels

Seifried, H.E., McDonald, S.S., Anderson, D.E., Greenwald, P., Milner, J.A., 2003. The

antioxidant conundrum in cancer. Cancer Res. 63, 4295–4298.

Shahidi, F., 2000. Antioxidants in food and food antioxidants. Nahrung. 44, 158–163.

Shahidi, F., Arachchi, J.K.V., Jeon, Y., 1999. Food applications of chitin and chitosans.

Trends Food Sci. Tech.10, 37–51.

Shanahan, F., 2003. Probiotics: a perspective on problems and pitfalls. Scand. J.

Gastroenterol. Suppl. 237, 34–36.

Shakila, R.J., Vijayalakshmi, K., Jeyasekaran, G., 2003. Changes in histamine and

volatile amines in six commercially important species of fish of the Thoothukkudi

coast of Tamil Nadu, India stored at ambient temperature. Food Chem. 82, 347–

352.

Shin, Y., Yoo, D.I., Jang, J., 2001. Molecular weight effect on antimicrobial activity of

chitosan treated cotton fabrics. J. Appl. Polym. Sci. 80, 2495–2501.

198

Sinanoglou, V.J., Batrinou, A., Konteles, S., Sflomos, K., 2007. Microbial Population,

Physicochemical Quality, and Allergenicity of Molluscs and Shrimp Treated with

Cobalt-60 Gamma Radiation. J. Food Prot. 70, 958–966.

Sinead, C.C., Cormac, G.M.G., Colin, H., 2007. Impact of selected Lactobacilli and

Bifidobacterium sps. on Listeria monocytogenes infection and mucosal immune

response. FEMS Immunol. Med. Microbiol. 50, 380–388.

Singh, A., Fei-Peng, Y., Mc-Feters, G.A., 1990. Rapid detection of chlorine induced

bacterial injury by the direct viable count method using image analysis. Appl.

Environ. Microbiol. 56, 389–394.

Singh, R.P., Chidambaramurthy, K.N., Jayaprakasha, G.K., 2002. Studies on antioxidant

activity of pomegranate (Punica granatum) peel and seed extracts using in vitro

models. J. Agric. Food Chem. 50, 81–86.

Skerget, M., Kotnik, P., Hadolin, M., Hras, A.R., Simonic, M., Knez, Z., 2005. Phenols,

proanthocyanidins, flavones and flavonols in some plant materials and their

antioxidant activities. Food Chem. 89, 191–198.

Smid, E.J., Gorris, L.G.M., 1999. Natural antimicrobials for food preservation. In M. S.

Rahman (Ed.), Handbook of food preservation (pp. 285–308). New York: Marcel

Dekker.

Smirnoff, N., Cumbes, Q.J. 1989. Hydroxy radical scavenging activity of compatible

solutes. Phytochem. 28, 1057–1060.

Solomon, E.F., Sharada, A.C., Uma Devo, P., 1993. Toxic effects of crude root extract of

Plumbago rosea (Raktachitraka) on mice and rats. J. Ethnopharmacol. 38, 79–84.

Song, Y., Babiker, E.E., Usui, M., Saito, A., Kato, A., 2002. Emulsifying properties and

bactericidal action of chitosan–lysozyme conjugates. Food Res Int. 35, 459–66.

Songisepp, E., Kulisaar, T., Hutt, P., Elias, P., Brilene, T., Zilmer, M., 2004. A new

199

probiotic cheese with antioxidative and antimicrobial activity. J. Dairy Sci. 87,

2017–2023.

Soni, K.A., Nannapaneni, R., 2010. Bacteriophage Significantly Reduces Listeria

monocytogenes on Raw Salmon Fillet Tissue. J. Food Prot. 73, 32–38.

Stachowicz, W., Burlinska, G., Michalik, J., 1988. EPR detection of foods preserved with

ionizing radiation. Radiat. Phys. Chem. 52, 157–160

Stavikova, L., Polovka, M., Hohnova, B., Zemanova, J., 2008. Multi-experimental

characterisation of grape skin extracts. Czech. J. Food Sci. 26, S43–S48.

Stone, W.L., Leclair, I., Ponder, T., Bagss, G., Barret-Reis, B., 2003. Infants discriminate

between natural and synthetic vitamin E. Am. J. Clin. Nutr. 77, 899–906.

Stiles, M.E., 1996. Biopreservation by lactic acid bacteria. Antonie van Leeuwenhoek.

70, 331–345.

Su, H.C.F., Horvat, R., 1987. Isolation and Characterization of Four Major Components

from Insecticidally Active Lemon Peel Extract. J. Agric. Food Chem. 35, 509–511.

Suhendanmol., Nuray Erkan., Didemucok., Syasemin tosun., 2007. Effect of

psychrophilic bacteria to estimate the fish quality. J. Muscle Foods. 18, 120–28.

Suyatma, N.E., Copinet, A., Tighzert, L., Coma, V., 2004. Mechanical and barrier

properties of biodegradable films made from chitosan and poly (lactic acid) blends.

J. Polym. Environ .12, 1–6.

Swain, S.M., Chandrasekar, S., Arul, V., 2009. Inhibitory activity of probiotic bacterium

Streptococcus phocae PI80 and Enterococcus faecium MC13 against vibriosis in

shrimp Penaeus indicus. World J. Microbiol. Biotechnol. 25, 697–703.

200

Szabo, E.A., Cahill, M.E., 1999. Nisin and ALTATM 2341 inhibit the growth of Listeria

monocytogenes on smoked salmon packaged under vacuum or 100% CO2. Lett.

Appl. Microbiol. 28, 373–377.

Taguri, T., Tanaka, T., Kouno, I., 2004. Antimicrobial Activity of 10 Different Plant

Polyphenols against Bacteria Causing Food-Borne Disease Biol. Pharm. Bull. 27,

1965–1969.

Tang, S.Z., Sheehan, D., Buckley, D.J., Morrissey, P.A., Kerry, J.P., 2001. Anti-oxidant

activity of added tea catechins on lipid oxidation of raw minced red meat, poultry

and fish muscle. Int. J. Food Sci. Technol. 36, 685–692.

Tepe, B., Sokmen, M., Akpulat, H.A., Daferera, D., Polissiou, M., Sokmen, A., 2005.

Antioxidative activity of the essential oils of Thymus sipyleus subsp. Sipyleus var.

sipyleus and Thymus sipyleus subsp. Sipyleus var. rosulans. J. Food Engg. 66, 447–

454.

Thayer, D.W., Rajkowski, K.T., 1999. Developments in irradiation of fresh fruits and

vegetables. Food Technol. 53, 62–65.

Thayer, D.W., Boyd, G., Fox, J.R., Lakritz, L., Hamson, J.W., 1995.Variations in

radiation sensitivity of food borne pathogens associated with the suspending meat.

J. Food Sci. 60, 63–67.

Thayer, W., Christopher, J.P., Campbell, L.A., Ronning, D.C., Dahlgren, RR., Thomson,

G.M., Wierbicki., E., 1987. ‗Toxicology studies of irradiation-sterilized chicken‘.

J. Food Prot. 50, 278–288.

Theron, Rykers Lues., 2011. Organic acids and food preservation.CRC press, Taylor and

francis group, London.51–84.

201

Tortuero, F., Rioperez, J., Fernandez, E., Rodriguz, M.L., 1995. Response of piglets to

oral administration of lactic acid bacteria. J. Food Prot. 58, 1369–74.

Tsai, G.J., Su, W.H., Chen, H.C., Pan, C.L., 2002. Antimicrobial activity of shrimp chitin

and chitosan from different treatments and applications of fish preservation. Fish Sci.

68, 170–7.

Työppönen, S., Petäjä, E., Mattila-Sandholm, T., 2003. Bioprotectives and probiotics for

dry sausages. Int. J. Food Microbiol. 83, 233–244.

Ueckert, J.E., Ter Steeg, P.F., Coote, P.J., 1998. Synergistic antibacterial action of heat in

combination with nisin and magainin II amide. J. Appl. Microbiol. 85, 487–494.

Valko, M., Dieter Leibfritz, D., Moncol, J., Cronin, M.T., Mazur, M., Telser, J., 2007. Free

radicals and antioxidants in normal physiological functions and human diseases. Int. J. Biochem.

Cell biol. 39, 44–84.

Van Cleemput, O., Debevere, J., Debevere, P., Baert, L., 1980. Gamma irradiation of

tropical shrimps. Lebensm. Wiss. U. Technol. 13, 322–323.

Van Kraaij, C., Breukink, E., Rollema, H.S., Siezen, R.J., Demel, R.A., De Kruijff,

B., Kuipers, O.P., 1997. Influence of Charge Differences in the C-Terminal Part of

Nisin on Antimicrobial Activity and Signaling Capacity. Eur. J. Biochem. 247, 114–

120.

Van Luyen, D., Huong, D.M., 1996. Chitin and Derivatives, in: Polymeric Materials.

Encyclopedia, Salamone J.C. (Ed), CRC, Boca Raton (Florida), 2, 1208–1217.

Vanderzant, C., Splittstoesser, D.F., 1992. Compendium of methods the microbial

examination of food. American public health association, Washington. 1219.

202

Vareltzis, K., Koufidis, D., Gavriilidou, E., Papavergou, E., Vasiliadou, S., 1997.

Effectiveness of a natural Rosemary (Rosmarinus officinalis) extract on the stability

of filleted and minced fish during frozen storage. Food Res. Technol. 205, 93–96.

Variyar, P., Limaye, A., Sharma, A., 2004. Radiation-Induced Enhancement of

Antioxidant Contents of Soybean (Glycine max Merrill). J. Agric. Food Chem. 52,

3385–3388.

Vasiljevic, T., Shah, N.P., 2008. Probiotics From Metchnikoff to bioactives,

Int. Dairy J. 18, 714–728.

Veciana-Nogues, M., Vidal-Carou, M., Marine-Font, A., 1990. A research note:

Histamine and tyramine during storage and spoilage of anchovies, Engraulis

encrasicholus: relationships with other fish spoilage indicators. J. Food Sci. 55,

1192–1193.

Venugopal, V., 1990. Extracellular proteases of contaminant bacteria in fish spoilage: a

review. J. Food Prot. 53, 341–353.

Venugopal, V., Doke, S.N., Thomas, P., 1999. Radiation processing to improve the

quality of fishery products. Crit. Rev. Food Sci. Nutr. 30, 391–440.

Vescovo, M., Scolari, G., Zacconi, C., 2006. Inhibition of Listeria innocua growth by

antimicrobial-producing lactic acid cultures in vacuum-packed cold-smoked

salmon. Food Microbiol. 23, 689–693.

203

Vignolo, G.M., Suriani, F., Pesce de Ruiz Holgado, A., Oliver, G., 1993. Antibacterial

activity of Lactobacillus strains isolated from dry fermented sausages. J. Appl.

Microbiol. 75, 344–349.

Wada, S., Fang, X., 1994. Synergistic antioxidant effects of rosemary and α-tocopherol at

different storage temperatures and its application for inhibiting dried sardine meat

oxidation. J. Jap. Oil Chem. Soc. 43, 109–115.

Wang, Y., Yang, J.X., Qiu, K.Y., 1994. Graft copolymerization onto chitosan, Acta.

Polym. Sin. 2, 188–195.

Ward, D.R., Baj, N.J., 1988. Factors affecting microbiological quality of sea foods, Food

Technol. 42, 88–89.

Weiss, A., Hammes, W.P., 2006. Lactic acid bacteria as protective cultures against

Listeria spp. on cold-smoked salmon. Eur. Food Res. Technol. 222, 343–346.

Wessels, S., Huss, H.H., 1996. Suitability of Lactococcus lactis subsp. lactis ATCC

11454 as a protective culture for lightly preserved fish products. Food Microbiol.

13, 323–332.

Whitford, M.F., McPherson, M.A., Forster, R.J., Teather, R.M., 2001. Identification of

bacteriocin-like inhibitors from rumen Streptococcus spp. and isolation and

characterization of bovicin 255. Appl. Environ. Microbiol. 67, 569–574.

Whittle, K.J., Hardy, R., Hobbs, G., 1990. Chilled fish and fishery products. In: Gormley,

T. R., Ed., Chilled Foods: The State of Art, Elsevier, Amsterdam, 87–117.

WHO., 1969. Specifications for identity and purity of some antibiotics. World Health

Organization/Food additives. 69 , 53–67.

204

WHO., 1981. Wholesomeness of Irradiated Food, World Health Organization Technical

Report Series 659, Geneva.

WHO., 1999. High-dose irradiation. Wholesomeness of food irradiated with doses above

10 kGy. Report of a Joint FAO/IAEA/WHO Study Group on High Dose Irradiation.

WHO Technical Report Series. Geneva: World Health Organization; Food Irradiation

Clearances Database. http://nucleus.iaea.org/apps/FICDB/Browse.aspx. Assessed

17.03.10.Food Irradiation Clearances Database. http://

nucleus.iaea.org/apps/FIFDB/Browse.aspx. Assessed 15.10.09.

Wirawan, R.E., Klesse, N.A., Jack, R.W., Tagg, J.R., 2006. Molecular and genetic

characterization of a novel nisin variant produced by Streptococcus uberis. Appl.

Environ. Microbiol. 72, 1148–1156.

Witschi, H.P., 1986. Enhanced tumour development by butylated hydroxytoluene (BHT)

in the liver, lung and gastro-intestinal tract. Food Chem. Toxicol. 24, 1127–1130.

Xie, W., Xu, P., Liu, Q., 2001. Antioxidant activity of water-soluble chitosan derivatives.

Bioorg. Med. Chem. Lett. 11, 1699–1701.

Xie, W.M., Xu, P.X., Wang, W., Liu, Q., 2002. Preparation and antibacterial activity of a

water-soluble chitosan derivative. Carbohydr. Polym. 50, 35–40.

Xu, G., Ye, X., Chen, J., Liu, D., 2007. Effect of heat treatment on the phenolic

compounds and antioxidant capacity of citrus peel extract. J. Agric. Food Chem. 55,

330–335.

Yamazaki, K., Suzuky, M., Kawai, Y., Inoue, N., Montville, T.J., 2003. Inhibition of

Listeria monocytogenes in cold-smoked salmon by Carnobacterium piscicola CS526

isolated from frozen surimi. J. Food Prot. 66, 1420–1425.

205

Yerlikaya, P., Gokoglu, N., 2010. Inhibition effects of tea and grape seed extracts on lipid

oxidation in bonito fillets during frozen storage. Int. J. Food Sci. Technol. 45, 252–

257.

Yilmaz, Y., Toledo, R.T., 2004. Major flavonoids in grape seeds and skins: antioxidant

capacity of catechin, epicatechin and gallic acid. J. Agric. Food Chem. 52, 255–260.

Yongjin, H., Wenshui, X., Xiaoyong, L., 2007. Changes in biogenic amines in fermented

silver carp sausages inoculated with mixed starter cultures. Food Chem. 104, 188–

195.

Yu Shengfu., Zhang Yuanhu., Cheng Bingsong., Zheng Suqin., 1993. Effects of cobalt-60

gamma ray irradiation on fresh-keeping and storage of kiwi fruits. Radiat. Phys.

Chem. 42, 339–341.

Zhong-Yi, L., Zhong-Hai, L., Miao-Ling, Z., Xiao-Ping, D., 2010. Effect of fermentation

with mixed starter cultures on biogenic amines in bighead carp surimi. Int. J. Food

Sci. Technol. 45, 930–936.

Zhou, J.S., Shu, Q., Rutherford, J., Prasad, J., Gopal, P.K., Gill, H.S., 2000. Acute oral

toxicity and bacterial translocation studies on potentially probiotic strains of lactic

acid bacteria. Food Chem. Toxicol. 38, 153–161.

Zhou, Q., Li, K., Jun, X., Bo, L., 2009. Role and functions of beneficial microorganisms

in sustainable aquaculture. Bioresour. Technol. 100, 3780–3786.