Report on Quality of Preserved Fresh Water Giant Prawn

Introduction:

Bangladesh is an agro-based riverine country enriched with enormous fisheries resources. The total area of inland water in general estimated about 53, 32,657 hectares (DOF, 2008). Water area of inland fisheries including rivers, beels, Kaptai lake, floodplains and polder/enclosure as open water body (capture based ) comprise about 4,12,341 hectares; ponds and ditches, oxbow lake and coastal shrimps farm as closed water body (culture based ) comprise about 49,20,316 hectares (DOF, 2008). The republic has a 710km long coastline and approximately 68,480 square nautical miles of seawater area (DOF, 2005-2006). The nation’s exclusive economy zone contains up to 41,040 square nautical miles from the coastline (DOF, 2008). Thus the nation’s total area of water having fish production potential is of very great importance. Fish contributes about 63% of the available protein in the diet and the rest 37% protein comes from livestock and poultry. It indicates the importance of fish in contributing to the level of nutrition of the people of Bangladesh (DOF, 2008).

The fisheries sector generates  socio-economic development, nutrition, employment and poverty alleviation of large number of population and foreign exchange earning in the economy of Bangladesh. It is the perfect provider of fat and important minerals like calcium, phosphorus, iron etc. including vitamin and iodine. It has been estimated that about 1.28 million people are directly related with fisheries activities and fish farmers in Bangladesh are about 3.08 million. Another 12 million people indirectly earn their livelihoods from fisheries related activities (Chowdhury, 2001). The prawn and shrimp sector currently provides two million jobs directly and indirectly. The sector provides another millions jobs within the next five years and account for at least of the countries exports if handing right.

Bangladesh is blessed with favorable conditions for prawn farming. 2.5 percent of the global production of prawns and shrimps is supplied by Bangladesh and the prawn and shrimp sector generating US$ 301 million annually (DoF/MoFL, 2002). The importance of fisheries sub-sector in our national economy has been shown by its contribution. In 2006-2007, 4.91% of GDP and 5.71% of the total export earnings came form this sub-sector (DoF, 2008). The total fish production in Bangladesh was estimated at 2440011 mt where production of shrimp was 129160 mt in 2006-2007 (DoF, 2008).  Total export of fish and fish product was 73704 mt and value was Tk.3352.89 crore where frozen shrimp and prawns was 53361 mt and value was Tk.2992.33 crore that was 89.24% of all exported fish and fish products (DoF, 2008). Prawn and shrimp is the principle foreign exchange carrying crop among the frozen sea food of Bangladesh. The fishery sector has been ranked in the 2^nd position among the export item of the country.

Mostly fish is sold and preserved at the traders and consumers level with ice and freezing condition respectively in Bangladesh. All the prerequisites like proper handling, different techniques of preservation are essential since fish is an extremely perishable. Rigor mortis started as soon as the fish dies and a large amount of fish is lost due to spoilage every year in Bangladesh. (1982) estimated that about 8% of the total catch equivalent to 4.25 million tons never reached the market and is wasted due to spoilage. So it is essential to preserve the surplus fish. The cost of preservation should, however be low to ensure profitable return. The method of preservation should be simple, such as many are applicable on a commercial scale and suitable for the climatic condition.

It is not necessary to preserve fish in Arctic and temperate zone because of cold climatic condition. In temperate zone the need for preservation is not so pressing as fish can remain fresh few days without any preservation. Different picture in the tropics, here the hot weather favors the rapid spoilage of fish. For example, in India the landed fish can ordinarily remain fresh for not more than 8 hours after capture, after which decay is imminent (Srivastava, 1985). Fish must therefore be preserved soon after landing.

Two main ways of reducing and stopping spoilage are preservation and processing Preservation methods keep the fish in afresh state so that the changes in texture, taste and appearance, etc. are minimized. Bacterial and autolytic spoilage are biological systems which operate under certain optimum conditions. Therefore, altering these conditions can prevent or reduce spoilage. As bacteria require water and are sensitive to temperature, salt concentrations and pH, a number of  approaches can be used. Control of autolysis is affected by controlling enzyme activity. The most commonly used and practiced way of reducing autolytic action is to lower temperature (Clucas, I.J. and Ward, A. R., 1996).

Types and load of microorganisms present are related to environment in which fish is caught and processed. Generally, fish from tropical water have more bacteria than those from temperate zone and marine fish has higher bacteria than freshwater fish (Shewan, 1976).

The genera of bacteria most frequently found in freshwater fishes are- Bacillus, Staphylococcus, Enterobacter, Klebsiella, Serratia, Citrobacter, Aeromonas, Escherichia, Micococcus (Jay, 1977).

Difficulties with the international quality standard are being faced by some tropical prawn exporting countries including Bangladesh. The difficulties in meeting those standards are evident from the high level of rejection of  prawn and shrimp exports by the importing countries with problem such as decomposition, high total bacterial counts, filth, unexpected foreign matter and food borne microbial organisms. The rejections represent not only the monetary loss to the exporter but also more significantly, injury to the factory. In the year 1997 EU Commission imposed a temporary ban on our export for hygienic quality of our product. Eventually this ban became a blessing to our processing plants because, each factory hurriedly developed to a considerable extent through the introduction of new facilities in the factory.  The successful development of a prawn industry depends not only on improved supply of raw material but also on the quality of exports. Poor quality of the raw material and poor sanitary performance of freezing plant affect in frozen prawn and shrimps by filth and also the freezing of decomposed prawns are major problems encountered by the seafood industry of Asian Countries (Dziezak, 1997).

Preservation method are designed to inhibit the growth of organisms and killing the organisms in fish and kept it fresh minimizing the changes in texture, taste and appearance etc. There are several methods of preservation such as freezing, drying, by chemical preservative, irradiation etc. The chief methods of preservation of fish and fleshy products are low temperature, drying and irradiation and combination of the above.

The bacterial flora of fish and enzymes present in the tissue are adapted to the temperature at which fish lives i.e. around 5-10˚C for fish from cold water and 25-30˚C for tropical fish. By lowering or raising the temperature, bacteria and autolytic spoilage rate will be reduced (Frazier and Westhoff, 1988).

In general, enterocococci grow better at refrigerator temperatures than do coliform bacteria. The lowest reported temperatures for growth of staphylococci and salmonella in foods is 6.7˚C. Clostridium botulinum has been reported to grow at temperature lower than all other food poising microorganism. The effect of chilling on the micro flora in a particular food will depend on the temperature. Growth of micro organism at temperature below optimum can cause a number of morphological and physiological changes.

In most case different retardation of the activity of enzymes at low temperature can changes metabolic pathway and end products. Lipase and proteinase production by pseudomonas and certain other genera occur preferentially at low temperature. However in food chilled and held at proper chill temperature (below 1˚C) only psychrotrops will cause spoilage. The long periods of refrigerated storage common for many foods permit psychrotrops population to reach many million per gram of product in only a few days often resulting in objectionable changes in color, taste and texture.  In broads terms it can be said that the lower the temperature, the slower the bacteria and enzyme activity and consequently the longer the storage life. Thus fish can be chilled or frozen.

Objectives:

The objectives of the present study were:

• To determine the proximate composition and some mineral contents of freshwater prawn;
• To investigate the effect of low temperature on spoilage rate;
• To determine the microbiological, chemical and organoleptic changes in the samples before and storage at -20˚C temperature; and
• To enhance the shelf-life of freshwater prawn.

Literature Review:

Freshwater Prawn: General Aspects:

The main species is Macrobrachium rosenbergii – Giant prawn locally it is called Golda Icha. They are euryhaline species i.e. normally live in freshwater and estuarine region. Its rostrum is concave and the rostral formula 13-15/12-13. Bluish carapace, straight body shape and long chilled legs present. This species can reach a length of over 34 cm and a weight of over 170 g (Shafi, 1982). According to De Man, 1879 the systemic position of freshwater prawn

Kingdom: Animalia

Phylum: Arthropoda

Class: Crustacea

Order: Decapoda

Family: Palaemonidae

Genus: Macrobrachium

Species: M. rosenbergii

 Proximate composition of fresh fish:

Al-Habib (1990) estimated the protein contains of six fresh water fishes and observed that those fishes were contained 11% to 16.75% protein.

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.

Aykroyd el 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.

Banu et al. (1985) studied on Ompok pabda, Puntius ticto, Heteropneustes fossilis, Mystus tengra and reported that the protein contents of them were 14.3%, 12%, 18.62% and 14.24% respectively.

Begum and Hoque (1986) studied on the effects of temperature on the composition, color, 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 shrimp.

Brogstorm (1961) reported that the variation of the fat and protein contents in fish depend on some factors such as size, age, species, sex and seasonal changes.

Burgess et al. (1965) recorded a total liquid of fish is about 80%.

Chandrashakar and Deosthale (1993) found that a wide variation existed between species in protein content (marine 8%-21% and fresh water fish 13.5%-17.3%) and fat content (marine 0.7%-14.7% and freshwater fish 0.6-13%).

“Deshio Khadyodrbyer Pustiman” published by Institute of Food Science (1980) reported that fresh water fishes contained 70-80% moisture, 15-18% protein, 0.1-8% fat.

Gopalan et al. (1978) studied on the biochemical composition of body muscle and its contents of Mystus vittatus. They reported 70% moisture, 18.75% protein and 2.37%fat.

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 range was 16-19%.

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.

Malek et al. (1966) determined the moisture and fat contents in Puntius stigma and the result were 72.65% and 2% respectively.

Johnstone (1918) anslyzed the amount of fat in Halibut and the values range from 0.5-9.6%, whereas the protein content remained constant at 18%.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.

Kamal et al. (2000) studied on changes of proximate composition of freshwater prawn (Macrobrachium rosenbergii) and found the initial moisture, protein, lipid and ash content were 78.34%, 18.46%, 1.8% and 1.15% respectively.

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-278mg/100g.

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.

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.

Rubbi et a. (1987) studied the proximate composition of 27 species of fresh water fishes both scaly and non-scaly and found that 72.1-836% moisture, 11.9-21.9% protein, 0.8-15% fat, and 0.8-5.11% ash.

Stansby (1954) analyzed the proximate composition and macronutrient from the edible flesh of certain fresh water fishes those were 76.8% moisture, 1.2% ash, 5%fat, and 19% protein.

Sensory, Chemical and Microbial changes of fishes:

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-odors such as musty, causing sensory rejection within 7-10 days of chilled storage.

Chang et al. (I 998) 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 ≥0°C.

Islam (1995) found that total viable bacterial count of lotia fish (Harpodon_ nehereus) ranged between 7.8xl0⁶ to l.0x10⁸ cfu/g and total coliform count ranged between l.0xl0² to 3.0xl0² cfu/g.

Leitao and Rios (2000) studied the microbiological and chemical changes in freshwater (Macrobracium rosenbergii) stored under refrigeration. They extended the shelf life freshwater of prawn 10 days at stored temperature 0°C wherein shelf life reduced 5 days when stored at 5°C temperature..

Rashid et al. (1996) reported about microorganisms associated with fine different 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 x10⁶ cfu/g total coliform count varied from 2.8 x 10¹ to 7.5 x10² cfu/g and psychrophilic bacterial count from 1.3 x 10² to 7.0 x 10⁵ cfu/g.

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.

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 odors were produced on boiled shrimp by the typical shrimp spoiler (Alteromonas).  Psedomonas putrefaciens, Pseudomonas spp. and Moraxella spp.bacteria produced less offensive odors.

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 mgN/100g of sample.

Yaoet 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.

Fish preservation at low temperature:

Chang et al. (1998) reported fresh sea bass (Lateolabax japonicus) under temperature ranging from -3 to 10°C. The shelf-life of stored sea bass at 5°C was 3 days; it was extended to about 2 weeks at 0°C, partial freezing storage at -3°C increased the shelf-life to a 4 weeks.

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 -20°C 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 I week for the air-stored samples was extended.

Materials and Methods:

Laboratories investigations:

All investigations were carried out in the laboratory of Food Processing & Preservation Division, Institute of Food & Radiation Biology (IFRB), Atomic Energy Research Establishment (AERE), Savar, and Dhaka. There the research was initiated through the collection of specimen fish.

 Specimen selected for the present study:

Specimen shellfish, freshwater prawn, Macrobrachium rosenbergii (De Man,1879) which  locally known as “Galda Chingri” was selected in this study.

Collection of specimen:

The specimen freshwater prawn, Macrobrachium rosenbergii were collected from the Karwan Bazar Fish Market, Dhaka. Usually, collection were made early in the morning then samples were taken in polyethylene bag with ice and immediately brought in the laboratory of Food Processing & Preservation Division, IFRB, AERE, Savar, Dhaka.

Preparation of samples:

The entire samples were at first randomly divided into two lots for proximate composition, microbiological and other studies. Between the two lots, one was taken in polypropyl polythene bags for preservation purpose and it was kept at -20°C.

All samples were examined before storage and after storage at the 15, 30, 45, 90 days. The samples before storage were examined for proximate composition according to AOAC (1965). The samples before and after storage were evaluated organoleptically for odor, appearance, color and texture by a panel of judges on a 9 point hedonic scale as described by Peryam and Pilgram (1957). Trimethylamine (TMA) and Total volatile nitrogen (TVN) were examined by using Conway micro diffusion technique method (Farber and Ferro, 1965)The total bacterial count (TBC), total mould count (TMC), Yeast, Salmonella and Coliform were estimated by the method of Sharf (1966).

 Analytical methods for determination of proximate composition:

The carapace of shellfish detached first and muscles were collected for determination       of biochemical composition.

Determination of Moisture:

Principle

The change of weight is estimated under certain temperature. Moisture of fish is commonly determined by drying a sample at some elevated temperature and reporting to loss in weight as moisture (AOAC, 1975)

Apparatus

Analytical balance, Drying Oven, Petri dishes, Desiccator, and Laboratory Grinder

Procedure

Two steps of experiments were done for each time of estimate of moisture content. About 5g (accurately weighed by loading Top balance, AL-1800) of fairly minced fish sample was taken in weighted crucibles (pre washed & dried at 100⁰C). Then the crucibles with fish sample were placed in oven at about 105⁰C for 5-6 hours.

Each time before weighing the crucible containing the fish muscle was cooled in a desiccator. The difference in the weight of fresh fish and content dry weight gave the moisture content.

Calculation

The percentage of moisture was calculated by the following equation:

Moisture content (g/100g of the sample) =

        (Wet weight of the sample) – (Dry weight of the sample)

                                                                                                                      X 100

                              Weight of the sample

Determination of Protein:

Principle

The Universally accepted method for the determining total nitrogen of crude protein in fish is the “Micro-Kjeldahl” method. This involves the oxidation of organic matter with sulphuric acid in the presence of catalyst and then the formation of ammonium salts and amines from the nitrogen components of fish. The solution is then alkaline and the amines and ammonia is distilled into standard acid. The solution then back titrated with standard alkali and the amount of nitrogen of ammonia calculated. The nitrogen value is then multiplied by 6.25 to give the value for crude protein.

Reagents

• Concentrated sulphuric acid
• Digestion mixture : 98 parts of anhydrous K₂SO₄ is mixed with 2 parts of CuSO₄
• 30% NaOH ( Sodium Hydroxide)
• 0.01N HCl ( Hydrochloric Acid)
• 2% Boric acid
• Mixed indicator: 0.1% Bromocerosol green and 0.1% of methyl red indicator was dissolved in 95% alcohol separately. Then 10ml Bromocerosol green was added with 2 ml of methyl solution.

Apparatus

Kjeldahl flask, filter paper, Pumic stone

Procedure

At first 1 g sample was taken for each experiment and was poured in a cleaned and dried “Micro-Kjeldahl” flask (100ml) to which 2 g of digestion mixture and 25 ml of pure concentrated sulphuric acid were added and the mixture digested by heating at 315⁰C for 5-6 hours till the mixture became clear (in “Kjeldahl Nitrogen and distilling apparatus” model No. OSK  8417). Glass beds were added to prevent bumping during digestion. The digested products were the cooled and after that 30 ml of digested product were transferred to 100 ml volumetric flask by distilled water. Then 5 ml from diluted digest was transferred in Kjeldahl dilution apparatus and distilled with 10 ml of 30% NaOH. The distillate was collected in excess of 2% boric acid solution with indicator and was titrated by 0.01 N HCl. A similar digestion and titration was carried out without samples (blank).

Calculation

The percentage of nitrogen was calculated by the following formula:

                                   (S-B) x N x 14 x C

Nitrogen (g/100g) =                                         x100

                                                A x W

Where,

          S = Titration reading for sample

          B = Titration reading for blank

          N = Strength of HCl (0.01N)

          C = Volume made up to digest

          A = Aliquots of digest taken

          W = Weight of the sampleing

The protein content was obtained by multiplying the nitrogen value by 6.25

Therefore,   Protein (g/100g) = Nitrogen x 6.25 (Bellomonte et al., 1987)

 Determination of Lipid:

Principle

The lipid content was determined quantitatively by extraction with a mixture of chloroform methanol (2:1) to a little amount of sodium chloride (0.9%) as recommended by AOAC, (1975). The mixture was allowed to stand over night and lower lipid protein was transferred to a pretreated and weighed beaker and heated to dryness. The difference in the two weights of the beaker gave the weight of the fat (Floch, 1957).

Reagents

• Chloroform-methanol (2:1) mixture
• 4% Calcium chloride (CaCl₂) solution

Apparatus

Mortar and pestle, Stoppered measuring cylinder, water bath

Procedure

At first 2 g of fish muscle were taken in a mortar and homogenized. An adequate amount of sands were added and grinded gently by a pestle. 10 ml of chloroform-methanol (2:1) mixture was added into the above sample and homogenized properly. Then filtered through a filter paper (11cm) and was collected into a pre-weighed test tube. Add 1 ml 4% Calcium chloride (CaCl₂) solution and kept it over night. The supernatant from the upper portion of the test tube was separated. The test tube was kept into the oven till drying the mixture. After drying out the test-tube was again weighed.

Calculation

                                              (Final weight-Initial weight of test tube)

Lipid content (g/100g) =                                                                         x 100

                                                   Weight of sample taken

Determination of Ash:

Principle

The ash content of a sample is the inorganic residue left after the organic matter has been burnt away at about 550-600⁰C for hours, depending on the method used. Then the residue is weighed and reported as ash.

Apparatus

Porcelain crucible, Crucible furnace and Desiccator

Procedure

About 5 g (accurately weighed by loading Top BalanceAL-1800) of macerated sample were taken in each pre weighed crucible. The crucible with the contents was first burnt over a low flame until it became charred .Then charred sample kept in an Electrical Muffle Furnace (NABER, Model No. L 51/S) at about 550-600⁰C for 4-5 hours with a view of ashing completely. To ensure the completion of ashing the crucible were again heated for half an hour. The crucibles were cooled in a desiccator and weighed. This was repealed until two consecutive weights for each sample. Ash was almost white in color.

Calculation

The percentage of ash content was determined as follows:

                                                                        Weight of the ash

Ash content (g/100g of sample) =                                                           x 100

                                                                Weight of the sample taken

Determination of Minerals:

Preparation of mineral solution

The ash (obtained from the previous experiment) in the crucible was moistened with 1 ml of distilled water and 5 ml of concentrated HCl. The moisture was then evaporated to dryness on a boiling water bath. Another 5 ml of HCl was added and the solution was evaporated to dryness as before. 4 ml of HCl and a few ml distilled water then added and the solution heated over a boiling water batharid filtered into a 100 ml volumetric flask using “Whatman” filter paper (11cm). After cooling the volume was made up to 100 ml with distilled water. The prepared mineral solution suitable aliquots were taken for estimation of calcium and phosphorus.

 Determination of Calcium:

Principle

Calcium content was determined by precipitating it as calcium oxalate and titrating the solution of oxalate in dilute sulphuric acid against standard potassium permanganate (KMnO4) solution.

Reagents

• Ammonium oxalate (6%)
• Methyl red indicator
• Strong ammonia (33%)
• Diluted sulphuric acid (2N)
• N/100 potassium permanganate ( KMnO₄)
• Glacial acetic acid, and
• Calcium chloride.

Procedure

25 ml of the mineral solution was diluted to about 150 ml in 250 ml conical flask with distilled water. A few drops of methyl red indicator were added and the mixture was then neutralized with ammonia till the pink color changes to yellow. Then added 10 ml of 6% ammonium oxalate and the solution was heated to boiling point. The mixture was then allowed to boil for a few minutes and glacial acetic acid added till the color was distinctly pink. The mixture was then kept in an oven at low temperature to settle down the precipitate. A drop of ammonium oxalate solution was added to the supernatant to ensure that the precipitation was completed. The precipitate was filtered through the filter paper and washed gradually by pouring water over funnel with filter paper with its contents, till it was free from oxalate, which was again ensured by observing that the water washing the precipitate was absolutely colorless.

The precipitate was transferred into a beaker by piercing a hole in the filter paper and washing it down gradually pouring 10 ml of 2 N sulphuric acid. After washing, the solution was heated to about 70⁰C and titrated against N/100 KMnO_4, 1 ml of N/100 KMnO₄  =0.2004mg of calcium

 Determination of Phosphorus:

Principle

Determination of phosphorus is carried out by measuring the intensity of blue color spectrophotometrically which is developed by the addition of color reagent, ammonium molybdate.

 Reagents

• Ammonium molybdate: 25g ammonium molybdate was dissolved in 300 ml distilled water; 75 ml of concentrated H₂SO₄ diluted to 200 ml was then added to ammonium molybdate solution.
• Hydroquinone solution: 0.5g hydroquinone dissolved in 100 ml distilled water and 1 drop of concentrated H₂SO₄ was added to retard oxidation.Sodium sulfite solution: 200g Na₂SO₃ dissolved in distilled water and diluted to 500 ml and then filtered.
• Monopotassium dihydrogen phosphate (KH₂ PO₄): 0.4393g pure dry KHPO₄ was dissolved in distilled water and diluted to 1000 ml. 10 ml of this solution was taken to 100 ml volumetric flask and was diluted to 100 ml. This was the standard phosphorus solution. (1 ml = 0.01mg phosphorus)

Procedure

1 ml of mineral solution was taken in a test tube and was added 1 ml of ammonium molybdate, 1 ml of hydroquinone and 1 ml sodium suphite (Na₂SO₃) solution and after addition tubes were shaken for well mixing. The volume was then made up to 15 ml with distilled water and the solution thoroughly mixed up. After 30 minutes, the optical density of this solution was measured in a spectrophotometer (KLB, Biochom, ULTROSPEC-4050) at a wave length of 660 nm against a blank solution (prepared in the same way expect the sample solution ).

The phosphorus content of the sample was read of from a standard curved prepared with standard solution (0.01-0.1mg phosphorus) following the same procedure as describe above. 

Methods for the estimation of freshness or shelf life:

1. Physical Changes by organoleptic score sheet
2. Chemical Changes by modified micro-diffusion technique
3. Microbial Changes enumerating bacterial, mould and yeast population

Organoleptic analysis:

Sensory evaluation for the detection of freshness or shelf-life of the stored fish and consumer’s acceptance was performed with high degree of reliability by organoleptic evaluation. Peryam and Pilgrim (1957) had developed a useful method for assessing the overall acceptability of food products. Nine points’ hedonic scales were used for sensory evaluation by five judges Miyauchi et al. (1964).

The hedonic scale was as follows:

9- Like extremely

8- Like very much

7- Like moderately

6- Like slightly

5- Neither like nor dislike

4- Dislike Slightly

3- Dislike moderately

2- Dislike very much

1- Dislike extremely

Incase of organoleptic analysis, the fishes were judges into 4 scales

1. Appearance

2. Color

3. Odor

4. Texture

The panel scoring sheet which was used is given below:

     Name of the panelist……………………                        Date……………………….

This analysis was performed by some expert tasters. The average score of 5 was consider to be the border line of acceptability (Miyauchi et al. 1964)

Determination of chemical changes:

Certain chemical changes in spoiling fish appear to run parallel with change in odor, texture, appearance etc. Various attempts have been made to measure freshness by establishing the quantities of some of the end product that were the result of both the extra cellular and intracellular enzymatic actions. The products were-

1. Total volatile nitrogen (TVN)
2. Trimethylamine (TMA)

The spoilage of stored fish occurred due to bacterial enzymatic action, which results in the production of various volatile compounds; among the commonest is total volatile and volatile acid. The amount of TVN and TMA were used for assessing the freshness of fish.

Determination of Total Volatile Nitrogen (TVN):

Principle

Measurement of TVN was probably the first chemical method to be used as a practical index of freshness (Tillmans and Otto, 1924) and it is still the most popular. TVN has widely been used as an index for freshness of fish (Stansby et al., 1944). According to Burgess et al. (1965) the upper limit of TVN was 30mg N/100g for acceptable condition for fresh raw fish.

Conway micro-diffusion technique was (Conway and Byrne, 1933) employed for the estimation of TVN and also TMA. This technique was carried out in the special glass dish called “Conway Dish” which had second concentric wall inside, thus having two separated chambers.

Reagents

• 10% trichloroacetic acid (TCA) solution
• 2% boric acid solution for 100 ml

1. 2 g boric acid
2. 22 ml alcohol
3. 1 ml mixed indicator
4. 77 ml distilled water
5. N/10 NaOH was added up to a color of reddish was obtained

• Mixed indicator

1. 0.1% bromocresol green in 95% alcohol
2. 0.1% methyl red in 95% alcohol
3. 10 ml(a) + 2 ml (b) mixed in a bottle

• N/70 NaOH solution: 0.571 g NaOH in 1000 ml distilled water
• Saturated K₂CO₃ (4 part K₂CO₃ in3 part distilled water)
• N/70 H₂SO₄ (0.392 ml H₂SO₄ in 1000ml distilled water).

Procedure of sample preparation:

2 g sample was taken separately in conical flask from each batch. Then 10 ml of 10% TCA solution was added with the sample and kept overnight. Then next day it was grounded in mortar and pastel and filtered through filter paper and volume made up to 50 ml in volumetric flask.

2 ml of 2% boric acid solution was taken into the inner chamber of the Conway dish. Glass lid, which was applied with grease surrounding the side way covering the Conway dish in such a way that outer chamber of the Conway dish was partially open. This was done during transferring the solution to the outer chamber to reduce little drops of solution from jumping out. Then 2 ml of sample extract was taken into outer chamber a finally 2 ml of saturated K₂CO₃ solution was added with sample. Then the lid was fixed immediately and left it over night at room temperature. On following day titration at the residual boric acid solution was done by standard N/70 H₂SO₄ solution through micro-pipette.

Blank sample:

The procedure followed to prepare blank was as TVN but only the exception was that 2 ml of the sample was not transferred to outer chamber of Conway dish.

 Determination of Trimethylamine (TM

Determination of Trimethylamine (TMA)

 Determination of Trimethylamine (TMA):

Beatty and Gibbon (1937) suggested that TMA could be used as an index of freshness of fish. During fish spoilage Trimethylamine oxide (TMAO) is reduced by acid bacteria to TMA (Poller and Linnewch, 1920). TMA contents fluctuate with seasons as well as species (TMA contents also fluctuate with habitat of fish. It’s level lower in freshwater fish and higher in marine water fish. It’s level proportional to the salinity of water (Shewan and Jones, 1957).

Procedure

In case of TMA, same procedure (procedure of TVN) was followed, except that 1 ml of 40% formaldehyde solution was added to outer chamber of Conway dish before adding K₂CO₃.

Blank Sample

The procedure was same as TMA to carryout this experiment only one exception was that 2 ml sample solution was not to the outer chamber of the Conway dish.

Determination of Microbial Changes:

The microbiological changes in raw fishes are due to bacterial, mould and yeast growth. It degraded the product quality and acceptability. The total bacterial count (TBC) was determined by decimal dilution technique followed by pour plate technique (Sharf, 1966).

 Estimation of total bacterial count (TBC), total mould count (TMC), total yeast count (TYC), total coliform count (TCC) and Salmonella count:

The microbial changes were estimated by total bacteriological count technique following Withfogel (1962). TBC, TMC, TYC, TCC and Salmonella are the valuable measures to assess the degree of freshness of fish.

In present investigation the changes in total bacteria, mould, yeast, coliform and Salmonella count was estimated by the pour plate technique in Petri dishes.

Selection of suitable media:

Dehydrated nutrient agar (Difco) consisting of peptone was used as the media for the bacterial growth at the ratio of 2.3 g per 100 ml of water and potato dextrose agar media at the ratio of 3.1 g per 100 ml of water was used for the mould growth. Yeast extract media was used at the ratio of 4.6 per 100 ml of water for yeast growth and SS media used for Salmonella count.

Sterilization of media and glassware:

The cleaned Petri dishes were sterilized in the oven (Grieve, Eocene Thermal oven) at 160⁰C temperature for 3 hours. All the glassware such as conical flasks containing media, test tubes with distilled water, tips of micropipettes and media were sterilized by autoclave (OSK-8870, OWAGA SEIKI Co. Ltd, Japan) at 15lbs pressure for 20 minutes at 121⁰C temperature before the experiment started.

Preparation of homogenized fish sample:

To prepare fish sample, 1 g fish muscle of each batch was taken and homogenized in mortar and pastel. It was then transferred to 50 ml distilled water contained in a conical flask and the whole volume was made uniform by shaking

Plating procedure:

1 ml of homogenized samples was poured into Petri dishes with micropipette. Sterilization media (Nutrient agar media for TBC, PDA media for TMC and Yeast media) of conical flask was then poured in Petri dishes in duplication and stirred carefully to spread out the sample uniformly over the media. The lids of the Petri dishes kept few minutes partially closed for solidification of the media. Then their lids covered the Petri dishes when the media got down cooled. Then different agar plates i.e., nutrient agar plates were incubated at 37⁰C for 24-48 hours, PDA plates and Yeast plates at 30⁰C for 72 hours in  incubators. All the operations were carried out aseptically in a laminar air cabinet.

Counting method:

Colonies that developed on the plates after incubation at 37⁰C and 30⁰C for 24 and 48 hours were counted with the help of colony counter (Stuart Scientific Counter- S.S Co. Ltd.). The number of bacterial, mould, yeast, coliform and Salmonella colonies per gram of the sample were obtained by multiplying the number of colonies with dilution factor. The count was expressed as colony forming unit (cfu) per gram (cfu/g).

 Data Analysis:

All data were studying using ANOVA, HSD; P<0.05.

 Proximate composition of freshwater prawn:

The levels of protein (19.08 ± 0.05 %), lipid (1.20 ± 0.03 %), ash (1.32 ± 0.04 %), moisture (78.93 ± 0.24 %), calcium (190 ± 6.46 mg/ 100g) and phosphorus (88.02 ± 7.82 mg / 100 g) were found in freshwater prawn before storage. However the moisture content was high and lipid content was low. Of the minerals calcium was higher than phosphorus.

Table 1 Proximate composition of freshwater prawn, Macrobrachium rosenbergii

Figure 1 Levels of Proximate composition in freshwater prawn, Macrobrachium rosenbergii before storage

Organoleptic Scores (OS)

The levels of organoleptic scores were gradually decreased with the progress of storage period (Figure 2). The level (6.22 ± 72.65.04) of organoleptic scores found 90 days after storage was two third the level (9.00 ± 0.00) before storage. However, levels of organoleptic scores found in other sampling duration treatments were also significantly different.

Table 2 Organoleptic Scores of freshwater prawn, Macrobrachium rosenbergii

Figure 2 Levels of organoleptic scores in freshwater prawn, Macrobrachium rosenbergii sampled before and after storage (-20ºC) at 15, 30, 45 and 90 days. Bars (mean ± SEM) with different letters are significantly different (ANOVA, HSD; p<0.05).

Total volatile nitrogen (TVN):

TVN (mg N/100 g) levels elevated over durations (Figure 3). The level (4.46 ± 0.11 mg N/100 g) of TVN found 90 days after storage was near three times the level (1.50 ± 0.07 mg N/100 g) before storage. However, levels of TVN found in other sampling duration treatments were also significantly different.

Table 3 Values of TVN (mg N/100 g) in freshwater prawn, Macrobrachium rosenbergii

Figure 3 Levels of TVN (mg N/100 g) in freshwater prawn, Macrobrachium rosenbergii sampled before and after storage (-20ºC) at 15, 30, 45 and 90 days. Bars (mean ± SEM) with different letters denote significantly different (ANOVA, HSD; p<0.05).

Trimethylamine (TMA):

TMA (mg N/100 g) levels increased over durations (Figure 4). The level (3.80 ± 0.08 mg N/100 g) of TMA found 90 days after storage was more than four times the level (0.84 ± 0.11 mg N/100g) before storage. However, levels of TMA found in other sampling duration treatments were also significantly different.

Table  4 Values of TMA (mg N/100 g) in freshwater prawn, Macrobrachium rosenbergii

Figure 4 Levels of TMA (mg N/100 g) in freshwater prawn, Macrobrachium rosenbergii sampled before and after storage (-20ºC) at 15, 30, 45 and 90 days. Bars (mean ± SEM) with different letters indicate significantly different (ANOVA, HSD; p<0.05).

Total Bacterial Count (TBC):

TBC (cfu/g) levels raised over durations (Figure 5). The level (2133.33 ± 60.09 cfu/g) of TBC found 90 days after storage was near nine times the level (233.33 ± 16.67 cfu/g) before storage. However, levels of TBC found in other sampling duration treatments were also significantly different.

Table 5 Values of TBC (cfu/g) in freshwater prawn, Macrobrachium rosenbergii

Figure 5 Levels of TBC (cfu/g) in freshwater prawn, Macrobrachium rosenbergii sampled before and after storage (-20ºC) at 15, 30, 45 and 90 days. Bars (mean ± SEM) with different letters are significantly different (ANOVA, HSD; p<0.05).

Total Moulds Count (TMC):

TMC (cfu/g) levels elevated over durations (Figure 6). The level (600 ± 28.87 cfu/g) of TMC found 90 days after storage was more than five times the level (116.67 ± 16.67 cfu/g) before storage. However, levels of TMC found in other sampling duration treatments were also significantly different.

Table  6 Values of TMC (cfu/g) in freshwater prawn, Macrobrachium rosenbergii

Figure 6 Levels of TMC (cfu/g) in freshwater prawn, Macrobrachium rosenbergii sampled before and after storage (-20ºC) at 15, 30, 45 and 90 days. Bars (mean ± SEM) with different letters are significantly different (ANOVA, HSD; p<0.05).

 Total Yeast Count (TYC):

TYC (cfu/g) levels elevated over durations (Figure 7). The level (1616.67 ± 72.65 cfu/g) of TYC found 90 days after storage was more than five times the level (316.67 ± 16.67 cfu/g) before storage. However, levels of TYC found in other sampling duration treatments were also significantly different.

Table 7 Values of TYC (cfu/g) in freshwater prawn, Macrobrachium rosenbergii

Figure 7 Levels of TYC (cfu/g) in freshwater prawn, Macrobrachium rosenbergii sampled before and after storage (-20ºC) at 15, 30, 45 and 90 days. Bars (mean ± SEM) with different letters are significantly different (ANOVA, HSD; p<0.05).

Total Coliform Count (TCC):

Coliform was absent during 90 days of preservation.

Table 8 TCC (cfu/g) sampled from different storage period

Salmonella Count:

Salmonella was absent before and after storage.

Table 9 Salmonella (cfu/g) sampled from different storage period

Proximate composition:

In this study the values obtained from the analysis of proximate composition, which includes moisture, protein, lipid, ash and mineral contents such as calcium and phosphorus. The proximate composition and mineral contents of the freshwater prawn, Macrobrachium rosenbergii was analyzed on wet-weight basis.

Moisture:

The major constituent of freshwater prawn was moisture. Average moisture content was 78.93 ± 0.24% that agreed with the findings of Rubbi et al. (1987) and Kamal et al. (2000) in Macrobrachium rosenbergii.  Finding of Aykroyed et al. (1951) and NIN (1996) in prawns was similar with present findings.

Stansby (1954) and Rubbi et al. (1987) showed that moisture of fresh fish varied from 72.1-83.6% with an average 77.64%. Brogstorm (1961) reported that fish contain 66-84% moisture. “Deshio khaiddodrober postiman” by Institute of Nutrition and Food Science (1980) reported that fresh water fishes contained 70-80% moisture. Rao (1967) observed that the muscle of Johnius curata contained 70.05 to 80.75% of moisture. Rubbi et al. (1987) reported that three species of tengra (Mystus bleekeri, M. vittatus, M. tengra) contained 76.71%, 78.51% and 73.48% of the moisture respectively. They also revealed the moisture of other fresh water fishes like fresh water eel (Macrognathus aculeatus, Mastacembelus armatus and M. pancalus) contained 76.58%, 75.34% and 75.75% of moisture respectively. Chewa (Trypauchen vagina and Gobiodes rubicundus) contained 80.11% and 82.28% of moisture respectively. Besides these, catfish (Heteropneustes fossilis), snake head (Channa punctatus, Channa striatus) contained 77.58%, 79.52% and 77.27% of moisture respectively. Tilapia nilotica contained 74.86% of moisture (Ahmed et al., 1981). Moisture content was 70% in Mystus vittatus. Kamaluddin et al. (1977). A report on Malek et al. (1966) found

moisture content 72.65% in Puntius stigma. However, moisture contents varied seasonally and also varied from species to species.

All these finding coincide well with the present results of moisture content.

Protein:

The protein content of freshwater prawn was crucial position. Average protein content was 119.08 ± 0.05%. Rubbi et al. (1987) was found 18.40 ± 0.25% in prawn and Kamal et al. (2000) found 18.46% in Macrobrachium rosenbergii.  Aykroyed et al. (1951) and NIN (1996) found 19.1% in prawns that were quite similar with the present findings.

Banu et al, (1985) studied on papda (Ompak pabda), punti (Puntius ticto), catfish (Heteropneustes fossilis) and tengra (Mystus cavasius) and reported that the protein contents of them were 14.03%, 20.12%, 18.62% and 14.24% of fish respectively. Stansby (1954) reported that freshwater fishes contained 19% protein. A statement “Deshio khaddodrober pustiman” published by Institute of Nutrition and Food Science (1980) reported that fresh water fishes contained 15-18% protein.

Oehlenschlager (1991) studied the protein content of Antarctic cods and showed that this fish contained 17-19% protein. Vlieg (1982) determined that the flesh of Jack mackerel (Trachurus declivis and T. novaezelandiae) and of blue mackerel (Scomber australasicus) contained more than 20% protein. Matto et al, (1990) estimated the protein content of 5 species of marine fish as Sardina pulchardus, Thunnus thynnus, Mugil cephalus, Atherina hepsetus and Lepidopus candatus and were 18.6%, 18.3%, 19.6%, 18.7°/o and 20.2% of protein respectively Al-Habib (1990) estimated the protein contents of six fresh water fishes and observed that these fishes contained 11%­-16.75% of protein. Akande, et al. (1991) estimated the protein content of mackerel (Scomber scombrus) which was 18.3. Chandrasheker and Deosthale (1993) observed that a wide variation exist in protein of fresh water fishes and it were 13.5% to 17.3%. Govindan (1985) analyzed the amount of protein content that was present in different fresh water and marine fishes contained 9% to 25% of protein, in most cases the limit was 16% to 19%.

The above discussion was also more or less similar to the present findings.

Lipid:

The immaterial constituent of prawn was fat or lipid. Average fat content was 1.20 ± 0.03% that was similar with the findings of Rubbi et al. (1987) in Macrobrachium rosenbergii was 1.37 ± 0.07% and Kamal et al. (2000) found 1.8% in M. rosenbergii. Finding of Aykroyed et al. (1951) and NIN (1996) both of them found 1.65% in prawns was similar with present findings.

Kamaluddin et al. (1977) reported that catfish (Heteropneustes fossilis, Clarias batrachus) contained 1% lipid. Stansby (1954) reported that certain freshwater fish contained 5% lipid. A report on “Deshio khaddodrober pustiman” published by Institute of Nutrition and Food Science (1980) reported that fresh water fishes contained lipid from 0.1-8% of fish muscle.

Akande, et al, (1991) estimated the lipid content of mackerel (Scomber scombrus) 13.2%. Mediterranean fish species like Sardina pulchardus, Thunnus thynnus, Mugil cephalus, Atherina hepsetus and Lepidopus candatus contained 5.8%, 5.2%, 5.9%, 4.04%, 0.8% lipid respectively (Matteo et.al., 1990. Vlieg (1982) determined the lipid content of jack mackerel (Trachurus declivis and T. novaezelandiae) and of blue mackerel (Scomber australasicus). The majority of jack mackerel samples had low lipid content (less than 5%) and blue mackerel had higher lipid content. Vlieg (1984) also determined the lipid content of the New Zealand marine fish species which was 0.8-3.6% that supported to the present findings.  Rubbi et al. (1987) observed that 27 species of both scally and non-scally fresh water fishes contained 0.8-1.55% lipid of fish. Chandrasheker and Deosthale (1993) showed that wide variation of lipid in fresh water fishes and it was 0.65-1.3% of fish.

The above conversation was more or less similar to the present result.

Ash:

In the current study average ash content was 1.32 ± 0.04% of prawn that was parallel with the judgment of NIN (1996) in prawns. However Rubbi et al. (1987) was found 1.02 ± 0.01% in prawn and Kamal et al. (2000) found 1.15% in Macrobrachium rosenbergii. Rubbi et al. (1987) reported that the ash of fresh water fishes both scaly and non-scaly fish ranged from 0.85% – 5.11% of fish muscle. So further investigation is requested.

Akande et al. (1991) estimated the ash content of mackerel (Scomber scombrus) which was 1.2% of fish. Matto et a1. (1990) found 1.2%, 1.3%, 1.3% and 1.1% ash content in case of Sardina pulchardus, Thunnus thynnus, Mugil cephalus, Atherina hepsetus and Lepidopus candatus respectively. A report on “Deshio khaddodrober pustiman” published by Institute of Nutrition and Food Science (1980) reported that fresh water fishes contained ash from 0.8-5.11%.of fish muscle.  Malek et al. (1966) reported that the ash content of Puntius stigma was 2%.

These values more or less supported the present findings.

 Minerals:

Minerals are indispensable constitute of fish flesh. Under mineral contents I observed calcium and phosphorus.

Calcium:

In the current study average calcium content was 190 ± 6.46 mg/100g of prawn muscle. Begum and Hoque (1986) were found 114.65 mg/100g in Shrimp which was lower than the present findings. Aykroyd et al. (1951) set up 430 mg/100g in prawn and NIN (1996) established 323 mg/100g in prawn which was more than the present findings but did not found exact similar result in case of calcium content.

Rubbi et al. (1989) found that calcium content of Chapila (Gudusia chapara) was 117.9mg, Foli (Nototerous notopterous) was 220mg, koi (Anabas testudineus) was 300mg and Kachki (Corica soborna) was 359mg. per 100g of fish muscle.  Murray and Burt (1969) reported that calcium and phosphorus vary 19 to 881mg/100g and 68mg/100gm of fish muscle respectively. Calcium contents of some local fish i.e., Clarius sp. 210 mg, Pangasius sp. 180mg and Mystus sp. 270mg per 100g of fish muscle (INFS, University of Dhaka, 1977) which were close to the calcium content of  Macrobrachium rosenbergii.

The above discussion was also more or less similar to the present findings.

Phosphorus:

In the present study average phosphorus content was 88.02 ± 7.82 mg/100g of fish muscle. Aykroyd et al. (1951) set up 310 mg/100g in prawn and NIN (1996) established 278 mg/100g in prawn which was more than the present findings. Rubbi et al. (1987) estimated the phosphorus content 50.25 ± 0.1 mg/ g which was plunge from present findings.

According to Rubbi et al. (1987) the phosphorus content of Koi (Anabas testudineus) was 75.2mg, Bele (Eleotris sp) was 75.22mg, Chapila (Gudusia chapara) was 143mg, Foli (Nototerous notopterous) and Kachki (Corica soborna) were 153.9mg and 172.30mg respectively.

Proximate composition of prawn are affected by several factors either of intrinsic and also environmental factors. Many investigations reported that seasonal variation affects the chemical composition of fish (Jacquot, 1961). It also differs due to types of food, age, size, stage of maturity, place of collection etc (Jacquot, 1964). Knowledge of the chemical value was important in order that fish could complete with other sources of animal protein food (Stansby, 1954). Processing method was dependent on the biochemical composition of fish and effects on the nutritional contents.

From the above discussion it is established that in respect of proximate composition the studied species freshwater prawn can be reported as a standard nutrition. Among the freshwater fishes it can be regarded as valuable and positively effective in human nutrition.

Quality assessment of preserved prawn:

Freshwater prawn, Macrobrachium rosenbergii stored at low temperature (-20°C) for 90 days. It is established that interaction between food and microorganism is dependent on both the intrinsic parameter like the composition of food, nutrient content for water activity, pH, oxidation reduction potential, presence of inhibitory compounds, biological status etc, and the extrinsic parameters like the temperature of storage, relative humidity and other handling, processing, storage, preservation condition etc. Fish belonging to perishable classes of food are subject to spoilage due to autolysis, oxidation and bacterial activity. Most fresh fishes are more perishable than meat, because of rapid activity of fish autolytic enzymes, presence of unsaturated oil, vulnerable to oxidative deterioration (Shewan 1975, William 1988). Bacteria invariably spoil freshly preserved fish.

Before and after storage period (90 days) at 15, 30, 45 and 90 days the organoleptic test, chemical analysis (Total Volatile Nitrogen, TVN; Trimethylamine, TMA), microbial analysis (Total Bacterial Count, TBC; Total Mould Count, TMC; Yeast, Salmonella and Coliform count) was carried out for assessing the degree of spoilage during the preservation.

Organoleptic scores:

Muscle of live fishes is more or less sterilized but after death autolytic, bacterial and other changes occur (Huss, 1986). The physical changes could be perceived with sense organs. These changes are the average of over all acceptability in respect of appearance, odor, color, texture. The average organoleptic scores of freshwater prawn, Macrobrachium rosenbergii decreased continuously and show a general trend of decreasing with storage time. Before storage the average organoleptic score was found 9.00 ± 0.00, the values were gradually degraded with storage time and after 90 days the average organoleptic score 6.22 ± 0.04 was recorded.

The degradation rate was higher in storage sample that stored at -20ºC than that of sample before storage. The average organoleptic score was 9.00 ± 0.00 at before storage sample, after 90 days the score come down 6.22 ± 0.04 which were stored at -20ºC. The sensory score due to colour, odour and texture were gradually decreased and more rapidly in samples which were stored at -20ºC temperature than that of samples before storage. Miyauchi et al. (1964) suggested that the average sensory score of 5 might be accepted in case of organoleptic test.

Thus the samples before storage and the samples stored at -20ºC was acceptable up to 90 days of storage period.

 Total volatile nitrogen (TVN):

Concerning the shelf-life of freshwater prawn, Macrobrachium rosenbergii stored at -20°C, the TVN values were found to be increased gradually with rising of storage period. TVN values are represented in Table 3. In present study the TVN value of the sample before storage was 1.50 ± 0.07 mg N/100g and the TVN value was4.46 ± 0.11 mg N/100g at the end of 90 days storage periods. It did not point out any degree of spoilage but this value depends on Non Protein Nitrogen (NPN) of fresh fish (DE and Nazrul, 1964).

It was practical that TVN value exceeds 30mg N/100g of muscle, the fish become unacceptable, Tanikawa (1935), Shewan (1975), Stansby et al. (1954) and Ota (1985) also found that the total volatile nitrogen (TVN) increased with the increase of time during spoilage and all of them suggested that 30 mg N/100gm of fish muscle should be taken as the upper limit for acceptability. Yamamura (1938) also suggested that the acceptable limit of TVN was 30 mgN/100gm of fish muscle. Wierzchowski (1956) estimated the acceptable limit of TVN in freshwater fish as 35mg N/100g of sample. According to Connel (1975) the acceptability of TVN was 30 to 35mg N/100g of sample. Khuda et al. (1964) estimated the TVN values of fresh and spoiled fish which were 19-33mg N/100g and 36-37mg N/100g of sample respectively. From all of above suggestions, 30mg N/100g of fish should be taken as the upper limit for acceptability.

From the present investigation it was found that were acceptable throughout the storage period. To detect the days when level of TVN exceed the acceptable limit research should be conducted long time.

The production of TVN was retarded considerably in the samples before storage as compared to the samples after storage. The length of storage period was well effective in the rate of TVN accumulation.

During the storage period of 90 days, the TVN value of the samples before storage was comparatively lower than the samples after storage at -20ºC. Such pattern of spoilage was also obtained from the findings of Ota (1985) and Ahmed et al. (1981). Increased value of TVN decreased the acceptability of fish. This phenomenon had been observed by other worker Venugopal et al. (1973). The present experiment also supported this result.

Trimethylamine (TMA):

TMA accumulation followed similar pattern of TVN. TMA is one of the volatile basis compounds, which is found in very low amount in freshwater fishes but this accumulates in spoiling marine fishes, as a result of bacterial reduction of TMAO (Trimethylamine oxide), reported by Ahmed et al. (1988). This means that the analysis does not give any information about early autolytic changes but only about the bacterial changes or degree of spoilage.

The TMA value of the sample before storage was 0.84 ± 0.11 mg N/100g and the TMA was found 3.80 ± 0.08 mg N/100g at the end of 90 days storage period.

Connel (1975) and Huss (1986) suggested that the TMA value ranged from 10-15mg N/100g of fish muscle was the upper limit of acceptability. But according to Yamamura (1938), in case of marine fish acceptable limit of TMA is 30 mg N/100gm of fish. Moreover, Balwin (1957) and De and Mea (1966) suggested that the presence of TMA in freshwater fish is very little and it did not give a proper indication of spoilage.

Sharma (1988) showed that TMA and TVN increase during the frozen storage of both fishes, Pink perch (Nemipterus japanious) and oil sardine (Sardinilla longiceps).

From the current research it was found that were acceptable throughout the storage period. To detect the days when level of TMA exceed the acceptable limit research should be conducted long time.

During the storage period of 90 days, the TMA value of the samples before storage was comparatively lower than the samples after storage at -20ºC.  It was found from the present study that effect of treatments differs significantly (p< 0.05).

Total bacterial count (TBC):

Total bacterial count (TBC) increased with the increase of storage period.  Shewan (1975) recommended that TBC 1.0 x 10⁶ cfu/g of fish flesh is considered as maximum allowable limit. In the present investigation TBC was found 233.33 ± 16.67 cfu/g in the sample before storage. At -20⁰C storage temperature TBC was obtained 2133.33 ± 60.09cfu/g of prawn muscle at the end of 90 days.

According to Kader et al. (1988) the acceptable limit of bacterial load was 2 million. According to international commission on the microbiological specification of foods (ICMSF, 1982) guideline, acceptable total bacterial count for fish is 10⁶cfu/g.

According to the above mentioned suggestions, preserved samples remained acceptable up to 90 days of storage period. To detect the days when level of TBC exceed the acceptable limit research should be conducted long time.

 Total mould count (TMC):

In case of total mould count (TMC), it was found that the population increased with the increase of storage period. In the present study TMC was found 116.67± 16.67 cfu/g in the sample before storage. At -20⁰C storage temperature TMC was obtained 600 ± 28.87cfu/g of prawn muscle at the end of 90 days.

Since, no work on total mould count (TMC) of fresh fish has been reported in the literature, so, the results as obtained could not be compared with the others.

From the present study it was found that the prawn samples before storage contained less mould than the samples which stored at -20˚C. And with the increase of storage time, the stored samples obtained more mould than that of the samples before storage.

Total yeast count (TYC):

Total yeast count (TYC) increased with the increase of storage period. TYC was found 316.67±16.67 cfu/g in the sample before storage. At -20⁰C storage temperature TYC obtained 1616.67 ± 72.65cfu/g of prawn muscle at the end of 90 days.

In view of the fact that, no work on total yeast count (TYC) of fresh fish has been reported in the literature, so, the results as obtained could not be compared with the others.

From the current investigation it was found that the prawn samples before storage contained less yeast than the samples which stored at -20˚C. And with the increase of storage time, the stored samples obtained more yeast than that of the samples before storage.

During 90 days of preservation period the TCC was absent in the samples before storage and the samples stored at -20˚C. According to (ICMSF, 1986) guideline, acceptable total coliform count for fish is less than 500 cfu/g. So the entire sample was acceptable during whole investigation period. To detect the days of storage when coliform developed in sample research should be conducted for long time.

 Salmonella count:

During 90 days of storage period Salmonella was absent in the samples before storage and the samples stored at -20˚C. According to (ICMSF, 1986) guideline, acceptable Salmonella for fish is 25 cfu/g. So the entire sample was acceptable during whole investigation period. To detect the days of storage when  Salmonella developed in sample research should be conducted for long time.

  Conclusions:

A significant role in the national economy is played by Prawn and shrimp industries in Bangladesh. 3% of global production of prawn and shrimp is contributed by Bangladesh. Prawn and shrimp industries have direct and indirect relationship with several million people of Bangladesh. Bangladesh prawn and shrimp having original texture and a mouth-watering taste has already been acclaimed in the world market.  Since fish is the main source of animal protein and freshwater prawn is one of the export goods, valuable source of foreign currency. So its shelf life extension is very much needed. But lack of proper techniques of preservation a significant amount of shrimps and prawns have been lost every year. So, inquest new and pertinent technology of preservation is essential to protect the national loss. Various factors contribute to the spoilage and deterioration in the quality of the products. So in the present study, attention was paid to investigation the effects of low temperature on freshwater prawn.

To preserve the freshwater prawn stored at frozen temperature. Proximate composition of freshwater prawn was studied. Contents of moisture, protein, lipid and ash were 78.93 ± 0.24%, 19.08 ± 0.5%, 1.20 ± 0.03% and 1.32 ± 0.04% respectively. Beside these, minerals were also estimated. The calcium and phosphorus contents were 190 ± 6.46 mg/100g and 88.02± 7.82 mg/100g respectively. In this view, freshwater prawn should be treated as highly nutritious fish.

To extend the shelf-life of the freshwater prawn were stored at low temperature (-20⁰C) for 90 days for determining the shelf life extension of these prawn sample some parameters such as organoleptic score, total volatile nitrogen (TVN), Trimethylamine (TMA), total bacterial count (TBC), total mould count (TMC), total yeast count (TYC), total Coliform count (TCC) and total Salmonella count (TSC) were used before storage and at 15, 30, 45 and 90 days of interval during the storage period.