2 REVIEW OF LITERATURE

2.1. Vacuum Packaging

Vacuum packaging has been shown to extend the shelf life of food products. The

shelf life extension of chill-stored vacuum packed and modified atmospheric packed cod,

from less than 3 days to about 2 weeks, is thus very short, when compared to the

extension obtained with meat products (Jensen et al., 1980; Cann etal., 1983; Daniels et

al., 1986; Jorgensen et al., 1988). Sediman and Durland (1983) had reviewed the

implication of vacuum packaging of fresh beef, suggesting the importance of conversion

of oxygen to carbon dioxide in meat package. Recognition of this has resulted in the

development of vacuum packaging technique for meats.

Such packaging method reduced the total psychrophilic microflora, since the

growth of most numerous species like Pseucionionas is reduced, when oxygen content

falls to less than one percent, increasing the level of CO 2 . Reduction in spoilage bacteria

in vacuum packed beef was reported by Steinhouser et al. (1988) and Bell and Garout

(1994)

Use of hurdle technology to reduce the hazards associated with minimally

processed foods packed in a modified atmosphere I vacuum packaging has been an

interest of food scientists over the past 10 years. Use of hurdle technology, that involves

combination of synergistic techniques like reduced water activity, packaging under clean

conditions, rapid cooling following cooking, removal of 02 from packaging and

replacement with CO 2 and other gasses, sealing under modified packaging, use of spices

with antimicrobial properties and maintenance of low temperature during distribution to

8

improve microbiological safety, shelf life and quality of packaged products and

packaging innovations (e.g. barrier packaging and packaging materials containing 02

scavengers, ethylene and antimicrobiological agents that diffuse into foods have been

reviewed by Conner et al. (1989) and Forcinio (1999).

Effectiveness of 02 excluding atmospheres (vacuum, CO2 and N2) in inhibiting /

suppressing spoilage bacterial flora was documented for a variety of meat products such

as pork, beef, ham, veal and pastrami (Christopher et al., 1980; Hanna ci al., 1981; Kemp

etal., 1983; Lee etal., 1984). Vacuum packaging of wholesale fresh meat is increasingly

being practiced by meat industry, as it reduces shrink loss, protect meat colour and delays

microbial spoilage (Medonca ci al., 1989). Reduced bacterial growth in vacuum packed

fish and changes in spoilage pattern compared to that in aerobic pack were reported by

several authors (Banks et al., 1980; Dalgaard ci al., 1993; Shalini ci al., 2001;

Shanmugam ci al., 2000).

Refrigerated foods certainly are not new. Dairy products, fresh and cured meat

have been successfully marketed this way for many years. The product of concern with

regard to microbiological safety are those designed for refrigerated storage that are

marketed with extended shelf life expectations. These types of products have been

referred to as new generation refrigerated foods. These products may include sauces,

soups, salads, pasta, seafood and meat salads, fresh pasta as well as complete meals.

Many of these products receive a heat treatment that reduces the microbial load but does

not produce commercial sterility: thus the products still require refrigeration to prevent

spoilage and ensure product safety (Conner ci al., 1989). Many of these products are

generally sealed in barrier packaging to prevent microbial contamination and reduce

oxidative and other chemical deterioration during distribution. The barrier packaging

treatment in conjunction with refrigeration is used to achieve extended shelf life

necessary to manufacture, distribute and market the new generation refrigerated food.

2.1.1. Vacuum packing material

A wide range of materials with varying properties have been used world wide in

vacuum packaging of fish and meat products for shelf-life improvements. Dalgaard et al.

(1993) used low permeable Riloten 40!70X plastic laminate film for packing cod fish

fillets. At 75% RH and 25°C the laminate has an oxygen transmission rate of 2.0

CC/m 2/24 hIl atm and a CO2 transmission of rate of 8.0 CC/m2124 hll atm. Packaging

film pouches such as (i) low density polyethylene (LDPE) monolayer with water vapour

transmission rate (WVTR) of 12.74 g/m 2!24 h and oxygen transmission rate (OTR) of

1800 cm3/m 2/24 h (ii) polyester! polyethylene (PET/PE) with WVTR of 10.09 g/m 2!24 h

and OTR of 140-150 cm 3/m 2!24 h (iii) multilayer consisting of LDPE + bonding agent +

nylon + bonding agent + LDPE with WVTR of 7.57 g/m 2!24 h and OTR of 95 to 150

cm 3/m2124 h have been studied for assessing the effect of vacuum packaging on the

chemical and microbial qualities of beef during storage (Dushyanthan et al., 2000).

An improvement in quality and shelf life of prepacked fish will be obtained

Jepending on the type of fish, packaging material and packaging method. Many works

)n vacuum packaging have been carried out using different packaging materials of

;uitable thickness. They include saran coated melinex - polyethylene laminate (Cann ci

i/., 1965), low density polyethylene and nylon film (Huss, 1972) 100 gauge nylon /2 mu

10

curpolymer - surlyn (Unda et al., 1990) and multilayer nylon barrier film of 100i

thickness with oxygen transmission rate of 65-3 3.2 CC/m2/24 h at 23°C at 80% relative

humidity (Shalini et al., 2000 and Shanmugam et al., 2000).

2.2. Preservatives

2.2.1. Chemical Preservatives

2.2.1.1. Sorbates

Sorbic acid (2,4-hexadienoic acid) was first recommended as a food preservative

by Gooding (1945). The use of sorbic acid as antifungal agent became widespread.

Products such as cheese, fruit, juices, frostings, cakes, and pie filings were preserved with

sorbic acid and this compound is included in the U.S. Food and Drug Administrations'

list of chemicals which are generally recognised as safe (GRAS) in foods (Troller and

Robert, 1967). One of the chief attributes of sorbic acid is its effectiveness throughout a

concentration range in which little off-flavour or odour is imparted to the preserved food.

In addition, this compound is relatively non-toxic, being metabolised in a manner similar

to longer chain fatty acids (Deucl ci al., 1954) and relatively inexpensive.

Sorbic acid is a straight chain, 13-unsaturated trans-trans, 2,4-hexadienoic mono

carboxylic aliphatic acid and has the molecular formula CH3-CH+CH-CIFCH-COOH.

The carboxyl group of sorbic acid reacts readily and forms salts and esters. The salts of

sorbic acid especially the potassium salt are very important in applications due to high

solubility in water. Solubility of sorbic acid in water increases with p1-I and temperature.

In vegetable oil. the acid form is more soluble than potassium salt. Increased

concertrations (>10%) of soluble components such as glucose, sucrose and NaCl reduce

It

the solubility of sorbic acid in water (Sofos and Busta, 1981). Practical applications of

sorbates include preservation of human food, animal feed, pharmaceutical, cosmetic

products and packaging materials. The practical aplications of sorbate as a food

preservative include dairy products (cheese, cheese products, yogurt, sour cream, cheese

spreads and chips), bakery products (cakes and cake mixes, pies and pie fillings,

doughnuts, icings, toppings) fruit and vegetable products (wines, beverages, fruit juices

and syrups, jams and jellies, dried fruits, salads, fermented and pickled vegetables) and

other food products (certain meat and fish products, mayonnaise, margarine, salad

dressing)

Methods of application of sorbate include direct addition into the product, dipping

in or spraying with a sorbate solution, dusting and incorporation in wrapping or

packaging material. The effectiveness of sorbate as an inhibitory agent against key

microorganisms is used to determine the use concentration of the compound and depends

on factors such as pH of the product, ingredients of the product, moisture content of the

product. product contamination, processing, packaging, storage temperature, storage

length and sanitation (Sofos and Busta, 1981).

It was reported that sorbate was more efficient and less toxic than benzoate (Sheu

t al., 1975). Among food preservatives, the World Health Organization has stipulated

'or sorbate, the highest acceptable daily intake, which is 25 mg/kg of body weight.

\llowable residual level of sorbic acid in fish products is 0. 1% and concentration of 5% -

10% is permitted in dipping solutions (International Sourcing, 1986 ; Joseph, 2003a).

orbate was three times more effective than benzoate in preserving fish and bakery

12

products (Smith and Rollin, 1954; Boyd and Tarr, 1955). Sorbic acid has enormous

applications in food products as sorbic acid inhibits certain dehydrogenates which are

involved in the n-oxidation of fatty acids (Boyd and Tan, 1955).

The effect of potassium sorbates on Clostridium botulinum, Clostridium

perfringens, Salmonella and Staphylococcus aureus in an uncured, cooked sausage,

temperature abused at 27°C was studied (Tomphin et al., 1974). The results indicated

that 0.'.% potassium sorbate a retarded total microbial growth, Salmonella and S.aureus

growth. Sorbate also retarded botulinal toxin production. Sorbic acid did not inhibit or

stimulate the growth of clostridia in microbiological media at a pH of 6.7. Certain mould

species were more resistant to sorbic acid and therefore resulted in occasional mould

spoilage of foods preserved with this compound (Vaughn and Emard, 1951; Bullerman,

1977). Sorbic acid has been shown to inhibit the growth of yeasts, moulds and many

bacteria. Its activity against bacteria, however, is not as comprehensive as against yeasts

and moulds. Brog et al. (1955) reported that 0.1% sorbic acid not only inhibited growth

of fermentative yeasts in cucumber fermentations but also retarted growth and acid

production by the acid forming bacteria. There are several reports on the inhibitory

effect of sorbate on a range of bacteria. Vaughn and Emard ( 1 95 1) reported that sorbate

inhibited several species of bacteria in laboratory media. Low concentration of sorbate

(0.075%) were active against Salmonella typhimuriuin and Escherichia coli (Doell,

1962). Studies showed that sorbate has also inhibited total microbial growth,

staphylococci, Pseudomonas sp., Vibrio parahaemolyticus, Bacillus etc (Bradley et al.,

39 Dog)), 1962; Gould, 1964 Mouafa and Collins. 1969; Raevuori, 1976; Robach,

13

1978, 1979a Robach and Hickey, 1978; Pierson et al., 1979). It was also documented

that the rate of spore germination of six Bacillus spp was depressed at sorbate

concentration of more than 0.04% at pH 6.0 (Gould, 1964).

Pseudomonasfluorescens is a major spoilage organism in fresh seafood, (Chai et

al., 1968). Robach (1978) studied the effect of potassium sorbate on the growth of

Pseudomonas Jluorescens in trypticase soy broth at 24°C. Potassium sorbate was more

effective in inhibiting the growth of P. Jluorescens at pH 5.5 than in pH 6.0. Addition of

0.05% sorbate inhibited the growth of this organism in p1-1 5.5 and 0.2% of sorbate

delayed the growth of the organism in pH 6.0. Robach (1979a) also reported that

addition of 0.2% sorbate to trpticase soy broth (pH 6.0) inactivated Pseudomonas

'utrefaciens and resulted in a 3 log reduction in number of viable cells of the organism

through 6 days of incubation at 24°C. A 30 sec dip treatment in 5% (w/v) solution of

Dotassium sorbate increased the shelf life of fresh whole broilers upto 19 days whereas

he control sample could be stored well upto 10 days only (Robach, 1979b). Treatment

jf potassium sorbate at 0.5% alone did not continue to inhibit the gram negative bacterial

ounts beyond 6-9 days (Kim and Ilcarnsberger, 1994). Kemp ci' al. (1983) reported the

ffect of potassium sorbate and vacuum packaging on the quality and microflora of dry

ured intact and boneless hams. Sorbate did not affect the tenderness and saltiness.

;orbate did not inhibit the staphylococci completely but it delayed the growth of

'taphylococcus aureus.

Kolsarici and Candogan (1995) evaluated the effects of 5% potassium sorbate and

% lactic acid applications on total mesophilic aerobic bacteria, total psychrotrophic

14

aerobic bacteria, lactic acid bacteria, Staphylococci, coliform bacteria and pH values of

vacuum packed chicken leg and breast meats during storage at 4±1°C. A decrease in

bacterial counts of chicken leg and breast meats was observed in the periods following

the treatments of potassium sorbate and lactic acid however, towards the end of the

storage period, the effectiveness of potassium sorbate was greater than that of lactic acid.

With prepacked cod fillets, addition of 0.135 or 0.4% of potassium sorbate

inhibits almost completely the bacterial spoilage (Debevere and Voets, 1972). Bremmer

and Statham (1983) investigated the effect of potassium sorbate on refrigerated storage of

vacuum packed scallops. The sorbate treated scallops stored well upto 28 days at 4°C.

2.2.1.2. Sodium benzoate

Benzoates - sodium and potassium are most effective in controlling yeasts and

bacteria and least effective in controlling molds. The sodium salt of the acid, being more

water soluble than the acid, is generally used as the antimicrobial agent in a variety of

Foods. When incorporated into a product, it has the advantage of being soluble, odourless

and colourless. Available as a dense flake or as a granule, sodium benzoate is

;liaracterised by a sweetish, astringent taste and is soluble to the extent of lg in 2 ml

water or in 50 ml of 90% alcohol.

According to the U.S. Food and Drug Administration (USFDA), Sodium benzoate

s affirmed as Generally Recognized As Safe (GRAS) for use as an antimicrobial agent

md as a flavouring agent (21. Code of Federal Regulations, CFR, 184. 1733). In

indissociated form, benzoic acid is more active. Fish dipped in sodium benzoate solution

for 30 seconds to 2 minutes can extend the shelf life of fish fillets for several days (Miles

15

Labs. Inc., 1985a; 1985b). Recommended level of sodium benzoate is 0.15% to 0.35% in

dipping solution for treating fish (Miles Labs, Inc., 1985b). Codex Alimentarius

commission has recognised sodium benzoate as a preservative for use in food products

(Codex Alimentarius, 1995). Recommended level of sodium benzoate for application in

food products is 0.1% maximum (Miles Labs, Inc., 1 985a; Madhavi ci' al., 1995).

Benzoic acid (C6 1-1 5 COOH) is used as such or as its sodium salt. Benzoic acid

also shows a certain growth inhibitory capacity in the dissociated state. Growth inhibitory

action of benzoic acid on bacteria has been reported (Eklund, 1989). Minimum inhibitory

concentration (MIC) of benzoic acid against Pseudomonas sp was reported to be 200 -

480 ppm at pH 6; Micrococcus sp, 50 —100 ppm at pH 5.5 - 5.6; Streptococcus sp, 200-

400 ppm at 5.2 - 5.6 p1-I; Lactobacillus sp, 300 - 1 800 ppm at 4.3-6.0 pH; Escherichia

coli 50 - 120 ppm at 5.2 - 5.6 pH and Bacillus cereus, 500 ppm at 6.3 pH. Sodium

benzoate (0.2-0.3%) inhibited the growth of 12 strains of Acinetobacter (Saha and

Chopade, 2002).

Benzoate has been found to influence enzymes controlling acetic acid metabolism

and oxidative phosphorylation and also to intervene at various points in the tricarboxylic

acid cycle, especially where the dehydrogenate of a-ketoglutanic acid and succinic acid

ire involved. Influence on the enzyme 6-phosphofructo-2-kinase has been found by

(rebs ci al. (1983) and Francois etal. (1986). It was assumed that inhibition of growth is

lue to the elimination of the electrochemical gradient across the cell membrane by

indissociated benzoate passing through the membrane (Freese et al., 1973; Cramer and

rcstegard. 1977). In addition, it has been proposed that benzoic acid, like other fatty

16

acids, influences the membrane either by interfering with membrane protein (Sheu et al.,

1972) or by changing the membrane fluidity (Gomez and Herrero, 1983).

2.2.13. Propyl gat late

Propyl gallate is a recognised food additive (Codex Alimentarius, 1995) and it is

used in food products as an antioxidant as well as antimicrobial agent. Propyl gallate is

the most sensitive to heat and undergoes degradation at frying temperatures. It is used at

the level of 0.001% to 0.02% directly in food products (Madhavi et al., 1995). Zhuang et

al. (1996) has reported the effect of propyl gallate on the microbial population in fresh

shrimp and catfish fillets during refrigerated storage. They observed that the propyl

gallate improved the shelf life of the products by controlling the microbial growth.

Literature on the application of propyl gallate could not be traced much in fishery

products.

2.2.2. Natural preservatives

2.2.2.1. Chitosan

Chitin, a -(1,4)-D linked polymer of N-acetyl glucosamine, is a common

constituent of crustacean, arthropod and fungal cell walls (Allan et al., 1978: Udgata and

Khuntia, 1994; Roller and Covill, 2000). Chitin is a highly hydrophobic material that is

insoluble in water and most organic solvents (Udgata and Khuntia, 1994). Chitosan is a

derivative of chitin. Hydrolysis under drastic conditions with concentrated acids gives

relatively pure aminosugar, D-glucosamine. Deacetylation of chitin with strong alkali

yields the free base 2-amino 2-deoxy-D-glucosamine commonly known as chitosan,

(Madhavan and Nair. 1974; Pangburn etal., 1984).

Chitosan is insoluble in water but soluble in dilute acids forming the

corresponding salts (Pangbum et al., 1984; Gopakumar, 1997) The possible potential

sources of chitin are krill, shellfish, lobsters, fungi, squid and insects (Allan et al., 1978;

Dutkiewicz et al., 1988). Chitin is the second most abundant organic compound next to

cellulose on earth (MPEDA, 1998). However, at present, the principal sources of chitin

are shrimp and crab waste from fish processing plants. Very high quality chitin and

chitosan can be obtained from squid, cuttle fish and diatoms in smaller quantities

(Gopakumar, 1997; Shahidi, 1997). It has been estimated that chitin is synthesised in

nature at a level of up to lO - 1010 tonnes per year (Roller and Covill, 2000). Most

commercial chitosans have a degree of deactylation that is greater than 70% (Li et al.,

1997).

The shrimp shell waste contributes nearly 5 0-60% of the body weight of shrimp.

The availability of shrimp shell waste in India is estimated to be 75,000 - 80,000 metric

tonne annually and it is the single largest fishing waste in our country (MPEDA, 1998).

The world wide production of crustacean waste has been estimated at 1.44 million tonnes

per annum on dry weight basis (Hall and De Silva, 1992).

Chitin and chitosan have immense application in various fields such as food

industry, agriculture, waste water treatment, biomedicine, biotechnology, textile industry,

aper industry, cosmetic etc. (Knorr, 1984; Brezeski, 1987; Michihiro et al., 1998;

Shahidi, 1994). Balassa and Prudden (1978) found that chitosan has higher wound

healing accelerated activity than the standard acid - pepsin digested cartilage

preparations. They also found that chitosan prepared from lobster had higher wound

ii:

healing activity (+75%) than from shrimp (+30%) and that surgical cotton gauze coated

with regenerated chitin was substantially more active than uncoated control. It is

possible to flocculate E. co/i cell debris with chitosan as a flocculant. Flocculation cause

an effective separation as 98% of the cell debris within 30 minutes by sedimentation

under gravity. The flocculation also contributes to the purification of protein solution

(Agerkvist et al., 1 988).

Chitosan becomes adhesive, viscous at low pH and binds acid. The high acid

binding capacity of chitosan accounts for antiulcer property of thin substance in

prevention of ulcers and induced gastric damage (Konturek et al., 1981). The

polycationic nature of the chitosan has led to its application in various fields including

encapsulation (Hwang etal., 1985) and immobilisation of microbial cell and mammalian

cell (Lim, 1983; Kim and Rha, 1988). The biological functions of chitosan in the

reorganisation of damaged corneal tissues were reported (Biagini ci al., 1987; Muzzarelli

et al., 1988). The disappearance of vascularization and over inflammatory event with

time indicates that the chitosan is a biocompatible and biodegradable polymer, that can be

safely applied to healthy and integer tissues as well (Biagini ci al., 1988). The contact

lenses, made from partially depolymerised and purified squid pen chitosan by spin caston

echnology, are clear tough films and possess physical properties desirable for bandage

ontact lenses (Markery C! al., 1988).

Chitosan is an effective agent for coagulation of suspended solids in various food

rocessing waste. It is also effective for dewatering of activated sludge suspensions

:struszczyk et al., 1988). Chitosan can be used for complete removal of mercury salts

19

from water or from industrial effluents (Nair and Madhavan, 1984). Mathew and Nair

(1988) reported that chitin and partially hydrolysed chitin are effective

hypochiosterolemic agents.

Chitosan has attracted much research attention in the last 20 years as a potentially

important renewable resource that is both non-toxic and biodegradable. Much of the

interest in the antimicrobial properties of chitosan has focused on its possible role in plant

defense mechanisms. Relatively little work has been reported on the antagonistic

properties of chitosan against microorganisms important in foods. The sensitivity of nine

bacteria, including Salmonella typhimurium, to chitosan glutamate and chitosan lactate

(at 2 g/liter) in phosphate buffer (p1-I 5.8) at 32°C has been tested and inactivation of

between 1 and 5 logs within I h of exposure has been reported (Sudarshan et al., 1992).

Chitosan was similarly bactericidal against gram positive and gram negative organisms,

indicating non-specific biocidal action. Non-specific biocidal action of chitosan against

Escherichia coli, Staphylococcus aureus, Yersinia enterocolitica, Listeria nionocytogens

and Saccharomyces cerevisiae in bufferes and/or laboratory media has been reported

(Papineau et al., 1991; Wang, 1992).

Kurigsuwan et al. (1996) has attempted to study the preservative effect of chitosan

n fish products. They found that salted sepat siam fish (Trichogaster spp) dipped in

hitosan solution in 1% acetic acid extend the shelf life of the product and also the total

bacterial load was comparatively lower in chitosan treated fish than that of control

sample. Darmadji and Izurnimoto (1994) investigated the effect of chitosan on the

development of spoilage in mixed beef patties stored at 30°C for 2 days and at 4°C for 10

20

days. A reduction of I to 2 log cycles of total bacteria, pseudomonads, staphyhlococci,

coliforms, gram negative bacteria and micrococci was reported in the presence of 1%

chitosan. Lower concentrations of chitsoan (0.2% and 0.5%) had no effect on the spoilage

flora. However, the number of viable organisms present in the meat before the start of

experimentation was generally high (>l0 cfulg) and it is possible that chitosan addition

could have been more effective, had lower initial population been present.

Fresh strawberries and bell papers dipped in acidic chitosan soultions and

inoculated with Botrytis cinerea or Rhizopus stolonfer have been reported as equally

resistant to spoilage at 13°C as fruits treated with the conventional chemical fungicide

iprodione (El-Ghaouth et al., 1991; 1997). Chitosan is reportedly used as a preservative

in foods in Japan in products such as Kamaboko, noodles, soy sauce, chineese cabbage

and sardines (Li et al. 1997). Chitosan glutamate was an effective preservative against

spoilage yeasts in apple juice and that the antimicrobial activity was concentration,

temperature and pH dependent (Roller and Covill, 1999).

Fang et al., (1994) investigated the use of chitosan as an antimicrobial agent

against mold spoilage in candied kumquat and found that 6g/litter of chitosan was

-equired to maintain a mold free shelf life of 65 days at pH 4. Roller and Covill (2000)

as also studied the antimicrobial properties of chitosan in mayonnaise - based shrimp

;alads. They also observed in shrimp salads stored at 5°C. the presence of a coating of

thitosan (9 mg/g of shrimp) inhibited growth of the spoilage flora from approximately

og 8 cfu/g in the controls to log 4 cfulg throughout 4 weeks. However, at 25°C, chitosan

as ineffective as a preservative.

21

2.2.2.2. Spices

Spices are vital culinary addendums enhancing organoleptic characteristics of

food. In addition, they possess various preservative and antimicrobial properties and also

nutritional benefits which make them an inevitable food accessory. Spices have been

highly desirable products ever since the ancient civilizations of India. India is the spice

bowl of the world. India is the world's largest exporter of spices and has also emerged as

a major supplier of spice products such as ground spices, spice mixes, spice pastes and

curry powders (Subbulakshmi and Naik, 2002). India is reputed for the production of a

range of important spices like pepper, cardamom, ginger, turmeric, chillies, clove,

cinnamon, fenugreek, garlic, onion, coriander, cumin, saffron, etc. India produces

annually about two million tonnes of different spices. India contributes 25-30% to the

world production. Ginger and garlic produced in several states of India. India with a

35% share in world production, ranks first among the ginger producing countries. The

major markets for the Indian spices are Saudi Arabia, UAE, Kuwait and USA. The

export of spices from India during 1997-98 is 2187500 metric tonne to a value of US$

36362 million (Subbulakshmi and Naik, 2002). India's share in the world trade of spices

is 48.6% in volume and 24.26% in value over the last four decades (John, 1999).

The most important property shared by many spices is the antimicrobial property.

Synergistic inhibition by one or more combinations was evident against each microbe.

pices and its essential oils / oleoresins are one of' the components of the extremely

ffective naturally occurring antimicrobial systems. While naturalness alone is not

ecessarily a sufficient objective for Ibod preservation the use of natural inhibitors as

omponents of systems that can together enhance the effectiveness of preservation with

22

advantages in product quality and safety (Subbulakshmi and Naik, 2002). Lewis et al.

(1974) reported 10-12% of oleoresin yield in pepper, 12-16% in chilli, 5-7% in ginger

and 6-7% in turmeric. He has also reported the important principal in oleoresin: piperine

(40-45%) in pepper, capsaicin (2-3%) in chilli, gingerol (25-30%) in ginger, curcumin

(3 5%) in turmeric.

Menon et al. (2002) examined antibacterial property of cinnamon powder in meat

and cheese. It exhibited bacteriostatic action on Listeria monocytogenes in both the

foods. Food treated with 6% cinnamon showed 1-2 log less Listeria counts than in

control sample holding at 30°C for 7 days.

2.2.2.2.1. Garlic

Prasad and Seenayya (2000) have evaluated the effect of 20 spices including

garlic on the growth of red halophilic cocci isolated from salt cured fish and solar salt.

Excellent growth restriction was observed with garlic against halophilic isolates

S'alinococcus roseus, Halococcus turkmenicus and Halococcus morrhuae. Garlic is also

nown for its antifungal effect on candida albicans and other pathogenic fungi (Yamada

md Azunia, 1997). Various garlic preparations have been shown to exhibit a wide

;pectrum of antibacterial activity against Gram-negative and Gram-positive bacteria

ncluding species of Escherichia, Salmonella, Staphylococcus, Streptococcus, Klebsiel!a,

roteus, Bacillus and Clostridium (Ankri and Mirelman. 1999). Even acid fast bacteria

uch as Mycohacteriurn tuherclosis are sensitive to garlic (Uchida et al. 1975).

Cavillito and Bailey (1944) were the first to demonstrate that antibacterial action

)f garlic is due to allicin. Sreenivasamurthy (1974) studied the garlic for its antimicrobial

23

role and identified allicin as the active compound. Mucoid strains of Pseudomonas

aeruginosa and Enterococcus faecium were found to be resistant to the action of allicin.

Garlic extracts also have a strong antifungal effect and inhibit the formation of

mycotoxins like the aflatoxin of Aspergillus parasiticus (Lawson, 1996). Inhibition of

certain thiol-containing enzymes in the microorganisms by the rapid reaction of

thiosulfinates with thiol groups was assumed to be the main mechanism involved in the

anitimicrobial effect (Cavallito and Bailey, 1944).

2.2.2.2.2. Ginger

Antibacterial activity of ginger against red halophilic bacteria was investigated by

Prasad and Seenayya (2000). They reported that ginger exhibits very good inhibitory

effect on halophiles such as Salinococcus roseus, J-Ialococcus iurkmenicus and

1-lalococcus ,norrhuae. Effect of spices including ginger on growth and survival of

Escheric/zia coli 0157 and Salmonella enterica serovar in broth model system and

nayonnaise was investigated and was found that compared to garlic and clove, ginger

;howed less bacteriostatic activity and E.coli was found more sensitive than S. enterica

:Leuschner and Zamparini, 2002).

Mendiratta ci al. (2000) evaluated tenderizing and antioxidant effect of ginger

xtract on sheep meat. The study indicated that ginger extract at the level of 3% could be

ffectively used for improving the sensory and keeping qualities of mutton chunks.

avecna ci al. (2001) investigated the effect of incorporation of ginger extract in curing

;olution on the microbial and organoleptic quality of smoked spent hen meat. They

)bserved that ginger extract treated sample had lower bacterial count than the control

;ample which had contributed for extension of shelf-life.

24

2.3. Bacteriological quality

23.1. Bacterial characteristics of live fish

Microorganisms are found in skin, gills and intestine of live and newly caught

fish. Normally on skin surface microbial load ranges from 102_ 107 cfulcm 2 and the gills

and intestine contain 103 T 109 cfu / g (Shewan, 1977). The bacterial flora on newly

caught fish depends on the environment in which it is caught rather than on the fish

species (Shewan, 1977). Psychrotrops or psychrophiles are usually dominant in

temperate fish. In tropical fish, higher numbers of mesophiles can be isolated. The

microflora on temperate fish is dominated by psychrotrphic Gram - negative rod shaped

bacteria belonging to the genera Pseudomonas, Moraella, Shewanella, Acinetobacter,

and Flavohacterium. Gram-postivie organisms such as Bacillus, Micrococcus,

Clostridiuni, Lactobacillus and coryneforms are also found in different proportions

Mortia, 1975). Shewan (1977) reported that Gram - positive Bacillus and Micrococcus

1ominate on fish from tropical waters. Surendran et al. (1989) have reported the

rnicrofiora consisting of Pseudomonas, Acinetobactei; Moraxella and Vibrio in freshly

aught fish from Indian marine waters. Liston (1980) stated that microflora on tropical

fish often carry a slightly higher load of Gram-positives and enteric bacteria but

Dtherwise similar to the flora on temperate fish.

1.3.2. Changes in the microflora during chill storage

Freshly caught fish may not be immediately subjected to further processing like

Freezing, canning, curing, refrigeration or for products developments due to time factor.

T'herefore, until processing, the fishes are stored in iced condition in order to reduce the

rate of proliferation of microbes. During ice storage of temperate fish. the bacteria will

25

grow with doubling time of approximately 1 day and after 2-3 weeks, it will reach 108

i09 cfulg or cm skin. The bacteria on fish caught in tropical waters will often pass

through a lag-phase of 1-2 weeks if the fish are stored in ice, whereafter exponential

growth begins. At spoilage, the bacterial level on tropical fish is similar to the levels on

temperate fish species (Gram et al., 1990). Under aerobic iced storage, the flora is

composed almost exclusively of Pseudomonas spp and S. putrefaciens after 1-2 weeks.

(Mortia,1975). Photobacterium phosphoreum which can be isolated from the surface

can also be isolated in high numbers from intestinal tract of some fish species (Dalgaard

et al., 1993). Shewanella putrefaciens has been identified as the specific spoilage

bacteria of marine temperate marine fish stored aerobically in ice. If the product is

vacuum packed, P. phosphoreum is prominent in the spoilage. In ice stored tropical

freshwater fish, Pseudomonas spp are the specific spoilers (Gram ci al., 1990)

2.3.3. Quality characteristics of vacuum packed meat

2.3.3.1. Trimetylamine (TMA) and Total volatile base nitrogen (TVBN)

The Nitrogen containing extractives can be defined as the water soluble, low

molecular weight, nitrogen containing compounds of non-protein nature. This Non-

protein nitrogen (NPN) fraction constitutes from 9-18% of the total nitrogen in teleosts.

The major components in this fraction are volatile bases such as ammonia and

trimethylamine oxide (TMAO), creatine, free aminoacids, nucleotides and purine bases in

teleost fishes (Shewan, 1974). TMAO constitutes a characteristic and important part of

the NPN - fraction in marine species. The component is found in all marine fish species

in quantities from 1-5% of the muscle tissue (dry weight) but is virtually absent from

freshwater species and from terrestrial organisms (Anderson and Fellers. 1952; Hebard ci

26

al., 1982). However, Gram et al. (1989) reported that Nile perch and tilapia from Lake

Victoria contain as much as 150-200 mg TMAO / 100g of fresh fish. Stroem et al.

(1979) have shown that TMAO is formed by biosynthesis in certain zooplankton species.

TMAO is reduced to TMA which is one of the dominant components of spoling fish, has

a typical fishy odour. The level of TMA found in fresh fish rejected by sensory panels

varies between fish species, but is typical around 10-15mg TMA-N/lOOg in aerobically

stored fish and at a level of 30 mg TMA-N/lOOg in packed Cod (Dalgaard etal., 1993).

The TMAO reduction is mainly associated with genera of bacteria typical of the marine

nvironment (Alteromonas, Photohacteriurn, Vibrio and Shewanella putrefaciens), but it

ilso carried out by Aeromonas and intestinal bacteria of the Enterbacteriaceae

Sakaguchi etal., 1980; Ringo et al., 1984; Huss, 1995). Gram etal. (1990) reported that

FMA is not necessarily a characteristic component during spoilage of such fish because

;poilage is due to Pseudomonas spp. The formation of TMA is accompanied by a

ormation of ammonia during anaerobic storage of fish resulting in vigorous production

)f NH 3 owing to further degradation of the amino acids. The very strong NH 3 producers

vere found to be obligate anaerobes belonging to the family Bacteroidaceae, genus

usobacterium (Storroe et al., 1975, 1977).

TMA formation increases considerably under the influence of packing. It is

ssumed that the inhibition of trimethyl amine oxide reducing bacteria increases the

eeping quality of the fish to a large extent (Debevere and Voets, 1972). Various TMA

vels from 5 to> 26 mg/1 OOg of fish flesh have been reported for different spoiled fish

pecies (Castell etal., 1958; Sengupta and Mitra, 1972).

27

Debevere and Voets (1972) reported that TMA formation was completely

inhibited in the presence of 0.4% potassium sorbate in prepacked cod fillets. TMA is a

product of spoilage and its content is often used as an index to assess the keeping quality

and shelf life of seafood products (Hebard el al., 1982). Meekin et al. (1982) reported

that TMA-N content of vacuum packed untreated sand flathead fish was found to be

above 30 mg/100g after 14 days storage at 4°C. Dalgaard et al. (1993) reported that

TMA concentration at spoilage was approximately 30 mg/100g in vacuum and modified

atmosphere stored cod fillets.

2.3.3.2. Free fatty acids (FFA)

The two distinct reactions in fish lipids of importance for quality deterioration are

oxidation and hydrolysis. They result in the production of a range of substances among

which some have unpleasant rancid taste and smell, and some may also contribute to

texture changes by binding covalently to fish muscle proteins. Lipid autolysis, a

enzymatic hydrolysis, result in the formation of free fatty acids (Huss, 1988). The various

'eactions are either non-enzymatic or catalysed by microbial enzymes or by intracellular

r digestive enzymes from the fish themselves (Huss, 1995). During storage, a

onsiderable amount of free fatty acids (FFA) appears. The phenomenon is more

rofound in ungutted fish than in gutted fish probably because of the involvement of

ligestive enzymes. In lean fish also, for example Atlantic cod, free fatty acids are

)roduced, even at low temperatures. The enzymes responsible are belived to be cellular

thospholipases (Huss, 1995). The fatty acids themselves may cause a soapy off flavour.

28

Vacuum packaging has been found to substantially reduce oxidative deterioration

in frozen fish and fishery products (Yu etal., 1973; Lindsay, 1977). Huang etal. (1991)

reported that although no effect on lipid oxidation was found, vacuum packaging did

show the cat fish lipid hydrolysis. There was no significant difference on free fatty acid

content of cat fish packed in different methods of polyvinyldene chloride film over

wrapping, vacuum packaging with ethylene vinyl acetate bag and vacuum skin packaging

(Haung et al., 1992). Fresh cat fish stored either on ice or refrigeration showed little lipid

degradation during the expected market shelf life. FFA content remained low in cat fish

fillets at the end of the 13 days of storage at 4°C (Haung et al., 1994).

2.3.4. Sensory evaluation

The methods for evaluation of fresh fish quality may be conveniently divided into

two catagorics: sensory and instrumental. Since the consumer is the ultimate judge of

quality, most chemical or instrumental methods must be correlated with sensory

valuation, before being used in the laboratory. However, sensory methods must be

)erformed scientifically under carefully controlled conditions so that the effects of test

nvironment, personal bias, etc., may be reduced. The ultimate goal of sensory

lvaluation of seafood is to assess its acceptance for consumption. The actual consumer

ppeal can be reflected only in sensory analysis.

Sensory evaluation is defined as the scientific discipline used to evoke, measure,

nalyse and interpret reactions to characteristics of food as perceived through the senses

if sight, smell, taste and touch (Huss, 1995). Sensory methods are used for measuring

he properties that can not be evaluated directly by physical or chemical tests.

29

Many schemes have been developed for sensory analysis of raw fish. The first

modern and detailed method was developed by Torry Research station (Shewar et al.,

1953). They designed a scale for sensory quality of fresh fish having a fundamental idea

that each quality parameter is independent of other parameters. Later, the assessment

was modified by collecting a group of characteristic features to be expressed in a score.

The success of sensory testing depends on the proper identification of panel members,

appropriate training for them, selecting the appropriate test procedure depending on the

Droduct to be formulated and deriving reliable conclusions (Joseph, 2003b). In sensory

analysis appearance, odour, flavour and texture are evaluated using the human senses.

cientifically, the process can be divided into three steps, namely detection of a stimulus

y the human sense organs; evaluation and interpretation by a mental process; and the

esponse of the assessor to the stimuli (Huss, 1995). Howgate etal. (1992) suggested a

;cheme for assessing the freshness of fishery products where special schemes for white

ish, dog fish, herring and mackerel were developed. This scheme has been widely used

n the European Union. A new method, the quality Index method (QIM) developed by

he Tosmanian food research unit (Bremmer etal., 1987) is still used for fresh and frozen

od, herring and saithe.

QIM is based on the significant sensory parameters for raw fish when using many

arameters and a score system from 0 to 4 demerit points (Jonsdottir, 1992). QIM uses a

ractical rating system, in which the fish is inspected and the fitting demerit point is

ecorded. Descriptive test, a method of sensory evaluation, can be used for quality

etermination and shelf life studies applying a structure scaling method (Meilgaard et

30

cii., 1991). Structure scaling gives the panelist an actual scale showing several degrees of

intensity

Fey and Regenstein (1982) reported the freshness qualities of red hake stored in

ice. Regenstein (1982) reported freshness qualities for iced cod fish. Bremmer and

Statham (1983) reported changes in odour of raw scallops which were either air, vacuum

packed or treated with potassium sorbate. Kim and Hearnsberger (1994) evaluated the

:hanges in flavour, odour and appearance of refrigerated cat fish fillets treated with

:ombination of food preservatives and/or lactic acid bacterial culture. Kim et al. (1 995a)

evaluated the sensory characteristics of catfish fillets treated with sodium acetate and

nonopotassium phosphate and found that the fillets were sensorily accepted until twelve

Jays. Fruity, rotten, sulphdryl odours and flavours are typical of the Pseudomonas

;poilage of iced fish. Pseudornonas produce a number of volatile aldehydes, ketones,

sters and sulphides (Miller et al., 1973a, 197' b; Edwards et al., 1987).

31