Effect of Preservation Methods on Giant Freshwater Prawn

ACKNOWLEDGMENT

All appreciation to Almighty Allah who creates us such a beautiful earth, manages each and every things soundly. From the core of my heart, I would like to reveal my earnest awe, loyalty and reverence to him who has enabled me to complete the thesis work in spite of numerous tribulations.

I wish to express my deep gratitude and sincere appreciation to my honorable teachers Dr. Md. Abdus Salam, Associate Professor, Department of Zoology, Jahangirnagar University, Dr. Md. Khorshed Alam, Senior Scientific Officer, IFRB, AERE and Mr. Md. Kamrujjaman, Lecturer, Department of Zoology, Jahangirnagar University for their scholastic guidance, valuable suggestion and steady help throughout the thesis work.

I am grateful to Dr. Tahmina Afroz, Chairman Professor, Department of Zoology, Jahangirnagar University and Dr. Md. Mosharrof Hossain, Director Chief Scientific Officer, Institute of Food Radiation Biology for their kind permission and continuous support during thesis work.

I offer my special thanks to Dr. Abdul Jabbar Hawlader, Professor, Dr. Md. Mofizul Kabir, Associate Professor and Mr. Md. Mansurul Haq, Lecturer, Department of Zoology, Jahangirnagar University for their encouragement. I also acknowledge my best regard to all of my honorable and respected teachers of Department of Zoology, Jahangirnagar University, Savar, Dhaka for their academic collaboration and blessing to well wishes.

I am thankful to M. Samsul Islam, Principal Scientific Officer, Food Technology Division, IFRB, AERE; Dr. Rehena Begum, Chief Scientific Officer, Microbiology Industrial Irradiation Division, IFRB, AERE for their precious implication along the thesis work.

My cordial gratitude to Md. Afzal Hossain, Junior Experimental Officer, Food Technology Division; Shah Md. Safiqul Islam, Senior Scientific Assistant, Industrial Irradiation Division; Md. Faejur Rahaman, Scientific Assistant and Dilip Kumer Mohontho, Scientific Assistant of Food Technology Division, IFRB for their cordial help during conducting this research work.

My gratitude to Debabrata Chowdhury, Chief Photographer, BLRI; Md. Anisur Rahman, Senior Photographer, Jahangirnagar University and Md. Abdur Rob Sharif, Museum Assistant, Department of Zoology, Jahangirnagar University for their support.

I owe much to my parents and elder brothers due to their financial support as well as the well wishes of them. Very special thanks to S. Zaman for her good wishes and helping hand in my thesis work. All of their helps, inspiration, and encouragement have facilitated me to reach at this stage of education.

August, 2004

ABSTRACT

In order to inquest a pertinent technology of preservation the current concerned. Seasonal (summer, autumn, winter) nutritive values of edible-part and head-on; effects of potassium sorbate - 2%, gamma radiation - 2kGy and their combinations preserved at low temperature (00C, 40C) on sensory, chemical and microbial properties of freshwater prawn (Macrobrachium rosenbergii, de Man-1879) as well as isolation and identification of associated micro-flora with their sensitivity to potassium sorbate and gamma radiation were investigated during February 2003 to April 2004. On nutritive analysis marked seasonal as well as body-part variations in moisture, protein, lipid, ash, calcium and phosphorus contents i.e., summer, autumn and winter as 80.07%, 80.37%, 76.99%; 19.20%, 18.03%, 21.30%; 1.23%, 1.07%, 2.99%; 1.48%, 2.00%, 1.21%; 0.19%, 0.21%, 0.14% and 0.10%, 0.08% 0.10% in edible-part while 77.27%, 76.27%, 73.34%; 16.45%, 17.03%, 19.79%; 1.40%, 1.02%, 4.33%; 6.60%, 7.33%, 6.25%; 2.24%, 2.78%, 1.67% and 0.17%, 0.15% 0.21% in head-on respectively.

Quality appraisal of treated (potassium sorbate-2%, gamma radiation-2kGy and their combination) and preserved (00C and 40C) samples were done by organoleptic (colour, odour and texture), chemical (total volatile basic nitrogen and trimethylamine nitrogen) and microbial (total bacterial count and total mould count) evaluation at an interval of 7 days. The shelf life of freshwater prawn at 40C was 14 - 21 days which was extended to 21 - 28 days at 00C. Maximum shelf-life (28 days) was found in combined treated freshwater prawn that was stored at 00C.

Associated micro-flora were isolated and identified on the basis of their cultural, microscopic, biochemical physiological characteristics. Among thirty bacterial strains - thirteen (40.33%) were collected from control, nine (30%) from chemical treated, five (16.67%) from radiation treated and three (10%) from combined treated samples. Total nine bacterial species were identified in which Staphylococcus aureus (23.33%), Bacillus subtilis (20.33%), Micrococcus varians (16.67%) and Escherichia coli (13.33%) were frequent species. Among ten mould strains - seven (70.00%) were collected from control, three (30.00%) from radiation treated while no colonies were found from chemical and combined treated samples. Total four genus of moulds were identified in which Aspergillus (40.00%) was dominant. Potassium sorbate (0, 1, 2, 3, 4 5 %) and gamma radiation (0, 2, 4, 6, 8 10 kGy) were applied for the sensitivity on identified major bacterial species. Staphylococcus aureus, Bacillus subtilis, Micrococcus varians Escherichia coli were completely eliminated at 4, 5, 3 4 % potassium sorbate and 6, 8, 4 6 kGy gamma radiation doses.

Key words: nutritive values, low temperature, potassium sorbate, gamma radiation, freshwater prawn, Macrobrachium rosenbergii, bacterial-flora, mould-flora

ABBREVIATIONS

Abbildung in dieser Leseprobe nicht enthalten

1.1 General introduction

As a riverine deltaic country Bangladesh with its warm tropical climate is supremely located and offers an enormous reserve of fresh water, brackish water and marine water fishes as well as the prawns and shrimps. Bangladesh has 0.33 million hectors of inland water and 480 km coastline. During 2000-01 fiscal year about 1.61 million metric ton of fish including prawns and shrimps were produced from different sources (Choudhury, 2001). Bangladesh earns a lot of foreign currency in every year through the export of fish and fishery products that contribute about 5.14% of total export earnings (BBS, 2002) from which 85% of this earning coming from prawns and shrimps (Choudhury, 2001). Bangladesh is exporting frozen shrimps to about 30 countries of the world of which USA, Japan, Belgium, UK, Netherlands and Germany are the most foremost buying countries. Bangladesh offers scientific process and unique combination of natural resources and export friendly infrastructure, thereby making country’s fisheries items genuinely popular all over the world, due to their original taste of the products.

Bangladesh is one of richest country in the world in fresh water fish diversity (Shafi and Quddus, 1982). Fisheries sector plays an imperative role in the economic maturity of Bangladesh contributing about 5.5% of the countries GDP during 2000 - 01 fiscal years (Choudhury, 2001). This sector also acts an important role in rural employment and poverty alleviation. About 8% people of our country are directly or indirectly dependent on fish and fisheries (Islam, 1995). Fish and fishery products have got their supremacy to human consumption for its excellent protein source. About 63% animal proteins come from fish alone (Choudhury, 2001). Supply of fish was 32.8 g out of 57 g protein per person per day in our country (Ali, 1970). It has reported that the daily intake of our protein per person in Bangladesh is around 7-9 g which is 32% of the normal commitment for an adult which indicate there is massive protein deficiency (Haque, 1974).

Fish spoilage often causes sober problem in our economy. About 8% of the catch equivalent to 4.25 million tons never reached the market and is wasted (James, 1982). Due to bacterial, enzymatic and biochemical spoilage a great deal of fishery products are loss in every year, that effects on our national economy and adequate nutritional supply. So it is essential to investigate proper preservation method. Fish and fishery product is the cheapest and commonest source of animal protein for the diet in Bangladesh. So, it needs a careful handling and processing after its harvesting to consumption. All the prerequisites like proper handling, techniques of preservation are essential because fishery products are extremely perishable foodstuff easily damaged of all fresh food.

The country’s domestic marketing infrastructure facilities - fish landing, handling, preservation, distribution, transportation and quality control has by for lagged behind as compared to other developing countries of Asia (Khan et al., 1994). It is unfortunate that Bangladesh faced with food scarcity and technologically underdeveloped. So, investigation of proper preservation technology is crucial.

1.2 Fundamentals of fish biochemistry

Biochemical composition of fish or prawn and shrimp shows very wide variations from one species to another, within the same species, in different portions of the body in the same organisms from season to season, according to age, size and growth etc.

The important constituents of the fish and prawn/shrimp muscle in their order of magnitude are moisture, protein, fat, minerals. Fish and fishery products are main source of animal protein with all essential dietary amino acids. Except these, many other micronutrient substances like minerals and vitamins are present in fish chemical composition of fish are affected by numerous factors either of intrinsic in nature or environmental.

Flesh from healthy fish contains 60-84% water, 15-24% protein and 0.1-2% fat - these proportions of the constituents are species specific. The protein content of shellfish is lower than that of finfish. Thus, fish is important in the diet of the developing world. Within developing countries there is a recognizable trend for the poor to spend proportionally more of their household expenditure for animal protein on fish rather than on other meat products (James, 1984). The demand of fishes for human consumption depends on the type of the species and it varies from species to species, as because different fishes have different nutrient and taste value. So, identification is necessary before the selection of a species for consumption, because all fish are not edible.

1.3 CONTAMINATION OF FISH AND FISH PRODUCTS

Flora of living fish depends on the microbial contents of waters in which they live.

The slime of fish has been found to contain the bacterial genera Pseudomonas, Acinetobacter, Moraxella, Alcaligenes, Micrococcus, Sareina, Serratia, Vibrio, Flavibacterium and Bacillus; in the intestine of fish from both sources are found bacteria of the genera Alcaligenes, Pseudomonas, Flavibacterium, Vibrio, Bacillus, Clostridium and Escherichia (Frazier and Westhoff, 1988). In live fish, the muscle is sterile, i.e. free from any bacteria (Shewan, 1976). But its surface slime, intestinal tracts and gills harbor are the host of bacterial strains, such as Achromobacter, Pseudomonas, Vibrio, Bacillus, Aeromonas, Serratia etc. in marine fishes. In freshwater fishes, besides most of the above genera Lactobacillus, Brevibacterium, Alcaligenes, Streptococcus are generally met with (Jay, 1977).

Shrimps, crabs, lobsters and similar seafood have a bacteria-laden slime on their surfaces that probably resembles that of fish. Species of Bacillus, Micrococcus, Pseudomonas, Acinetobacter, Moraxella, Flavibacterium, Alcaligenes and Proteus have been found (Frazier and Westhoff, 1988). However, the following genera of moulds such as Aspergillus, Oospora, Penicillium, Wallemia, Seopulariosis, Sporendonema (Jay, 1977) which are responsible to spoilage also found on the fish and fish products.

1.4 SPOILAGE OF FISH AND FISHERY PRODUCTS

Country has an abundance of fisheries resources but lack of technological uses of fishes due to unavailability of suitable preservation techniques also contributes to fish spoilage (Hussain et al., 1994). Spoilage is the result of a series of complicated postmortem changes in fish mainly by enzymes and bacteria (Dyer et al., 1946). Fish and fishery products may be spoiled by autolysis, oxidation or bacterial activity or by combination of these. The kind and rate of spoilage of fish vary with a number of factors:

-Kinds of fishes: Fish spoiling depends on their kinds of fish. Some flat fish spoil more rapidly than round fish because they pass through rigor mortis more rapidly.

-Condition of the fish when caught: Fish that are exhausted as the result of struggling, lack of oxygen and excessive handling spoiled more rapidly.

-Kind and extent of contaminations: These may come from mud, water, handlers and the exterior slime and intestinal content of the fish.

-Temperature: The warmer the temperature, the shorter the storage life of the fish. Prompt and rapid freezing of the fish is still more effective in its preservation.

Spoilage of fish means being the fish unconsumed to human. Handling, preservation and transportation are considered the actual problem of fish spoilage. There are three types of spoilage as follows:

Enzymatic Spoilage

The natural enzymes as well as those produced by bacteria attack the muscle once the fish is dead; breaking down the proteins into simpler compounds as ammonia (NH3). These changes in the death tissues affect odour (flavour), texture (stiffness) and sometimes the appearance (colour) of the fish.

-Colour: Some of the discolorations commonly found in frozen fish are probably attributable to autolytic action, in that sugars produced by enzymatic action can interact with amino-compounds.

-Flavour: The breakdown of inosinic acid through autolysis results in a loss of this sweet, meaty flavor of fresh fish (Bremner and Statham, 1983). Hypoxanthene is also produced from the breakdown of inosinic acid, contributes to the bitter flavor.

-Texture: Rigor is of great importance in fish processing, particularly in freezing operations with very fresh fish, i.e. freezing at sea. In rigor, the fish can stiffen into distorted shapes and they can be difficult to load between freezer plates.

Chemical Spoilage

Chemical or biochemical spoilage consists of oxidation of the fat in the case of fatty fishes by atmospheric oxygen, resulting in rancidity, melanosis in shell fishes and hydrolytic changes in fats and proteins. Some representative food spoilage process and their products are:

Products

Amino acids, amines, ammonia, H2S etc Acids, alcohols and carbon dioxide Fatty acids and glycerol

Microbial Spoilage

When the fish dies, bacteria present on the surface and in the guts multiply rapidly and invade the flesh, which provides an ideal medium for growth and multiplication (Govindan, 1985). The increase in number of bacteria results in heavy slime on the skin and gills and an unpleasant ammonia-cal, sour odor; eventually they cause the

flesh to soften. The bacteria can breakdown the muscle itself and will also feed on the smaller units produced by autolytic action (Jay, 1986).

Changes in microbial population are a traditional quality index of fresh fish (Martin et al., 1978). Spoiled fish carries bacteria or their toxins or both. The species of the pathogen, the state of their activity and the quantity of their toxin present in the fish- food determine the nature of food poisoning in the patient. Following three common types of fish poisoning:

-Staphylococcus poisoning: These bacteria enter fish after capture by way of contamination during handling and processing. These bacteria multiply and produce toxin above 60C which are heat resistant.
-Clostridium poisoning: These heat resistant bacteria causing botulisms that produce a protein like toxin and acts like enzyme. It causes immediate paralysis of muscles involved in breathing, leading to instantaneous death (Srivastava, 1985).
-Salmonella and Shigella poisoning: These bacteria may attack from polluted waters in freshwater fishes while contamination during handling on ship or on shore in marine fishes.

1.5 Fundamentals of fish preservation

Preservation means keeping the fish after it has landed, in a condition wholesome and fit for human consumption. To prevent spoilage of fish, some form of preservation is necessary. Followings are intended to fish preservation:

-To prevent rigor-mortis (microbial, biochemical and enzymatic spoilage).
-To maintaining a steady supply of fish in the market throughout the year.
-To keeping a control over the hike and fall in prices by brawny supply.

Several spoilage indicators have commonly been used to assess the quality of stored fish products. Some widespread principles of fish preservation are cleaning, lowering temperature, raising temperature, dehydration, salting, fish preservatives and irradiation.

Preservation by Low Temperatures

Preservation by low temperature is very common and easy method of fish preservation that worn to retard chemical reactions, enzyme action and slow down growth and activity of microorganisms in food. The lower the temperature, the slower will be chemical reactions, enzyme action and microbial growth; a low temperature will prevent the growth of any microorganism. (Frazier and Westhoff, 1988). Many terms used in connection with low-temperature storage are applied, e.g., the term “cellar storage” refer to the use of temperatures is lower than 15 0C; the term “cold storage” refer to the use of temperatures is above or below 00C and the term “frozen storage” refer to the use of temperatures are at or below -180C.

A cooler temperature has a diverse effect on the various organisms present. The decrease of temperature would stop growth of most organisms and toward 40C or 00C, the fewer organisms that can grow and the slower will be their multiplication (Frazier and Westhoff, 1985). The increase in storage period, the total bacterial count increased for both 00C and 40C temperature but slowly (Karim et al., 1988). The main organisms of concern on refrigerated foods are the psychrophiles. These organisms can grow at temperature as low as -15 0C and some reportedly have an optimum growth temperature as low as 10 0C. A reduction in temperature from 30C to 00C doubles the storage life and a reduction from 100C to 00C increase the storage life 5-6 folds (Banwart, 1979).

Preservation by Chemical Additives

Chemical additive represents an important means of preserving food that kills the microorganisms or inhibiting their growth and activity. Addition of chemical additives (preservatives, antioxidants, colourants etc.) has frequently been associated with certain health problems, including allergies, and other more serious illness such as the initiation of carcinogenesis (Halliwell et al., 1995). So, to avoid such problems and to increase the shelf life of fish, the effect of carbon dioxide (Statham, 1984), potassium-sorbate (Ahmed et al., 1988 and Shaw et al., 1983) have already been studied.

Potassium-sorbate is the salt of sorbic acid, a naturally occurring organic acid that has been used extensively as an antimicrobial agent for foods that ionizes to form sorbic acid and is effective against bacteria, mould yeast (Frazier and Westhoff, 1985). It is widely used 250-1000 ppm levels in fish, cheeses and sour etc. as preservative. In many cases, sorbate and sodium benzoate are used together to provide greater protection against a wider variety of microbes. The organoleptic and hypoxanthine test results show that the treatment of potassium sorbate can slow the processes of spoilage by about 5 days (Gelman et al., 2001). In developed countries preservation of fish is widely practiced but unfortunately chemical preservatives for fish preservation are not practiced in Bangladesh. In Bangladesh, there are only two radiation sources, which are sited in Dhaka and Chittagong that’s why the chemical preservation is essential.

Preservation by Radiation

In general, radiation can be considered to be a form of energy (Mourthy, 1983). Radiation for decontamination are non-ionizing and ionizing (Banker, 1983). Non­ionizing radiation such as ultraviolet radiation is bactericidal between the wavelengths of 290 to 210 nm; 253.7 nm is most effective. Ionizing radiation such as gamma rays wavelengths is less than 2 angstrom emitted from the excited nucleus of 60Co or electrons emitted from a hot cathode.

Many terms used in connection with irradiation are applied, e.g., radappertization is a term used to define “radiation sterilization” which would implies high-dose treatments, with the resulting product shelf-stable; radurization refers to “radiation pasteurization” low-dose treatments, where the intent is to extend a products shelf-life and radicidation also is a low-dose “radiation pasteurization” treatment, but with the specific intent being the elimination of a particular pathogen (Board, 1983).

Regulation of food irradiation

The researchers at MIT have done pioneering work in radiation sterilization of food (Proctor et al., 1950) that x-rays are suitable for the sterilization of dry food materials. More penetrating radiation such as gamma rays can feasibly be applied to bulky materials or larger packages (Farkas, 1981). Over past 40 years, several national food control authorities have extensively studied this food process under a variety of condition and found it to be safe and effective. Now worldwide, some 170 industrial 60Co irradiators and 100 of electron accelerators have been processing variety of goods including industrial, medical and food. In USA Food Drug Administration (FDA), United States Department of Agriculture (USDA), Department of Defense (DOD) and National Aeronautics Space Administrations (NASA) among governmental organizations have been studied the irradiation process to determine possible risks to public safety (IFGFI-IFIC, 2002).

With the likely acceptance of irradiation for use in ready-to-eat foods, irradiation technology will continue grow interest with manufacturers; and with the increased focus on security and safety for the food supply, new technologies, such as irradiation for food pathogens and pests, will be at the fore font (Messick, 2002).

Mechanism of action of radiations

The gamma rays (ionizing radiation) ionize the path in the irradiated substance. This kind of activity excites chemical group in DNA, causing production of highly reactive short-lived chemical radicals that may alter chemical group in DNA, breaks DNA strands causing mutation and with high dose cause death of cell.

Ionizing radiation or energy exerts its lethal effects on bacteria mainly through direct action. With the progress of radiological research, it became obvious that the direct action is not the sole mechanism in the destruction of microorganisms by ionizing radiation. Inactivation of bacterial cells or loss of their capacity for reproduction is due to the energy deposition in the critical cell components (Alper, 1978)

Sources of gamma radiation

Gamma radiations are emitted during the de-excitation of a nucleus (Mourthy, 1983). The gamma ray originates from the nucleus itself. The chief sources of these rays are radioactive fission products, the coolant circulated in nuclear reactors and some fuel elements. A number of radioactive isotopes naturally or artificially emit γ-rays during their decay process. Cobalt-60 (in present study) and Cesium-137 have been used as a source of these rays.

Cobalt-60 decays to the nickel-60, which is a stable isotope, with the emission a ß- particle and two γ-rays of 1.17 meV and 1.33 meV in cascade. The half-life of Cobalt- 60 is 5.26 years. Cobalt-60 is produced by the neutron bombardment of 59Co in a nuclear reactor.

Abbildung in dieser Leseprobe nicht enthalten

In their search for new, improved methods of food preservation, investigators have paid special attention to the possible utilization of radiations of various frequencies, ranging from low-frequency electrical current to high-frequency gamma rays. Much of this work has focused on the use of ionizing radiation.

Advantages of food irradiation

Food irradiation ensures better public health and offers greater economic advantage to the producer. Radiation process is of importance in developing countries, which can achieve a reduction in post-harvest spoilage of agricultural and other commodities. A number of countries such as Brazil, Bangladesh, Ghana, India, Indonesia, Korea, Pakistan, and the Philippines are known to be carrying out extensive programs of research and development in food irradiation (Krishnamurthy, 1986).

The most significant public health benefit of food irradiation is that it stops the spread of food borne disease. It greatly reduces or eliminates the number of disease causing
bacteria and other harmful organisms including Salmonella, Escherichia, Staphylococcus, Listeria, Camphylobacter and Toxoplasma have caused many outbreaks of food borne illness. Perhaps one of the potent arguments for the irradiation of food is that there is no alternative to eliminate E. coli bacteria in raw foods (Tiernan, 2000). Irradiation is the only method to destroy Salmonella under cold conditions (Diehl, 1972).

An added advantage is that food can be irradiated in its final packaging - fresh or frozen, which prevents the possibility of contamination in the distribution system, at the store, or even in the home, prior to the package being opened (ICGFI-IFIC, 2002). Irradiating food does not appreciably warm the food, but still kills bacteria, fungi and insect (Diehl, 1972). Therefore, it is essential that irradiation be used in conjunction with all other established safe food handling and good manufacturing practices (ICGFI-IFIC, 2002).

Consumer reaction to food irradiation

Consumer also indicated that, for irradiated foods, safety and taste were more important than extended shelf life. Market test conducted in the past decade, have indicated that consumers were willing to purchase irradiated foods when they understood the benefits. Consumers learned to accept quickly the safety benefits, which irradiated food, brought to them.

Some segments of the population such as astronauts, hospital and immune compromised individuals have been taking advantage of the safety benefits of irradiated foods to protect them from potential food borne disease. As the conscious of consumer’s irradiation is unable to maintaining the quality of food, nearly all vitamins are destroying by the influence of radiation and enzymes are also destroyed.

The interest in food irradiation is fast growing both in developed and developing countries. Now, more than 70 countries are engaged in research and development programs in the field of food irradiation. About 20 countries have already cleared, unconditionally or provisionally, irradiated foods as safe for human consumption. International bodies such as IAEA, FAO, WHO are now actively engaged in bringing out recommendations for facilitating the acceptance of food irradiation process, by all UN member states (Krishnamurthy, 1986).

Biological effects of radiation

The lethal action of ionizing radiations on living cells is due to either a direct action on genetic material or to an indirect effect. With indirect action, there is an initial change in the suspending medium or some non-genetic molecule. Generally, the more complex the organisms, the more sensitive it is to ionizing radiation:

Microorganisms are affected by radiation. The energy of radiation is expressed in rad unit. One rad is equal to 100 erg or 0.01 Grad or 0.001 Krad or 0.000001 Mrad. The new SI system uses the Gray (1 Gy = 100 rad). 1Gy equals a released energy of 1 Joule/kg radiated matter (Banwart, 1979).

There are many reports on the effect of radiation on microorganisms. Ionizing radiation can alter the structures of organic and biochemical compounds essential to normal life when these radiation are received in high dosage; thus, they may stunt, alter or kill plants, animals and microorganisms (Potter, 1973). Some least resistant bacteria are Pseudomonas, Proteus, Escherichia and Vibrio (Banwart, 1979).

1.5.4. Preservation by Combined Process

Among all methods of food preservation, only rarely is a single method effective, and usually several or combined (Frazier and Westhoff, 1988). There are many advantages to using two or more processes in combination that means the attainment of adverse environment for growing the microorganisms of the particular foods. This well-known principle has often been called the “hurdle effect” (Harringan and Park, 1991).

Combinations of two or more chemical preservatives are more effective than indicated by testing each one individually. Combination of chemicals and other treatments also may increase the potential for control of microbial spoilage of food (Banwart, 1979). The effects of combination of low-temperature, potassium sorbate and gamma radiation of extending the shelf-life extension have practiced in present investigation.

1.6 Freshwater prawn: general aspects

Family Palaemonidae (Decapoda, Crustacea) is of great interest to fishery science, as it contains true prawns, quite a large number of which are important in the capture and culture fisheries scene. The family includes two subfamilies, namely Palaemoninae and Pontoniinae. The prawns belong to the former inhabit inland water bodies, from brackish waters to hill streams, and are very rarely marine. Many are large in size and have potential for aquaculture. The latter subfamily contains prawns which are exclusively in marine water (Jayachandran, 2001).

The shrimps and prawns of the family Palaemonidae constitute a major group among freshwater crustaceans for aquaculture. However, production of freshwater prawns accounts for 50 percent of freshwater aquaculture production of crustaceans (FAO, 1999). The systemic position of freshwater prawn mention below:

Kingdom: Animalia (actively move but unable to amass their own food)

Phylum: Arthropoda (metamerically segmented body with jointed appendages)

Class: Crustacea (thorax and abdomen bear pair biramous appendages)

Order: Decapoda (thoracic appendages modified as maxillepedes)

Family: Palaemonidae (1st mixilliped with caridean lobe of exopod)

Genus: Macrobrachium (robust body with developed rostrum)

Species: M. rosenbergii (2nd chelate strongly spinulose)

Prawn and shrimp crustaceans related to crabs and lobsters, of the Decapoda order, found in all types of water body throughout the world. As many as 21 valid genera and around 300 species from different parts of the world have been reported under subfamily Palaemoninae (Jayachandran, 2001). Bangladesh has very rich source of prawns. A total of 60 species is reported, of which 36 are marine water and 24 are fresh water in habitats (Choudhury, 2001). Generally, freshwater fishes are more popular than marine fishes in Bangladesh. The freshwater prawn, M. rosenbergii (de Man, 1879) is renowned for the tasty, nutritious properties resultant got the high market price and exported huge amount every year. The major sources of prawn and shrimp are the shrimp farms located in the costal belt of Khulna, Bagerhat, Satkhira and Cox’s Bazaar. The collection of raw material passes through a number of steps and finally delivered to market and exporting industries using road, rail and water transport. So, considerable quantity of post-harvest loss of prawn and shrimp are reported in Bangladesh (Uddin and Das, 1994). /Fishes and shellfishes are relatively perishable protein sources for human consumption (Chang, 1998). Scientists have been constantly searching for improved methods to preserve or extend the shelf-life or various aquatic food products. In contrast, little information is available on the quality changes and the causes of spoilage of freshwater prawn, (M. rosenbergii). Among the various techniques of storage, icing is most popular and normally practiced for short time preservation which permits in transportation of fresh fish in different parts of inland areas. So, maintenance of good quality of processed product is very important. Considering the inconvenience of other techniques (fish preservation techniques) gamma radiation is best for shelf-life extension of fish. At the present investigation freshwater prawn was treated with 2% potassium sorbate, 2 kGy radiation and both the above treatment and preserved at both 00C and 40C to revise its freshness at different storage period or shelf-life extension. However, seasonal nutritive values, isolation and identification of micro-flora associated with preserved freshwater prawns with their sensitivity were also investigated.

1.7 Aims of present study

Both the fish fishery products are nutritious, tasty but high perishable that’s why can’t keep for long time for human consumption. Thus, in the question of preservation, spoilage of fish fishery products has drawn the attention of peoples and had put effort to know the reason of spoilage.

1.8 OBJECTIVES OF PRESENT STUDY

The present study was undertaken on M. rosenbergii with the following objectives...

Appraisal seasonal nutritive values:

-To evaluate the proximate composition in peeled and deveined as well as whole body of freshwater prawn.

-To scrutinize the variations of nutritional value in peeled and deveined and whole freshwater prawn at different seasons.

Extension of shelf-life or storage-period:

-To enhance the microbiological safety and self-life extension by using combination of low temperature, potassium sorbate and gamma radiation.

-To substantiate the efficacy of ionizing radiation (60Co) to eliminating the bacteria those are responsible for spoilage.

Isolation and identification of micro-flora:

-To isolate and identify of bacterial-flora from treated freshwater prawns.
-To isolate and identify of mould-flora from treated freshwater prawn.

Chemical and radiation sensitivity of micro-flora:

-To perceive the potassium-sorbate sensitivity of major bacterial-flora associated with freshwater prawns.
-To detect the gamma-radiation sensitivity of major bacterial-flora associated with freshwater prawns.

REVIEW OF LITERATURE

Fishes and shellfishes are relatively perishable protein sources for human consumption (Chang et al., 1998). Scientists have been constantly searching for improved methods to preserve or extend the shelf-life of various aquatic food products. There are numerous scattered reports which are painstaking to have to some extent relationship with the present work. A brief review of literature pertinent to the present investigation has been presented below.

2.1 Proximate composition of fish and fish products

Milory (1908) estimated the amount of fat and protein contents in the reproductive period of herring and derived the values as 3.52% fat and 18.29% protein. Aykroyd et al. (1951) conscious the nutritive values of Indian foods and the planning of satisfactory diets and found that prawn contains moisture-77.4%, protein-19.1%, fat - 1.0%, carbohydrate-0.5%, calcium-0.43% and phosphorus-0.31% in edible-parts. Venkataraman and Chari (1951) studied on seasonal variation in biochemical composition of Mackerel where they found ash and protein remains constant while moisture and fat subjected to seasonal variation and reciprocal relationship. Stansby (1954) found the macro-nutrient content such as moisture, protein, fat and ash of the edible flesh of certain fresh water fish and those were 76.8% moisture, 19% protein, 5% fat and 1.2% ash. Thurston (1958) studied the relationship between different nutritive components and reported that Alaska pink salmon maintained inverse relationship between fat and moisture and positive correlation between protein and moisture.

Borgstoom (1961) reported that the variation of the fat and protein contents in fish depend on some factors such as size, age, species, sex, seasonal changes and seasons. Jacquot (1961) studied on some fresh water and marine fishes and recorded that the nutritive values or the proximate composition of fish were related to the seasonal changes. Stansby and Olcott (1963) investigated that the nutritive food value depended on proximate composition of fish, which varied widely from species to species and from one individual to another. Malek et al. (1966) determined the moisture contents and ash contents in Punti fish, Puntius stigma and found that the fish contained 72.65% and 2% of moisture and ash respectively. Adhikari and Noor (1967) studied the seasonal variation in fat, moisture contents and solid matters of Puntius puntius and observed higher oil contents in winter. They also recorded inversed relationship between fat and moisture. Rao (1967) worked with the muscle of Pseudoscieana aneus and Johnius curatta and found that the fish contained 70.05% - 80.75% of moisture and 1.5% - 12% of fat. Gopalan et al. (1978) studied on the biochemical composition of body muscle and its caloric contents of Mystus vittatus. They reported 70% moisture, 18.75% protein and 2.37%fat.

Ahmed et al. (1981) found that Tilapia nilotica contained 22.10% protein, 1.32% fat, 1.72% ash and 74.86% moisture. Female fish contained less protein, fat and ash than those of male fish. Rahman et al. (1982) studied proximate composition and nutritive quality of some small, medium and large size Zeol fishes and found small fished showed higher percentage of moisture and presence of lipids and proteins are inversely correlated. Banu et al. (1985) studied on Ompok pabda, Puntius ticto, Heteropneustes fossilis, and Mystus tengra and reported that the protein contents of them were 14.3%, 20.12%, 18.62% and 14.24% respectively. Govindan (1985) analyzed the amount of protein content that was present in different fishes from both the fresh water and marine environment and obtained the result as fish contained 9%- 25% protein and in most cases the limit was 16%-19%. Begum and Hoque (1986) studied on the effects of temperature on the composition, colour, texture and reconstitution of dehydrated shrimps and found moisture - 74.20%, protein - 17.35%, fat - 1.68%, ash - 2.60% and calcium 114.65 mg/100 g in raw shrimps. Rubbi et al. (1987) studied the proximate composition of 27 species of fresh water fish (both scaly and non-scaly) and found that the moisture ranged from 72.1 to 83.6%, protein ranged 11.9 to 21.9%, fat ranged 0.8 to 15% and ash ranged 0.8 to 5.11%.

Al-Habib (1990) estimated the protein contents of six different fresh water fishes and observed that these fishes were contained 11% to 16.75% proteins. Joseph et al. (1992) worked on the proximate composition of Cypselirus suttoni and reported that it contained 76.29% moisture, 20.88% protein, 0.55 to 1.7% fat and 0.84 to 1.18% ash. Joseph et al. (1992) also reported that the proximate composition of Hirundichthys coramendelensis contained moisture 77.17%, protein 20.38%, fat 0.30% - 0.50% and ash 0.92% -1.29%. Shaheen (1992) studied on biochemical composition of sword fish and showed that moisture had reversible relation with protein and there was no remarkable change of lipid, salt and ash content. Chandrasharkar and Deothale (1993) found that a wide variation existed between species in protein content (marine 8-21%, fresh water 13.5-17.3%), fat content (marine 0.7-14.7%, fresh water 0.6-1.3%). They also showed that the content per 100 gm muscle had 4.7-51.4 mg calcium 116-312 mg phosphorus. Martin et al. (1995) studied on European catfishes for proximate composition and reported that fat content varied with season to season and average content 3.3% (range 0.6% - 8.6%). NIN (1996) intended on the nutritive values of Indian foods and demonstrated that the edible-part of prawn contains moisture - 77.4%, protein - 19.1%, fat - 1.0%, ash - 1.7%, calcium - 323 mg/100 g and phosphorus - 278 mg/100 g. Irianto and Irianto (1997) studied on Nile Tilapia in Indonesia and reported that it contained percentage of moisture, protein, fat and ash 76.8, 20.1, 2.2 and 1.0 respectively.

Kamal et al. (2000) studied on changes of proximate composition of freshwater prawn (Macrobranchium rosenbergii) and found the initial moisture, protein, lipid and ash content were 78.34%, 18.46%, 1.8% and 1.15% respectively. Salam (2002) studied on seasonal changes in biochemical composition of freshwater catfish, Heteropneustes fossilis and found that proximate biochemical composition was varied seasonally in relation to reproductive cycle of the fish. He also recommended the moisture, carbohydrate, protein, fat and ash contents were highest in July, December, February, January and July respectively. Shahiduzzaman et al. (2004) studied on seasonal variation in proximate composition and mineral content of Clupisoma atherinoides. They found that body constituents of this fish changed seasonally and had reversible relation with moisture. Fat and moisture contents remained around 3% and 73%, respectively.

2.2 Fish preservation by low temperature, chemicals and radiation

Nickerson et al. (1954) reported that the use of ionizing radiation is an effective method for extending refrigerated shelf life of fish. They also defined gamma irradiation as the process of partial or complete sterilization of a substance by means of ionizing radiation. Maclean and Welander (1960), Miyauchi (1960) and Proctor et al. (1960) had shown that the low dose radiation reduced the spoilage of fish from microorganisms and extended their shelf lives. Mossel et al. (1966) examined the growth potential of numerous strains and found that only one stain, Salmonella panama could grow at 40 C. Liston et al. (1969) worked out that the actual extension of shelf life of fish depended on the level of irradiation dose and the condition of fish at time of treatment. Pointel and Sam (1969) irradiated the Dermestes maculates degree at a dose ranging from 10 to 30 Krads to find out the sterilization dose. None of the treated stages gave any progeny and their life span was shortened.

Diehl (1972) showed that low dose of radiation was effective for increasing the shelf- life of marine and fresh water fish and other sea-food. Venugopal et al. (1973) showed the effectiveness of radurization for extending the shelf-life of fresh mackerel fish (Cybium guttatum). Chung (1974) reported that 0.5-2.5% kGy were promising for preservation of Korean fishes and shellfishes. Ghadi et al. (1978) investigated on the fresh mackerel and had shown the effectiveness of radiation for extending shelf life.

Hussain et al. (1979) investigated that, low dose of ionizing radiation were known to reduce the spoilage causing micro-flora in foods and thereby extended the storage life of marine and fresh fish.

Anonymous (1981) reported that the recommended dose of radiation for preservation of dried fish is 1 KGy. Venugopal et al. (1982) also stated that radiation treatment of

1.5 kGy suppressed the rate of spoilage of fish stored at the 0, 5, 10 and 150C. Licciardello et al. (1986) suggested that treatments of fresh Atlantic cod, G. morhua, fillets with either 100 Krad gamma irradiation or a sorbate dip were found to be comparably effective in extending the iced storage life. Frazier and Westhoff (1988) reported Pseudomonas, Acromobacter and Flavobacterium and recognized as fish spoilage bacteria and grow well at low temperatures. Karim et al. (1988) demonstrated that with the increase in storage period, the total bacterial count increased for both 00C and 40C temperature. Maha et al. (1989) studied on shelf life extension of 3 kinds of cured fish products and was found that 0.1% potassium sorbate followed by irradiation up to 4 kGy retards mould growth on the products and extends the shelf life to a considerable length of time in comparison with the commercially prepared products.

Szulc et al. (1990) were irradiated the fish samples by gamma rays with the doses 1.0,

2.5 and 5.0 kGy. Control and irradiated samples were examined in order to compare the shelf-life during storage at 40C. It was noted that the irradiation of carp ( Cyprinus carpio) and trout (Salmo gairdneri) with 1.0, 2.5 and 5.0 kGy allows the extension of their shelf-life at 40C by about 20, 35 and 51% for carp meat and by about 20, 49 und 69% for trout meat, respectively to the dose. Venugopal and Nair (1992) concerned the several aspects on the use of low dose gamma radiation for shelf life extension of Indian mackerel in ice has been examined to understand its amenability for the radiation process. These included, apart from determining the optimum dose (1.5 kGy) for maximum shelf life of fish. Sikorski and Pan (1994) stated that partial freezing might cause drip loss and possible quality degradation. Among the freshness preservation techniques, the effectiveness of partial freezing received both suspicion and applause. Chang et al. (1998) reported fresh sea bass (Lateolabax japonicus) under temperature ranging from - 3 to 100C. The shelf-life of stored sea bass at 50C was 3 days, it was extended to about 2 weeks at 00C, partial freezing storage at -30C increased the shelf-life to 4 weeks. Lakshmanan et al. (1999) reported that non packaged irradiated fish were judged to maintain acceptable sensory and

microbiological quality for up to 17 days and irradiated packaged fish showed the largest shelf-life of 20 days which were stored at low temperature. Bari et al. (2000) studies on shelf life extended by a combination of irradiation and ascorbic acid treatment of fish contents prepared. They found that a dose of 5 kGy had extended the shelf-life up to 5 weeks at room temperature. Lopez-Caballero et al. (2000) studied on extension of shelf-life of prawns (Penaeus japonicus) by vacuum packaging and high- pressure treatment. He found that the viable shelf-life of 1 week for the air-stored samples was extended.

Gelman et al. (2001) found that the organoleptic and hypoxanthine test results show that the treatment of potassium sorbate can slow the process of spoilage by about 5 days on shelf-life of pond raised freshwater fish, silver perch (Bidyanus bidyanus).

Hossain et al. (2001) reported that whole fish control remain acceptable range up to 5 days at 40C (low temperature) whereas degutted irradiated (150 Krads) increase shelf- life 21 days. Jeevanandam et al. (2001) reported that eviscerated threadfin bream (Nemipterus japonicus) were exposed to γ-radiation at 0, 1 and 2kGy. After radiation at 0, 1, 2 kGy had shelf life of 9, 14 and 28 days respectively. Khatun et al. (2003) studied the effects of gamma irradiation (3 kGy), 2% potassium sorbate (1 min.) in shelf-life of Golsha tengra, Mystus cavasius and found that shelf-life was extended by one to two weeks. However, they also found that the degutted samples shown the more shelf-lives than that of whole samples and recommended that the irradiated samples were fresh more time.

2.3 Sensory, chemical and microbial changes of fishes

Yamamura (1933) demonstrated that TVN value increased with the onset of spoilage and it was an indicator of spoilage and the upper acceptable limit of TVN was 30 mg N/100 gm of sample. Tarr (1944) investigated that a number of bacterial species such as Micrococcus and Achromobacter reduced trimethylamine-oxide to trimethylamine by the action of their triamine oxidase enzyme. Ingram and Rhodes (1962) claimed that the higher dose of radiation in the vicinity of 300 Krads to 500 Krads had caused noticeable changes in odour, texture and appearance of fish. Stavin et al. (1966) reported that radiation dose of 100 to 300 Krads was effective for the reduction of bacterial load in fishes. Kajanas (1968) showed that radiation had been shown to eliminate the bacteria that are responsible to bacterial spoilage of fish.

van Spreekens (1977) reported that trimethylamine is produced by Pseudomonas putrefaciens, a “non-defined” group resembling Pseudomonas putrefaciens, Photobacterium spp. and some Moraxella-like bacteria. He also investigated that strong off-odours were produced on boiled shrimp by the typical shrimp spoiler (Alteromonas). Pseudomonas putrefaciens, Pseudomonas spp. and Moraxella spp. bacteria produced less offensive odors. Hussain et al. (1979) investigated the initial and final counts of bacteria in the 100, 200 and 300 Krads irradiated samples and they stressed out finally 8x108, 5x107 and 2x106/g of sample at the end of one month which was initially 9x104/g of sample. Guevara (1980) reported that gamma radiation offers a potential for improving the microbiological flora, sensory qualities and extending the storage life of fishery product.

Hye et al. (1990) stated that the total bacterial count rapidly increased after 72 hours and total volatile nitrogen increase rapidly with the increases of storage life. Vinh et ^ al. (1993) studied on irradiated semi-dried Bombay duck (Harpodon nehereus) and shrimps (Penaeus indicus) were subjected to sensory evaluation during storage 260C.

Gamma radiation resulted in insect-free, mould-free and acceptable semi-dried fish.

Islam (1995) found that total viable bacterial count of dried lotia fish (Harpodon neherius) ranged between 7.8x106 to 1.0x108 cfu/g and total coliform count ranged between 1.0x102 to 3.0x102 cfu/g. Rashid et al. (1996) reported about microorganisms associated with fine different types of frozen fish sample i. e., Shrimps, Hilsa, Catfish,

Perch and Indian Pellona. The total viable bacterial count of the frozen samples ranged from 2.8 x 104 to 3.3 x 106 cfu/g, total coliform count varied from 2.8 x 101 to

7.5 x 102 cfu/g and psychrophilic bacterial count from 1.3 x 102 to 7.0 x 105 cfu/g.

Chang et al. (1998) reported that a maximum microbial population of 3 x 106 cfu/g of fish muscle seemed to be a good shelf-life indicator only at storage temperature ≥

00C. Aytac et al. (2000) reported that radiation has beneficial effects in controlling bacterial growth. Maximum inhibition of M. morganii was achieved using radiation at 2 kGy. Leitao Rios (2000) studied the microbiological and chemical changes in freshwater prawn (M. rosenbergii) stored under refrigeration. They extended the shelf life of freshwater prawn 10 days at stored temperature 00C wherein shelf life reduced 5 days when stored at 50C temperature. Lopez-Caballero et al. (2000) studied on preservation of prawns (Penaeus japonicus) and they could be extended for 1 week by vacuum packaging and high-pressure treatment.

Gelman et al. (2001) shown that keeping the fish (silver perch) at 0-2 0C can prolong the storage prior to spoilage by 10 days compared with those kept at 50C. These results obtained through organoleptic tests are corroborated by both the chemical and physical tests. Bjorkevoll et al. (2003) studied on origin and spoilage potential of the micro-biota dominating genus Psychrobacter in sterile dehydrated salt-cured and dried salt-cured cod. He found that the micro-biota develops off-odours such as musty, causing sensory rejection within 7 - 10 days of chilled storage. Popper and Kroll (2003) conducts sensory research with pre-school children’s to acceptable food.

They also found the differences between the sensory threshold of children and adults during the acceptance of foods. Yao et al. (2003) studied a cross cultured study with American, Japanese and Korean consumers on the basis of structured and unstructured 9 point of hedonic scales for determination of sensory changes.

2.4 Micro-flora associated with fish fishery products

Tarr (1944) studied the micro-flora associated with marine and freshwater fishes. He reported that there is generally the bacterial flora responsible for the spoilage of fish that, existing in both of the marine and fresh water environment Achromonobacter, Pseudomonas, Flavobacterium and Micrococcus are usually predominant and Sarcina, Proteus and Bacillus are found to a larger extent. Larke et al. (1956) found some bacterial species are responsible to fish spoilage, the spoilers bacteria belong to the following genera Pseudomonas, Acinobacter and Moraxella. Joardar (1974) studied on 4 batches of Hilsa fish both in quantitatively and qualitatively and reported that the Pseudomonas were the most responsible organisms for spoiling fish at low temperature and the next dominant spoiling group was Micrococcus. Jay (1977) reported that the genera of bacteria most frequently found in fresh water fishes are Bacillus, Staphylococcus, Enterobacter, Klebsiella, Serratia, Citrobacter, Aeromonas, Escherichia and Micrococcus. The genera of moulds found Aspergillus, Oospora, Penicillium, Seopulariopsis, Wallemia and Sporendonema in the fish and fish products. van Spreekens et al. (1978) said that lesser role is attributed to ^ Achromobacter genus in fishes while Moraxella and Alteromonas were considered as shrimp spoilers. Banwart (1979) encountered Cytophaga, Micrococcus, Bacillus, Vibrio, Flavobacterium and Clostridium in fresh and spoiling fishes.

Frazier and Westhoff (1988) also found that Clostridium and Sporotrichum have been found growing on foods at 6-70 C and Penicillium and Monilia at 40 C temperatures. Joseph et al. (1988) reported the presence of Aeromonas hydrophila in water undoubtedly contributes to its common occurrence in fish and sea food. The bacteria are the common component of the intestinal micro-flora of healthy fish. Atapattu and Samarajeewa (1990) studied on the mycoflora of dried-salted fish from markets in Kandy, Sri Lanka was studied with emphasis on visibly spoiled dried fish. A total of 61 fungal isolates from 25 dried-fish were isolated and identified. The most prevalent fungus was Aspergillus niger.

Alur et al. (1991) isolated Pseudomonas, Proteus, Aeromonas, and Achromobacter from spoiled fish. Pseudomonas and Proteus cause putrid and ammonia-cal odors, while Acinobacter and Aeromonas are associated with unpleasant fruity odors. Hoque et al. (1992) isolated Aeromonas, Enterobacter, Escherichia, Klebsiella, Pseudomonas, Serratia, Pleseomonas, Bacillus, Vibrio naming bacteria from the intestinal tract of live freshwater diseased fish and argued that Escherichia coil, Aeromonas hydrophila, Enterobacter spp. and Plesiomonas shigelloides were common bacterial species. Akther (1993) observed on giant prawn, Macrobrachium rosenbergii and she reported that its surface slime, intestinal tract and gills had bacterial strains of Pseudomonus, Aeromonas, Micrococcus, Achromobacter, Flavobacterium, Sarcina and Bacillus.

Hussain et al. (1994) isolated some pathogenic bacteria from freshwater fishes such as Plesiomonas shigelloides (39.9%), Vibrio spp. (42.3%), E. coli (16.7%) and Aeromonas spp. (6.2%). Hossain et al. (1994) were isolated 135 bacterial strains from L. rohita, C. catla, C. mrigala, H. molitrix, C. idella, C. carpio, P. gonionotus, C. batrachus, C. gariepinus and M. cavasius fry and fingerlings from which Acrinibas represented 62.47%, Micrococcus 49%, Pseudomonas 25.68% and Edwardsiella trada 11%. Islam (1995) isolated bacterial strains from loita fish (Harpodon neherius) and identified Bacillus cereus, Pseudomonas sp., Micrococcus varians, Micrococcus luteus, Staphylococcus epidermis and Bacillus alvei. Rashid et al. (1996) reported that shrimps were more contaminated than that of other fish samples such as catfish, perch etc. Dominant bacteria in the frozen samples were Pseudomonas aeruginosa, Staphylococcus aureus, Micrococcus radiodurans, Bacillus subtilis, Moraxella osloensis, Escherichia coli and Klebsiella edwardsii etc. Blanch et al. (1997) determined the bacteria associated with reared turbot (Scophthalmus maximus) larvae and found that Gram-negative bacteria were predominant at the initial stage while Vibrio were frequent at final stages. Hatha et al. (1998) analyzed 1264 and 914 samples of raw and cooked shrimps for coliform count, Escherichia coli, aerobic plate count (APC), Salmonella and coagulate positive Staphylococci. Incidence of coliform was 2.9% and 14.4% in cooked shrimps and raw shrimps respectively. Rahman et al. (1998) were isolated Aeromonas hydrophila, Aeromonas sorbia, Pseudomonas sp., Flavobacterium sp., Micrococcus sp. and Staphylococcus sp. from the lesions and kidney of the affected fishes.

Gelman et al. (2001) found that bacterial composition fluctuated during storage. The initial load on silver perch fish surface was predominantly gram positive of Micrococcus, Bacillus and Cornebacterium. Later Pseudomonas fluroscens naming

gram negative rapidly increased. Nabi et al. (2001) considered Micrococcus, Coryneforms, Pseudomonas and Acromobacter were the dominant groups of bacteria from fishes spoiled at room temperature and iced stored condition. Nur (2001) isolated 74 bacterial strains from cooked fish sample most of them were Bacillus megaterium (40%), Aeromonas hydrophila (22%), Staphylococcus aureus (18.9%) and Escherichia coli (10.81%). Austin (2002) indicated that fish possess bacterial populations on or in the skin, gills, digestive tract and light-emitting organs. In addition, the internal organs (kidney, liver, spleen) of healthy fish may contain bacteria, but muscles sterility debated. Khatun et al. (2004) studied on Mystus cavasius and isolated the major fish spoilage bacteria; they argued that Staphylococcus, Bacillus and Micrococcus were most frequent bacterial group that caused spoilage of M. cavasius. Lalitha and Surendran (2004) studied on bacterial micro-flora with farmed freshwater prawn (M. rosenbergii). They isolated total 367 isolates and identified Micrococcus, Bacillus, Corynebacterium, Arthrobacter, Aeromonas and Streptococcus naming bacterial genus.

2.5 Sensitivity to potassium sorbate

Tompkins et al. (1974) studied on the effects of potassium sorbate on Salmonella, Staphylococcus aureus and Closridium botulinum in cooked, uncured sausage and inhibited these bacterial species. Shaw et al. (1983) applied potassium sorbate to gutted, head on cod and cod fillets for 10 or 30 seconds, with subsequent storage at 10C. Results indicate that sorbate dips (either 10 or 30 second) were effective in extending shelf life of cod fillets but were ineffective for gutted fish. Ahmed et al. (1988) reported that preservation of jewfish and shrimp by potassium sorbate is more effective against microbial growth that helps to extend their shelf-life. Chakrabarti and Verma (2000) reported that microorganisms like - bacteria, fungi etc. are more sensitive to potassium sorbate. Hossain et al. (2000) potassium sorbate dips were applied to beheaded and degutted nola fish (Labeo rohita) for one minute with subsequent storage at 4-50C for 5 weeks. They reported that the control sample remains accepted in 4-50C for 3 weeks, whereas treated samples remain acceptable for 4 weeks.

Shalini et al. (2001) reported that in low temperature shelf-life periods of control packs were about 7-8 days. Whereas the potassium sorbate treated fillets has an extended storage life of 20 days. Khatun et al. (2004) studied on the chemical and radiation sensitivity of major fish spoilage bacteria isolated from M. cavasius. They found that Staphylococcus reduced to 50% by 2% whereas Bacillus reduced 50% by 2.5% potassium sorbate solution.

2.6. Sensitivity to gamma radiation

Nickerson et al. (1954) reported that, the use of ionizing radiation was an effective method for extending refrigerated shelf-life of fish. They also defined gamma irradiation is the process of sterilization of a substance by means of ionizing radiation. Stavin et al. (1966) reported that the radiation dose of 100-300 Krads to be effective for the reduction of bacterial load in fishes. Kazanas et al. (1969) showed that radiation was effective to control or eliminate the bacteria as well as some other microorganisms which had been shown or related to fish spoilage. Diehl (1972) worked on the shelf-life extension of fish and fish products; he showed that, low dose of irradiation was effective increasing the shelf life of marine and fresh water fish and seafood. Banwart (1979) stated that the psychrophilic Pseudomonas was the main
spoilage organisms of protein goods (meat, poultry and fish). He argued that these bacteria were resistant to radiation dose 100 to 600 Krad. Hussain et al. (1979) investigated initial and final counts of bacteria in the un-irradiated and 100, 200 and 300 Krads irradiated samples and they stressed out finally 8x108, 5x107 and 2x106 cfu/g of sample at the end of one month and initially with low count about 9x104 cfu/g of sample.

Hau et al. (1992) were irradiated the frozen prawns (Penaeus monodon) with different doses at -10 plus or minus 2 degree C. The D sub(10) values for Vibrio cholera, Staphylococcus aureus, Escherichia coli, and Salmonella enteritidis were 0.11, 0.29, 0.39, and 0.48 kGy, respectively. Islam (1995) used different doses of irradiation at 1, 3, 5 and 7 kGy on dried loitta fish (Harpordon neherius), which resulted in reduction of total bacterial count from 7.8x106 cfu/g to 3.9x106, 2.0x105, 9.0x102 and 0 cfu/g respectively. Coliforms were eliminated completely at a radiation dose of 1 kGy, while all most of the bacteria were eliminated at dose of 7.0 kGy. Ghaly et al. (2000) concluded that irradiation dose of 3 kGy was most effective at reducing total bacteria; pathogenic Staphylococcus aureus and E. coli were completely eliminated at this dose. Khatun et al. (2004) studied on the sensitivity of gamma radiation to Bacillus and Staphylococcus and found that Bacillus need 8 kGy radiation dose to control while 6 kGy doses was needed to control the Staphylococcus. They also found that the D 10 values of the Bacillus were 2 kGy, whereas the Staphylococcus was 1.33 kGy. They also recommended that the Bacillus was more resistant than Bacillus.

MATERIALS METHODS

Appraisal seasonal nutritive values; investigation a relevant preservation process by combination of low temperature, potassium sorbate and gamma radiation; isolation, identification and sensitivity of micro-flora associated with freshwater prawn, Macrobrachium rosenbergii (de Man, 1879) were done according to following materials and methods.

3.1 Research affiliations and study periods

Research Affiliations: Most of the present investigations were carried out in the Food Technology Division and Microbiology Industrial Irradiation Division, Institute of Food and Radiation Biology (IFRB), Atomic Energy Research Establishment (AERE), Savar, Dhaka and partly in the Laboratory of Limnology and Fisheries Biology, Department of Zoology, Jahangirnagar University, Savar, Dhaka.

Study Periods: The present study seasonal variations of proximate composition of freshwater prawn, M. rosenbergii (Photograph 1) were done in three consecutive steps like - summer (March 2003 - May 2003), autumn (July 2003 - September 2003) and winter (November 2003 - January 2003) fewer than two batch like - edible portion and head-on. The preservation of M. rosenbergii, by combined treatment of low temperature, potassium sorbate and gamma radiation were done in September 2003 to November 2003. The isolation and identification of micro-flora (both bacterial and mould flora) was done during December 2003 to January 2004. The potassium sorbate and gamma radiation sensitivity of major bacterial-flora which is responsible for spoilage of M. rosenbergii was carried out during February 2004 to March 2004.

3.2 PROXIMATE COMPOSITIONS AND MINERAL CONTENTS

3.2.1 Proximate Compositions:

In every season, the specimen’s freshwater prawn, M. rosenbergii were collected from the Dhaka New Market. The specimens were identified in Limnology and Fisheries Biology Research Laboratory of the Department of Zoology, Jahangirnagar University. Usually, collections were made early in the morning then samples were taken in polyethylene bag with ice and quickly transported to the laboratory. After collection, all samples were washed in clean water then the divided into two batches:

Batch - 1: Edible-part (deveined, peeled and degutted) were taken

Batch - 2: Head-on (only degutted) were taken as a sample

3.2.1.1 Determination of moisture content

Moisture was determined by drying the samples at elevated temperatures and reported the loss in weight as moisture (AOAC, 1975).

Requirements: Analytical balance, drying oven, small-petridishes, desiccator, scissors and forceps.

Procedure: At first 2 small-petridishes were weighed. About 3 - 5 g of fairly minced sample was taken in pre-weighed petridishes. Then the petridishes with samples were heated in oven at about 1050C for 5 to 6 hours. Each time before weighing the petridishes containing the sample was cooled in a desiccator. The difference in the weight of the fresh sample and the content dry weight gave the moisture content.

Calculation: Moisture content (g/100g)

Wet weight of the sample - Dry weight of the sample = x 100

Weight of the sample

3.2.1.2 Determination of protein content

“Micro-Kjeldahl” is an accepted method for determining total nitrogen of crude protein in fish and fishery product. This involves the oxidation of organic matter with sulfuric acid in presence of catalyst and then formation of ammonium salts and amines from the nitrogen components of fish.

Requirements: Concentrated sulfuric acid, catalysts for digestion, 30% sodium hydroxide, 0.01 N HCl, 2% boric acid, mixed indicator, Kjeldahl flask, Pumic stones, exhausting chamber, digestion tube, burette, conical flask, gas burner and measuring cylinder.

Procedure: About 1 g macerated sample was taken in a cleaned and dried digestion tube (100 ml) to which 2 g of digestion mixture and 25 ml of pure concentrated sulfuric acid were added and the mixture digested by continuous heating till the mixture become clear. Glass beds were added to prevent bumping during digestion. After digestion, solution were cooled and made up 100 ml with water transferring in a volumetric flask. Then 5 ml diluted sample and 10 ml of 30% NaOH were transferred in Micro-Kjeldahl dilution apparatus. The essence was collected in excess of 2% boric acid solution with mixed indicator and was titrated by 0.01 N HCl.

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3.2.1.3 Determination of fat content

Fat (Lipid) content was determined quantitatively by extraction with a mixture of chloroform-methanol to a little amount of calcium chloride (AOAC, 1975).

Requirements: Chloroform-methanol (2:1) mixture, 4% CaCl2, mortar pestle, pipette funnel, Whatman filter paper (11 cm), small beaker (10 ml), water bath and magnetic stirrer.

Procedure: At first 5 g of fish muscle were taken in a mortar and homogenized. An adequate amount of sands were added and grinded gently by a pestle. About 10 ml of chloroform-methanol (2:1) mixture was added into the sample and homogenized properly. Then filtered through a filter paper and was collected into a pre-weighed small beaker. Add 1 ml 4% CaCl2 solution and kept it over night. Keep the beaker into the water bath on stirrer till drying the mixture. After drying out the beaker was again weighed. The difference in the two weights of the beaker gave the weight of the fat (Folch and Stanley, 1957).

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