INVESTIGATION ON DIFFERENT SALT CURING METHODS IN THE QUALITY AN FISHES

INVESTIGATION ON DIFFERENT SALT CURING METHODS IN THE QUALITY AN FISHES

1. INTRODUCTION

1.1. Status and importance of fish processing and preservation in Bangladesh:

Fish is one of the most useful and easily digestible the protein source in the diet of Bangladeshi people. About 85 to 90 % fish proteins is digestible and contain all essential dietary amino acid (Nilson, 1946). In the agriculture based economy Bangladesh, fish and fisheries play an important role in human nutrition, employment, income generation and export earnings. Fisheries provides 63% of animal protein; contribute 5.3% to GDP and 5.77% to foreign exchange earnings (DOF, 2005). Fish is highly perishable food item especially in a hot climate of Bangladesh where unsanitary environment and poor handling practice worsen the situation. And a high proportion of artisanal catches fails to reach the markets far from areas of production due to the poor handling practice and very often due to the high cost involved in transportation and due to poor storage facilities.

In Bangladesh as the transportation and the process of handling of fish and fish products are poor; a huge quantity is spoiled during transportation of product from one end of the country to the other. Moreover, fishermen do not get proper return for their harvest during the peak period of harvest due to the seasonal supply of fish and lack of preservation facility in Coastal area of Bangladesh. Even sometimes they just have to abandon the spoiled harvest, as it becomes unfit for human consumption The lack of adequate fish handling, preservation and processing methods and storage facilities contribute significantly to the low supply of fish to poor rural dwellers which form three quarters of the population in developing countries whatever may be amount of harvest, as it has got no value until it reaches the consumer as a food Therefore appropriate fish processing and preservation methods are very much essential in order to minimize the fish wastage due to spoilage and to improve the fish quality for proper consumption.

1.2. Status and Potential of fish preservation and processing in Bangladesh:

Dry and dehydrated fish is one of the most important export items in Bangladesh. The demand for dried and dehydrated fish as an export item is increasing day by day. According to the Export promotion Bureau in 2003-2004 the quantity of exported dry fish andfish product from Bangladesh was 472 tons which valued 4.16 crores taka and in 2004-2005 it was 272 tons and valued 3.71 crores taka. On the other hand, in 2003-2004 the quantity of exported salted and dehydrated fish was 377 tons and valued 1.38 crores taka and in 2004-2005 it was 770 tons and valued 28.97 Crores taka indicating increased trend in the earning of more foreign currency from previous year exporting salted and dehydrated fish products. It is also clear that the demand of these fish product is increasing than that of the unsalted dried fish products.

1.3. Fatty fish is a potential candidate for preservation and processing:

From the time immemorial hilsa fish have been found to play very important role in the field of national economic development, employment and in the supply of protein enrich food to the people of our country. At present the country produces near about 2 lacks metrictons of hilsa fish which cost about 2000 Crores taka. Chilled hilsa exported to India was 3584 metrictons and export earning valued about 51.95 Crore taka. Source for the year 2004- 2005 regular earning statement from certificate issued. (Jatiya Matsya Pakkah, ’05) hilsa fish alone contributes about 12% – 13% of the total fish production of Bangladesh and it also contributes about 1% to the GDP. It also earn 50-60 Crores taka each year through exporting of about 2000-8000 M. tons of hilsa fish to India and many other countries (Halder et al., 2004)

About 4.5 lacks of fisherman are engaged on a full time and part time basis in harvesting. In the same way 20-25 lacks people are involved directly or indirectly in distribution marketing, transportation etc.

Hilsa fish is a very popular and tasty fish among Bangladeshi people living at home and abroad. It has been found to contain higher level of protein, fat and minerals. The fish has been found to contain specialized type of fat having about 50% of unsaturated fatty acids out of which about 2% w-3 fatty acids exists. This fatty acid reduce the human blood cholesterol level thereby reducing the level of heart diseases (Rao et al., 1977). Hilsa fish protein also contains nine different types of fatty acids which human stomach cannot synthesize. Moreover it contains higher quality of Ca, P, Fe and Vitamin A, D including trace level of Vitamin B. Hilsa fish liver contains 120 IU of Vitamin A (Halder et al., 2004)

1.4. Role of salt in fish preservation and processing:

Salting is a process of fish preservation where the water content is reduced by the penetration of salt, whereby the activity of moisture of the spoilage bacteria is stopped or reduced, Sodium chloride diffuses into the fish flesh by a dialysis mechanism and water is diffused outside due to the osmotic pressure between the brine and fish muscle solution. Two basic principles describe the mode of action as well as significance of salting:

1. Removal of water from the deepest part of the flesh quickly enough to reduce water activity;
2. Penetration of salt quickly enough into the deepest part of the flesh to lower the water activity. A concentration of 6-10% salt in fish tissue can prevent the action of most spoilage bacteria. A saturated salt solution has a water activity of 0.75 (Horner, 1992). Maximum salt concentration attainable in fish tissue under saturated salt solution is around 26% under normal temperature.

Salting is done in combination with sun drying and smoking for variety of species. But in case of fatty fish species like hilsa fish salting is the choice of traditional fish preservation.

1.5. Problem statement:

Salting is a suitable processing and preservation technique for country’s greatest harvest of fatty fish. There is however several problems found to be associated with the process as discussed below:

1. The producers do not follow the regulation regarding public health and sanitation.
2. The fish on the onset of spoilage or already spoiled one are used. For salt fermented products however, partial spoilage may be necessary for the development of special flavor and texture.
3. The fish or the cut pieces are not washed in most cases.
4. The raw material is contaminated by bacteria and pathogens during scaling, gutting, dressing and cutting as unclean knives, containers or tools are used.
5. Low quality of pure salt is used that inhibits the development of good texture, attractive color and nice flavor of the product.
6. Salt: Fish ratio is not properly maintained.
7. In wet salting, cut pieces are floated on the surface of the salt solution and the fish product quality is deteriorated as they come in contact with the air.
8. Sometimes excess salting may denature protein and impact upon the sensory and biochemical properties of the final product.
9. Semi fermented hilsa is not always well protected and covered in the earthen hole. Rain water and mud may enter and insects and rodents may attack and spoil or contaminate the products.
10. Packaging and storage are not appropriate and hygienic.
11. Very often rancid off flavor develops in the product during prolonged storage for the purpose of marketing.

1.6. Activities and expected outcome

? Hilsa and sorpunti fish were collected in high fresh and extremely acceptable condition as raw material for the present problem study.

? Commercially salt cured hilsa fish were also collected from the local fish market for using it as a raw material to compare its quality and storage stability with that of the experimentally salt cured hilsa and sorpunti fish.

? Attempts were made to produce best quality salt cured hilsa and fish. By using different salt curing methods and to adopt the best quality product producing salt curing method for future use in the laboratory as well as the industrial level.

? Our activities also includes studying the present problem on the basis of analytical, technological, chemical and physical aspect with a view to find out the best processing method suitable for salt curing of his and serpentine fish.

? We have also tried to evaluate the storage stability of all the salt cured products produced in the laboratory as well as commercially salt cured product collected from the local fish

Market.

? All possible variable/ parameters related to quality, storage stability etc. were utilized as per facilities available in the fish technology laboratory IFST,BCSIR, Dhaka.

? we have also taken steps to make a comparative study on the technological and qualitative gradation among all of the salt cured products studied in the present thesis work.

? Attempts were made to compute and interpret all of the available results to give a crystal clear idea regarding the findings of the present study for its benefit with immediate effect and afterwards.

1.7. Research Needs

? To take research initiative for following regulation regarding public health and sanitation while producing salt cured hilsa and sorpunti fish in laboratory level.

? To ensure on the freshness and acceptable range of the raw material so that no spoiled or already spoiled fish taken for salt curing process.

? Research work are also needed in order to find out where there is any variation in quality and storage stability of the product if washing and cleaning of the whole fishes and cut fishes are ensured.

? Clean knives, containers or other tools used during the salt curing process have to be ensured to be clean and contamination free so that the raw material of the process may remain contamination free by bacteria and pathogens.

? Pure salt of standard quality, proper salt fish ratio are to be maintained in order to avoid the problems already faced by the existing system of salt curing of hilsa fish on commercial basis.

? During wet salting the salted fish are to be kept in the brine solution so that the fish cannot come in contact with the air in order to avoid development of rapid rancidity.

? Research activities are also needed in the field of proper packaging and storage of the finished product in order to ensure stable quality product during storage and marketing.

? Research works are also needed to avoid excess salting which are not favorable for producing a quality salt cured fish product of standard quality and storage stability.

? Research and development works are needed in order to verify all of the steps starting from collection of fresh acceptable fish samples and to the finishing of the salt curing process together with quality assessment and storage stability study. Simultaneously making comparison with that of the commercially salt cured hilsa fish product procured from the local fish market.

1.8. Scope and limitation

? There was a scope for analysis of the nature and category of free acid present in the lipid portion of the experimentally and commercially salt cured hilsa and sorpunti fish. We know that some of the free fatty acids are essential for maintaining sound health and preventing cardiac diseases.

But we could not work in this field of free fatty acid analysis due to lack of GLC in the fish technology laboratory.

? We could compare between smoke cured and salt cured hilsa fish products during the present assignment, but due to time constrain us could not make such effort.

It was our limitation.

? In addition to find out the level of Fe, Ca, P and sugar there were scope for finding the level of Zn, Cu, in the salt cured hilsa and sorpunti fish.

But due to lack of the required chemical we become unable to do this sort of analysis.

It was our limitation.

? There was a scope for field trial of the salt cured fish produced in the laboratory together with the commercially salt cured fish to have an ideas about the consumer level of acceptance.

A field trial of the experimentally salt cured hilsa and sorpunti fish with that of the commercially salt cured hilsa fish collected from the local fish market were given in the fish technology laboratory and in the Food Quality Control Laboratory and the result of the trial goes infavour of the salt cured hilsa fish (mixed), two types of salt cured hilsa fish with sugar and preservative.

? There was a scope to see the amino acid present in all of the salt cured fish samples studied in the present thesis work.

But it was not possible for doing so due to unavailability of the amino acid analyser in the fish technology laboratory.

It was our limitation for the present thesis work.

1.9. Objectives:

No research activities can be carried out without any aims and objectives. As such we have considered all of the works already carried out in this field and we have also taken into considerations the problem statements mentioned above regarding salt curing of hilsa fish in Bangladesh. Having in mind all of the factors mentioned in the different paragraphs of the introduction chapter we deem it fit for undertaking the present thesis work with the following aims and objectives.

? To take an attempt for producing better quality salt cured hilsa and sorpunti fish the laboratory level.

? To find out and adopt the best method of salt curing especially for hilsa fish.

? To observe the quality as well as the storage stability of the salt cured experimental fishes with that of commercially salt cured fish collected from the local fish market.

?To give the suggestion and recommendation so that the fish processing personnels consumers as well as the academic professionals may be benefited economically, nutritionally and for academic fitness.

2. LITERATURE REVIEW

It is well known to all of us that just after harvesting the fish spoilage of fish is caused by autolysis, enzymatic and by bacterial action. As a result quality of harvested fish is reduced due to spoilage loss during post harvest period. To minimize the spoilage loss of the fish, various traditional and old methods as well as modern ad updated technique of fish processing and preservation have been introduced by the fish technologist, fish processors and by the academic professionals. As a ready reference we would like to sight some of the research work done in the field of fish processing and preservation those have already been carried out at home and abroad.

The literature reviewed here includes few works on salting and salted products of those species which are considered to have direct relationship with the present research work.

· The main features of salting are the removal of sufficient water from the fish tissue and its partial replacement by salt. As a result, a condition is arrived when spoilage activities are slowed down. (Tressler and Lemon, 1951; Cutting, 1955; Cutting and Waterman, 1965)

· Shewan (1961) stated that though many methods of salting had been developed in different parts of the world, they were in general variants of the two: (1) dry salting and (2) Pickling

· According to the amount of salt use Ray (1936) used two terms – (1) Hard or heavy salting and (2) Light salting

· In brine salt curing method, the amount of salt required to make a saturated brine solution is to use 26% salt by weight with pure clear drinking water (Balachandran, 2001).

• The portion of salt varies from 10 – 35 % of weight of fish depending upon kind of fish (Tressler and Lemon, 1951).

• Salting starts from the movement of fish surface coming in contact with salt is not a question. (Voskresensky, 1953).

• If the fish is cured in non stirred brine, it caused the slower uptake of salt by the fish tissue (Berezin, 1948; Minder, 1948(b); Levanidov, 1948; Nevtonov, 1935; Voskresensky, 1953).
• Movement of water from or to the fish tissue is due to osmotic diffusion is the main cause that drained weight change (Ray, 1936; Voskresensky, 1953).
• During dry salting weight loss by the fish is the highest and in brine curing due to extraction of water from muscle tissue by osmosis fish becomes hard (Voskresensky, 1953).
• Extraction of water from muscle tissue by osmosis during salting contracts the muscle and it becomes hard (Voskresensky, 1953).
• The most of the brine formed and weight loss of the fish took place during initial stage of salting process and a change in weight was affected by salt concentration (Ray, 1936).
• Minder (1952) and Nevtonov (1935) reported that during final stage of salting an increase in the drained weight took place.
• During salting of Herring it was found that loss of soluble substance in brine was only about 0.5% of the original weight of the fresh fish (Levanidov, 1948).
• The principle of salt curing is the withdrawal of sufficient water form the fish tissue and simultaneous penetration of salt into the fish tissue, a phenomenon known as osmotic diffusion. (FAO report, 1976).
• Joseph et al. (1986) studied the chemical, bacteriological and organoleptic quality of cured fish of tamilnadu coast. They reported the main defect of the cured product, which were found to be unhygienic processing, inadequate salting, use of poor quality salt and incomplete drying.

• Doe, (1985) observed the spoilage of dried fish and the effect of water activity and temperature on spoilage organisms. Reported that spoilage of dried fish may be due to bacterial, fungal, brining and other reaction, all of which are temperature and water activity dependent.

• Shewan (1977) observed that storage characteristics of fresh water fish when kept in ice do indicate that patterns of spoilage are similar to those of marine species.

• Disney et al. (1974) observed that certain physical and chemical characteristics, including the shape, size and fat contents can all combine to influence the duration of iced storage.

• Poulter (1978) examined R. brachiosoma from South China Sea. At -10°C the fish remained in prime condition for three months and they were just acceptable after nine months. At -30°C the storage life was at least 12 months. The rate of freezing had no significant effect on storage life. The lipid content of fish was low, 1 – 3 %, but the changes in peroxide value indicated that lipid oxidation was the major factor influencing the storage life. The percentage of soluble protein nitrogen showed no large decrease during the storage trials.

• Dyer (1986) examined that water temperature, food supply, breeding cycle, size of the fish and the way in which they were killed affect the composition. For temperate fish species all these factors influence the frozen storage life.

• Cooper and Noel (1966) and Doe, (1982) studied the effect of salt content on the sorption isotherm was similar at constant water activity , quantity of sorbed water increase with decreasing salt content were highly significant and water activity ranged from 0.30 – 0.75.

• Muslemuddin et al. (1986) described the effect of temperature and salt concentration of brine during short term preservation of Mola fish. They suggested that shelf-life of fish found to increase with the increase of salt concentration in brine.

• The composition of fish varies widely from species to species and even within the same species from one individual to another (Stansby, 1963).

• Jacobs (1958) stated that fish also vary in composition at different sections of the body of the same fish.

• In general, oil and moisture content varies inversely with one another and most of the species has a range of total liquid (moisture + oil) from 80 – 83 % of the total body constituents (Thurston et al, 1959).

• The greatest fluctuation of oil content is found in dorsal, lateral and belly flap parts in decreasing order and the variation in condition seemed to be more important than either seasonal or size variation (Thurston, 1958).

• In general, dried fish contains more nutrients than fresh fish (Khuda, 1960).

• According to Shaha and Chowdhury (1961), the protein content of locally cured dried fish was lower than that of dried fish produced by Govt. Yard in India.
• Rahman et al. (1978) observed that there was no loss of protein and lipids as a result of drying of Rohu fish.

• Martenik and Jacobs (1963) and Doha (1964) indicated that nutritionally the dehydrated products are very good and neither the nutritive value nor the digestibility of the protein is adversely affected due to dehydration process.

• Beatty and Fougere (1957) demonstrated that thickness of flesh has a pronounced effect on salt uptake. Thicker fillets obtained lower amount of salt than that of thinner one when curing time in both the cases remained the same.

• Impurities of salt effect the color, texture, taste and flavour and have direct bearing on salt penetration. (Klaveren and Lagendre, 1965; Dyer and Gunnarsson, 1954; Carter, 1932).

• Salt penetration into the fish flesh was retarded by the impurities of salt especially by Magnesium and calcium (Berezin, 1948; Minder, 1948b).

• According to Waterman (1976), bacterial action stops at 25% water content and moulds ceased at 15% water content for unsalted fish and that for salted fish 35% – 45%,

• While moisture removal, drying rate was different at different stages. In the first stage (the constant rate period) evaporation from the surface controls the rate. Moisture migrates from interior to the surface by the capillary and diffusion forces and is vaporized. During this period the surface of the solid remain saturated with liquid water and drying rate depends on temperature, the speed of movement of water, air velocity, dryness of the air and thickness of the fish. In the second stage, (the falling rate period), the water diffusion rate from interior can not maintain a sufficient flow to the surface to sustain the initial maximum rate of evaporation. The evaporation rate starts to fall and the water content of the fish approaches an equilibrium value. The drying rate depends on the temperature, internal diffusion, thickness and leanness of the fish. Cutting reports that in an atmosphere of 75% RH, drying rate may be very low. In Bangladesh, the ambient RH is 85% in wet seasons and drying is not possible with this RH. (Charm, 1963; Burgess et al. 1965; Jason, 1965; and Waterman, 1976).

• Jason (1958) found that drying in the constant rate period was found to be controlled solely by the ambient atmosphere; drying in the falling period was found to be occurring in two distinct phases each of which was characterized by a Fickian water diffusion coefficient. Concerning these two diffusion coefficient, both were found to be isotrophic and to depend upon the fat content of the fish; both also followed an Arrhenius-type variation with temperature. The coefficient for the first falling rate period was found to be greater than that for the second falling rate period. Jason also studied drying of heavily- salted cod in the falling-rate period. found that diffusion coefficients for the first falling rate period were approximately equal for both heavily salted and unsalted fish, while coefficients for the second falling rate period were considerably lower for salted than for unsalted fish. also found that salted fish entered the second falling rate period sooner than unsalted fish and also noted the salt-protein crust which is formed during drying of heavily salted fish.

• Jason (1965) investigated the effect of salt content on the diffusion coefficients. Found that both were sensitively dependent upon the amount of fat present in the muscle.
• According to Doha (1964), low concentration of salt in dry fish offers better conditions for bacterial growth during storage.
• Cutting (1955) reports that in an atmosphere of 75% RH very little drying occurred.
• A few moulds grow at a RH of 65% and most moulds grow rapidly at a RH of 75 %.( Waterman, 1976).
• Wang et al. (1954) while working with characteristics of muscle tissue dehydrated by fish drying techniques observed that reconstitution properties were influenced by six factors which were.

1) Orientation of muscle fibres

2) Thickness

3) Temperature of rehydrating solution

4) Osmotic pressure

5) pH of rehydrating solution and

6) Rehydration under vaccum

• ? Von Loesecke (1955) reported that reconstitution means the replacing of water in dried foodstuffs though the process of reconstitution a large quantity of the water which was originally present is assimilated. The ability to reconstitute is one of the most distinctive qualities of dried fish products.

• ? Wodicka (1958) while worked with texture of foods reported that the ideal type of dried foods are specifically processed products which have rapid reconstitution properties and closely resemble the freshly prepared good items.

• ? Von Loesecke (1955) observed that not all products reconstitute to 100 % of their original state because of inherent differences in their chemical composition.

• ? Connell (1957) reported that dried fish produced in a one current of air attain properties which is different from those of the fresh fish and understanding of the difference in quality among various dried food products have been found to have a clear picture from the consideration of reconstitution properties. Considering the fact Smithies (1962) observed that unsatisfactory processing condition or prolong storage may give a quality to a food product that will require periods more and will be difference from the tenderness and juiciness from the control.

• ? According to Acker (1967); Rockland (1969) and Labuza (1972), the temperature, atmosphere, moisture and the relative nature of the components comprising the foods governed the chemical and physical reactions responsible for food alteration during storage.

• ? Taylor (1961) reported that equilibrium relative humidity of a foodstuff determines whether it will gain or loss moisture in a particular environment and so this property is more relevant to storage behaviour than in moisture content.

• ? Gur-ariech et al. (1965) showed that data on moisture sorption of foods are useful in the determination of techniques for processing and packaging of dehydrated foods and for the prediction of undesirable chemical, physical and microbial changes that might occur during storage of dehydrated foods.

• Fish are highly perishable, and they will spoil rapidly if improperly handled. Fresh iced fish generally are spoiled by bacteria, but dried fish are usually spoiled by fungi (Jay, 1992).

• The simplicity of the salting process, the low cost of production and the ease with which it combines with other preservation methods, such as drying or smoking, has led to its popularity and extensive use (Berhimpon et al., 1991).

• Curing salt (containing sodium nitrate) can be added to the pickle to delay spoilage and control microbial activity during storage (Pederson and Meyland, 1981).

· In manufacturing processed fish products, adding certain amounts of NaCl assists in the extraction of salt-soluble proteins and the formation of a sticky paste of fish meat. The development of the gelled paste might be due to the formation of a protein network structure or polymerization of myosin-heavy chains (Kumazawa, et al., 1995).

· Once fatty compounds are oxidized, the breakdown products of lipid oxidation potentially can react with proteins and vitamins, leading to a loss of nutritional value and quality of the fish (Pokorny, 1981).

· In breaded processed fish products, NaCl is used in the predusting step to enhance adhesion of the batter to the fish (Claus et al. 1994). Processing fish has created a niche market for products that otherwise would have been wasted because of overharvesting of species, low consumer appeal, high processing costs, or limited shelf life.

· Use of crude NaCl (which contains impurities such as chlorides, sulfates, calcium, and heavy metals) accelerates lipid oxidation during fish processing and will adversely affect the overall quality of the finished product (Yankah et al., 1996).

· Because spores of Clostridium botulinum are known to be present in the viscera of fish, any product that will be preserved by salting, drying, pickling, or fermentation must be eviscerated prior to processing. Without evisceration, toxin formation is possible during the process even with strict control of temperature. Evisceration must be thorough and performed to minimize contamination of the fish flesh. If even a portion of the viscera or its contents is left behind, the risk of toxin formation by C. botulinum remains. Small fish, less than 5 inches in length, that are processed in a manner that prevents toxin formation, and that reach a water phase salt content of 10 percent in refrigerated products, or a water activity of below 0.85 (Note: this value is based on the minimum water activity for growth of S. aureus) or a pH of 4.6 or less in shelf-stable products, are exempt from the evisceration requirement (FDA, 2001).

· According to FDA Guidelines, Controlling pathogen growth and toxin formation by drying is best accomplished by:

1. Scientifically establishing a drying process that reduces the water activity to 0.85 or below, if the product will be stored and distributed unrefrigerated (shelf-stable);

2. Scientifically establishing a drying process that reduces the water activity to below 0.97, if the product will be stored refrigerated (not frozen) in reduced oxygen packaging;

3. Designing and operating the drying equipment so that every unit of product receives at least the established minimum process;

4. Packaging the finished product in a container that will prevent rehydration.

3. MATERIALS AND METHODS

3.1. Basic materials necessary for fish salting process:

Pure common salt, turmeric and two varieties of fatty fishes were used as raw material for the present research study.

3.1.1. Fishes

Introduction of the fishes:

The two varieties of fatty fishes were used for this comparative study of different salting process. The fish varieties were:

Hilsa (Tenualosa ilisha, Hamilton-Buchanan, 1976)

Sorpunti (Puntius sarana, Hamilton-Buchanan, 1822)

Collection of the fishes:

The raw fishes had been purchased from the local fish market of Dhaka city in the early hours of the day and the fishes were brought to the Fish Technology Section, IFST, BCSIR, and Dhaka for conducting the research activities in the month of February, 2006.

The whole experimental period covered 10 months of duration started from February, 2006 to November 2006.

Feature of the External morphology of the fishes:

For each species, ten samples of fishes had been taken for the experiment. The length and weight of each sample of hilsa, sorpunti fishes were estimated and an average length and weight for each species were determined and recorded.

Table1: Showing average length and weight indicating the external morphology

For each species.

Fish species	Number of fishes	Average length (cm)	Average weight (g)
Hilsa	10	35	445
Sorpunti	10	22.5	190
Commercially salted Hilsa	10	27	150

3.1.2. Common Salt:

Sodium chloride (NaCl), also called salt, common salt, and table salt, is generally recognized as a safe, antimicrobial and incidental food additive. Salt has been used as a seasoning and flavor enhancer as well as a preservative or curing agent, had been purchased from the local market.

Salt Purity:

For salting, the purest salt with the finest grain available had been used. Salt which are virtually chemically pure (less than 1% impurities) results in fish with a milder, more pleasant flavor, which do not need prolonged freshening. The finer the salt, the more rapidly the brine forms, and thus the more rapidly the flesh is penetrated with salt. Standard curing salt was collected from the local market.

Plate 1. Showing hilsa fish in fresh and highly acceptable condition

during taking measurement in the laboratory

Plate 2. View of sorting of the pieces of hilsa fish for salt curing by

application different salt curing methods in the laboratory

Plate 3. Showing sorpunti fish in fresh and highly acceptable

condition during taking measurement in the laboratory

Plate 4. Showing pieces of sorpunti fish for curing by different

salt curing methods in the laboratory

3.1.3. Turmeric:

Fresh dried and powdered form of turmeric is used as a newly introduced element in salt curing method with a hypothesis that it would work as –

· A protection against insect, pest, fungus and other pathogens

· A longer term preserver

· Taste increasing element

3.1.4. Sugar: Sugar, salt, preservative treated hilsa fish product were used as a research material in the present study with a view to introduce a new dimension in the salting process and extend its shelf life with the preservative action of the sugar itself. To confirm regarding the enhancement of quality of the quality of the sugar, salt, preservative treated hilsa fish we have produced this product in the laboratory so that we can control all the influencing factors for improvement of quality. The added sugar have been considered to act as a preservative along with added preservative.

3.1.5. Preservative: Addition of preservative actually slowed down the liberation of soluble amino compounds in the salted product. The amount of amino acid residues in peptides decrease strongly than that of the free amino acids. Addition of preservative during storage improve the taste. Added preservative actually slowed down proteolysis activity which is based on the retarded growth of enzyme producing bacteria. The effective action of the preservative was to a certain extent slightly different in its use for instance benzoate and POB-OMe were relatively stronger in preventing the liberation of peptides and sorbate was the least effective.

3.2. Methods applied for the present experiment

· General salt curing methods:

3.2.1. Dry salting:

During this experiment the raw fishes were eviscerated, cleaned, washed, weighed and had been enrolled by dry salt (fish weight : salt weight = 3:1), stacked in containers and stored for a salting or curing period, at room temperature.

In this method, the extracted water of the fish due to salt action had been removed from the container. Thus the fishes are always allowed to remain in dry condition for the production of dry salt cured fish.

During this process of dry salting the change in weight, salt content, moisture content and physical acceptance of the fish have been determined and observed. Its quality was also observed by chemical method.

3.2.2. Pickle salting:

While applying this method, the raw fishes were eviscerated, cleaned, washed, weighed and had been enrolled by dry salt (fish weight : salt weight = 3:1), stacked in containers and stored for a salting or curing period, at room temperature.

The salt reacts with the fish and water is extracted out from the fish-body and a salt-solution is formed. Thus in this method, the fishes are always allowed to remain in such solution for the production of pickle-cured fish.

During this process, the changes of fish in weight, salt content, moisture content and physical acceptance of the fish have been determined and observed. Its quality was also observed by chemical method.

3.2.3. Brine salting:

During this experiment the raw fishes were eviscerated, cleaned, washed, and weighed. A 30% salt solution is prepared (30 gm salt in 100 ml water) which is called brine. The fishes are kept at this saturated brine solution stacked in containers and stored for a salting or curing period, at room temperature for the production of brine salted fish.

During this process of dry salting the change in weight, salt content, moisture content and physical acceptance of the fish have been determined and observed. Its quality was also observed by chemical method.

3.2.4. Mixed salting:

This method is a combined form of dry-salting and brine-salting method.

At first, the raw fishes were eviscerated, cleaned, washed, weighed and had been enrolled by dry salt (fish weight : salt weight = 3:1). Secondly, another 30% brine solution is prepared and added to that fish, stacked in containers and stored for a salting or curing period at room temperature.

Thus in this method, the fishes are always allowed to remain in such dense salt solution for the production of mixed-salted fish.

During this process, the changes of fish in weight, salt content, moisture content and physical acceptance of the fish have been determined and observed. Quality assessment was done by chemical method.

3.2.5. Preparatory salting:

Fish are scaled, eviscerated and soaked in dry salting vat for duration depending on the types and size of the fish (2-8 days). Salted fish is soaked in freshwater tank and thus preprocessed for a sundrying.

Methods applied for the salting of hilsa fish

1. Dry salting

2. Pickle salting

3. Brine salting

4. Mixed salting

Methods applied for the salting of sorpunti fish

1. Dry salting

2. Pickle salting

3. Brine salting

4. Mixed salting

5. Preparatory salting

A B

Plate 5. Showing salted fish products A) hilsa B) sorpunti fish produced by different

salt curing methods in the laboratory

3.2.6. A new dimension of preservation and processing of fatty fish by using salt, sugar and preservative

3.2.6.1. Pickle salting: In addition of the pickle salting method, 5% sugar and trace amount of preservative were added and the salted fish samples were kept them in refrigerator. The weight of the fish were taken after a certain time interval.

3.2.6.2. Mixed salting: In addition of the mixed salting method, 5% sugar and trace amount of preservative were added and fish samples were kept in refrigerator. The weight of the fish were taken after a certain time interval.

Plate 6. Showing the salted hilsa fish product treated with sugar and preservative

Salting of roe

1. Brine salting

2. Brine salting with sugar and preservative

Plate 7. Showing roe in fresh and highly acceptable condition

A B

Plate 8. Showing salt cured roe A) In brine salt curing method B) In salt, sugar and

preservative

Plate 9. Showing commercially salt cured hilsa fish product

collected from the local market

Plate 10. Showing salt cured hilsa fish product produced in the

laboratory by following commercial salt curing process

3.3. Fish storage

The processed and preserved sorpunti fish after completion of the salt-curing process have been found to attain a level of quality. But storage of the salt-cured product is very important to extend its shelf-life. Because in a high humid and high temperate weather of Bangladesh, the cured fish products losses its quality by adsorbing more water from the surrounding air and thus facilitating the growth of bacteria, moulds and so on. In order to verify extended shelf-life of the salt-cured fish product of the present study, three kinds of storage conditions were designed and developed in the laboratory which are mentioned as follows:

1. Storage plastic pots (in ambient temperature).

2. Storage in plastic package (in ambient temperature).

3. Storage in plastic package in refrigerator

Procedure:

After 7 days, when the fishes become dry and crispy, each kind of salt-cured fishes are subdivided into 3 portions of amounts. Each portion are then weighed and kept into 3 different storage conditions into the laboratory. The stored products are observed at intervals to record the sensory scores.

In order to reduce the adverse effects of NaCl on lipid oxidation, color, and flavor of fish, fish and fish products are handled, prepared, and processed under refrigerated temperatures. Low temperatures reduce the rates of oxidative reactions and retard microbial growth.

A B

C

Plate 11. Showing different storage conditions of salted sorpunti fish

kept in

A) Polythene at ambient temperature

B) Plastic pots at ambient temperature

C) Polythene at refrigerator

3.4. Biochemical composition analysis

3.4.1. Moisture determination:

Procedure:

Determination of moisture content of the raw as well as salt-cured fishes was conducted by AOAC method (AOAC, 1975). For this purpose, some washed and dried tarred porcelain basins of known weight were taken. Each type of salted fish samples were taken into each basin and weighed. The samples were dried into oven at 104°C for 24 hours in order to remove the moisture. Drying, cooling and weighing were continued until constant weight was established.

Calculation:

% of moisture = x 100

Plate 12. Showing an electronic balance while taking weight of

fish muscles samples.

Plate 13. Showing porcelain basins with fish samples during a step of

moisture content determination

3.4.2. Protein determination:

Micro-Kjeldhal method:

The crude protein of the fish was determined by Micro-Kjeldhal method. The basic principle of this method involves the converting of the nitrogenous protein into (NH₄)₂SO₄, when boiled with H₂SO₄. (NH₄)₂SO₄, on distillation with excess of sodium hydroxide (NaOH), gives ammonia (NH₃) which is absorbed in boric acid solution containing methyl red. The amount of N₂ absorbed in boric acid is determined by titration with N/70 H₂SO₄.

Procedure: the process involves the following steps:

Preparation of digested solution:

Some pieces of filter paper were taken and weighed in an electronic balance. The each type of fish samples were taken in each piece of filter paper and kept a record of the identification of the fish sample-type and they were also weighed. A mixture was prepared by adding 20 ml concentrated H₂SO₄ (20%) with the addition of digestion mixture (a white powder).This mixture was kept in the Kjeldhal flask Buchi-digestion unit. Then the weighed fish sample with filter paper also kept in that Kjeldhal flask of digestion unit until the mixture becomes clear. Thus a water-colored digested solution is prepared.

Preparation of sample solution:

Water was mixed with the digested solution at an amount to make the total solution of 100 ml. 5 ml of sample solution was added with 10 ml NaOH and 150 ml water. The prepared sample solution was kept into Micro Kjeldhal distillation unit for 50 minutes and the distillate were collected in 5 ml of 2 % boric acid and were titrated with N/70 H2SO4.The titration readings were recorded and calculated as follows

Calculation:

The percentage of nitrogen in the sample was calculated by the following equation:

% of N₂ = (Titration Reading – Blank Reading) X Strength of Acid X

0.0002 X 100/5 X

% of protein = % of total N₂ x 6.25

Plate 14. Showing various steps of protein content determination of the fish samples

by micro-kjeldhal method in the laboratory

3.4.3. Fat determination:

Procedure:

The fat determination procedure had been completed by following the Bligh and Dyer (1959) method. For this purpose, each type of salted fish samples was taken and muscles were cut into pieces and weighed. The samples were dried into oven for 24 hours in order to remove the moisture. Oven dried samples were finely mashed. A mixture of solvent (Chloroform: Methanol = 2 : 1) were added and kept in an airtight conical flask for 24 hours. Fat content reacts with that solvent and remains in the solution. After 24 hours, the solution of the flask is filtered in other weighed flask. Then these flasks were kept on a hot-water bath to dry up and remove the solvent and next it was kept into oven to get the actual fat content. Then the flasks were weighed to get the amount of fat content.

Calculation:

Weight of the extracted fat

% of fat = X 100

Weight of the sample taken

Plate 15. Showing different steps during determination of fat content of

the fish samples

3.4.4. Salt content determination:

Salt content of the salt-cured fish products were estimated by Mohor method (Alexiyev, 1978). Fillets of salted fish samples were ground in a mortarwith a pastel. The minced fishes were weighed accurately and salt was extracted with distilled water in a conical flask (250 ml). They were kept over night at 10°C. The contents were then made into volume of 100 ml and filtered. The filtrate with salt content was titrated against standard N/10 silver nitrate (AgNO₃) solution in micro burette using potassium chromate as an indicator. Finally, the percentage of salt content was calculated

Calculation:

VxSxNx25x100

% of salt content =

W x V1

Where,

q V = volume of standard N/10 silver nitrate (AgNO₃) required

q S = strength of N/10 silver nitrate

q N = conversion factor (1 ml of N/10 silver nitrate =0.00585 gm of sodium chloride (NaCl).

q V1 = volume of solution taken.

q W = weight of the sample.

Plate 16. Showing different steps during determination of salt content of the

salt cured fish samples

3.4.5. Ash determination:

The salted fish sample were minced, weighed and ignited in the crucible. Then it was transferred in the muffle furnace held at dull red 550° – 600°C for 6 –8 hours until the residue become white. The weight of the ash were taken. Finally, the percentage of ash content were calculated as follows

Calculation:

Weight of ash

% of ash = X 100

Weight of the sample taken

3.5. Preparation of Mineral solution

For determination of minerals like Fe, P and Ca a mineral solution is to be prepared first from the concerned fish sample. The mineral solution is prepared from the ash of the fish sample through several steps of treatment with acid, distilled water evaporation etc. and finally the volume of the mineral solution is adjusted to 100 ml with distilled water. The procedure for preparing mineral solution is narrated herewith for a clear idea.

The ash is moistened with a small amount of distilled water (0.5 to 1 ml) and 5 ml of HCl are added to it. The mixture is evaporated to dryness on boiling water bath. Another 5 ml of HCl acid are added again and the solution evaporated to dryness as before. Four (4) ml HCl acid and a few ml of water are then added and the solution warmed over a boiling water bath and then filtered into a 100 ml volumetric flask using what man No. 40 filter paper. After cooling the volume is made up to 100 ml and suitable aliquots are used for the estimation of P, Fe, and Ca.

3.5.1. Determination of Ca by titration method (Vogel, 1978): Calcium is determined by precipated it as Calcium oxalate and titrating the solution of oxalate in dilute sulphuric acid, against standard KMnO₄ solution.

 

Reagent:

1. Ammonium oxalate 6%
2. Methyl red indicator
3. Strong Ammonia (NH₃)
4. 2N sulphuric acid
5. N/100 KMnO₄ solution
6. Glacial acetic acid
7. Calcium chloride solution

Procedure

A known volume of mineral solution was taken in suitable glassware to which a few drops of methyl red indicator was added and red color develops which were neutralized with concentrated NH₃. The color changes from red to yellow. After that it was heated to boiling for few minutes with addition of few chemicals. At the end point a pink color were developed and ppt of Ca- oxalate were filtered out. The ppt were transferred to a conical flask with 2N H₂SO₄ and washed with hot water. The resultant solution of the ppt of Ca- oxalate were titrated with N/100 KMnO₄ solution at a temperature of 70º C.

Calculation:

1 ml of N/100 KMnO₄ = 0.2004 mg of Ca.

3.5.2. Determination of IRON

Iron content was determined spectra photo metrically by trio cyan ate method as described in practical physiological chemistry (Vogel, 1978).

Reagents

1. 30% H₂SO₄ A. R.

2. Saturated potassium persulphate solution

3. Potassium thiocyanate 40% solution

4. Standard Iron solution 0.7022 g A. R

Procedure

To an aliquot (6.5 ml or less) of the mineral solution enough water is added (if necessary) to make up to a volume of 6.5 ml. followed by 1 ml of 30% H₂SO₄, 1 ml of potassium persulphate solution and 1.5 ml of 40% KCNS solution. The red color that develops in measured within 20 minutes at 540 nm.

Plate 17. Showing few steps for the estimation of Iron and Phosphorous content

of the experimental fish samples.

3.5.3. Determination of Phosphorous

Determination of phosphorous (NIN Manual, 1976) was carried out by measuring calorimetrically the blue color formed when the solution was treated with ammonium molybdate and the phosphomolyb, date thus formed was reduced. The developed blue color was then measured at 660 nm against a standard solution.

Reagents

1) Ammonium molybdate

2) Hydroquinone solution

3) Sodium sulphite solution

4) Standard phosphate solution

Procedure:

To an aliquot (0.1 ml) of the mineral solution are added 1 ml of ammonium molybdate, 1 ml of hydroquinone and 1 ml of Na₂SO₃ solution in this order mixing well after each solution. The volume then made up to 15 ml with water and the solution is thoroughly mixed. After 30 minutes, the optical density of this solution is measured in a photoelectric colorimeter, against a reagent blank (Prepared in the same way as the test expect that the test solution is omitted) using a red filter 660 nm.

The phosphorous content of the same is read off from a standard curve prepared with standard phosphate solution (Range 0.01-0.1 mg phosphorous) following the same procedure described above.

3.6. Methods for detection and estimation of Vit. A

It involves two steps. In the first step Vit. A is extracted from the oil of the salt cured hilsa fish by addition of 2 ml. 95 % ethanol and 4 ml of petroleum ether. It is then shaken for 3 minutes and all of the petroleum ether layer is taken and the solvent with Vitamin is evaporated to dryness under nitrogen gas or by fan. The extracted Vitamin

Plate 18. Showing the estimation of Vit. A

A is then dissolved 1 ml chloroform to which 2 ml of TCA solution is added and the reading at 620 nm is taken immediately in spectrophotometer.

To a standard sample of Vitamin A solution cholera form is then added And the absorbency at 620 nm is read out against blanks which contain only the solvent. The method is established on the principle of Carr- price reaction (Khalil et al., 2001) where in Vitamin A in Chloroform reacts with TCA solution and forms the blue color. Measurement of the density of color helps to detect and to estimate Vitamin A in the supplied sample.

3.7. Determination of Total Sugar:

Sugar content of the salt cured hilsa fish is treated with sugar and preservative while processed in the Fish Technology Laboratory were determined by titration method described in AOAC (1970) which involves basically two steps

1) Extraction of the sugar by refluxing the fish sample with 40% ethanol, 7.5 M HCl and finally making the volume with distilled water.

2) In the second step – the sugar thus extracted is determined by titrating the extracted sugar with feeling solution with ethylene blue indicator.

Reagents Required:

1) 5 N HCl solution; 44.5 ml concentrated HCl in 100 ml

2) 7.6 M HCl : 63.36 ml

3) Correz solution I

4) Correz solution II

5) Fehling solution

6) Ethylene blue

Calculation :

W/100 X [(F X 1.08) + (P X 1.55)]