STUDY ON THE HISTAMINE CONTENT AND HYGEINIC QUALITY OF DRIED FISH IN SOME DISTRICTS OF NEPAL

BY

Food Research Officer Food Quatantine Laboratory

Prashanta Pokhrel

Submitted to

Planning, Management & Evaluation section

Department of Food Technology and Quality Control
Babarmahal, kathmandu 2013

1. Introduction Histamine is a biogenic amine produced during microbial decomposition of scombroid fish such as mackerel as well as non scombroid fish such as sardines. Consumption of spoiled fish results in the outbreaks of food poisoning and histamine fish poisoning is one such type of food poisonings. Scombroid poisoning is usually a mild illness with a variety of symptoms including rash, urticeria, nausea, vomiting, diarrhea, flushing, tingling and itching of the skin. Fish is a rich source of protein and provide a means to compensate the nutritional difficulties of man. Fisheries can contribute as a low cost supplementary source of protein. According to a report 100gm of table fish provides 17.0gm of protein. The percentage of protein varies from 15-20 in fishes. This protein is relatively of high digestibility, biological and growth promoting values for human consumption. Nutritional studies have proved that fish proteins rank in the same class as chicken protein and are superior to milk, beef protein and egg albumin. Fish protein comprises of all the ten essentials amino acids in desirable strength for human consumption. It is also rich in minerals and vitamins. Besides, protein fish contains Potassium, Calcium and Phosphorus in few amounts. The fish liver is well known as a source of vitamin A and D essential for the development of children. Fishes also contains vitamin B and B12 in the same degree as in meat. Fish flesh contains negligible quantity of carbohydrates. Fish is a quickly perishable commodity and is spoiled if it is not properly preserved. The place of consumption is generally at a distance from the landing or catching point, and fish if it is to be markettted fresh it needs fast transport and cooling facilities. During peak period, large quantities of fish are caught and need proper preservation so as to be available in lean period, and have to be transported to market at long distances for consumption. When alive in water, fish carries large number of bacteria on its body, gills and in the gut. Soon after the death of fish, these bacteria start reproducing and attack various organs so that the preservation of the fish has to be done without loss of time. The principal aim of fish preservation is to delay, reduce or inhibit the microbial spoilage. In the case of fatty fish, the preservation may also aim at reducing or inhibiting oxidation and other undesirable changes in the fish oils, which are highly unsaturated and capable of going rancid at various stages of processing. Some fish preservation methods such as canning and curing change the character of the fish substantially, while the sequence of freezing, cold storage and thawing normally aims at retaining the fresh fish character as much as possible (Timilsina, 2007).

Histamine Production Histidine exists naturally in many types of fish, and at temperatures above 16C (60F) on air contact it is converted to the biogenic amine histamine via the enzyme histidine decarboxylase produced by enteric bacteria including Morganella morganii (this is one reason why fish should be stored at low temperatures). Histamine is not destroyed by normal cooking temperatures, so even properly cooked fish can be affected. Histamine is a mediator of allergic reactions, so the symptoms produced are those one would expect to see in severe allergic responses. The production of histamine on fish can be fairly rapid. In one outbreak, threshold toxin levels were reached after only three to four hours of storage at room temperature. The greater the temperature abuse, the higher the level of histamine that can be expected. Levels in excess of 50 mg/100 g fish tissue are considered potentially dangerous. In Canada, imported fish is rejected if it contains more than 10 mg of histamine per 100 g of fish. Origin of Histamine poisoning/ Scombroid toxicity Scombroid toxicity, also known as histamine poisoning, results from ingesting fish incorrectly held at warm temperatures after capture or during subsequent handling and storage. Histamine is formed by bacterial decomposition of free histidine. Histidine is a naturally occurring amino acid found in the muscle tissue of fish harvested in tropical and temperate marine waters. The fish species most often implicated with histamine illness include mahi mahi, marlin, bluefish, mackerel, bonito and tuna. Fish Production in Nepal In Nepal the fish production per capita is 1676 gm. (MOA, Directorate of Fisheries Development 2004/2005). There are about 122 species of fishes in Nepal. The commonly cultivated are Labeo rohita, Catla catla. The exotic species are Cyprinus carpio, Hypopthalmichthys moltrig, Aristichthys nobility and Oncorhynchus mykiss (trouts). They are consumed fresh (Niroula, 2010) In many districts, ethnic grups, mainly the Tharus, Pode, Maghim, Danuvarm, Darai and Damaihas have been capturing fish from lakes, swamps, rivers and streams (Bhusal, 2011). They generally sell the fish fresh if the fish are big enough, while dry to make sidra. Even big fishes are dried when the fished quantity is big (Timilsina, 2007). Fish drying technology in Nepal Small fishes after washed to remove dirt and sand. After draining they are laid spread over bamboo mats, plastic mats gunny bags. Then they are left for sun drying for 2-3 days with a drying time of 8-10 hours daily. The dried fish are stored in plastic bags or gunny bags. The dried fish have a moisture content of 10-12 %. Regarding big fishes, bamboo skewer is inserted into the big fishes after degutting and dried over sunlight.

Alternatively removing the gut and cleaning, 25 % salt is rubbed and left for few hours. After that they are sundried (Kharel et al, 2007). The fish processing technology is traditional (Timilsina, 2007). Fish Marketing in Nepal Dried indigenous fish species in Nepal are kept in the market for selling in background Malekhu Bazzar, Koshi Barrage, Khadichour, and so on which are attached with highway. These places are near big rivers which are rich source of indigeous fish species. Big fishes are sold fresh while excess of fish and small fishes are sundried and kept in the market for selling. Fish species used for drying are Nakata (Garra gotyla), Buduna (Garra annandalei), Kabre (Glyptothorax sps,), Sahar (Tor putitora), Faketa (Barilius sps,) and Sidra (Puntius sps) ( Bhusal, 2011 ).

Fig 2: Dry fish being kept for selling in Nepal 2. Objective of the study To study the histamine content of dried fish found in different districts of Nepal. To study the hygienic condition of dried fish. To find the proximate composition of dried fish.

3. Problem Statement Fish have formed an important item of human diet from the time man appeared on the earth, and are primarily caught for this purpose. Fish diet provides proteins, fats and vitamins A and D. A large amount of phosphorus and other elements are also present in it. They have a good taste and are easily digestible. Fishes are eaten in various ways, either cooked or even raw. Some are dried, while some are salted or cured to preserve. In Nepal fresh and dried fish are available in the market. The processing aspect of fish in Nepal is traditional and unhygienic. Taking into account, the small fish, the gut is not removed /cannot be removed, and dried over sunlight. Similarly in the processing of big fishes, the cleaning water used is not potable. Hence the condition is very much favorable for the enteric bacteria to decompose free histidine into histamine. In the Terai belt of Nepal people are suffering from Allergic reactions and histamine poisoning due to the consumption of such fish. Even due to their negligence, severe cases of blurred vision, swelling of tongue, respiratory problems have arosed. In this context study on the histamine content and sanitary condition of dried fish marketed in Nepal is of value. 4. Expected output This study will find the histamine content of dried fish markted in Nepal. It will show the hygienic condition of the fish. Help the planning agencies and DFTQC to develop new fish processing methods Help DFTQC to develop ways to reduce histamine poisoning in Nepal
Dried Fish Sample Collection Sunsari Bagmati Chitwan Dhading Sindhupalchok

5. Materials and Methods (30 samples, 3 species) Experimental design:

Histamine content analysis

The whole study will be conducted at Food Quarantine Laboratory, Tatopani, Sindhupalchok. A total of 30 dried fish samples will be collected from 5 districts of Nepal. At least 3 species of fish will be included in the study. The histamine content of the fish will be analyzed.
and proximate Similarly, the hygienic Microbiological condition of fish will be studies through microbiological count. analysis Data Collection

Data Analysis

Result Interpretation, Report Writing and Submission

Fig 1: Methodology of the study. Materials Fish sample collection: Dried Fish Saples will be collected from 5 different markets of 5 districts of Nepal. They will be Sunsari, Chitwan, Dhading, Bagmati and Sindhupalchok. From each district 6 samples (at least 3 different species of fish) will be collected. Chemical Equipment and glassware requirements Chemicals 1. Benzene 2. Butanol 3. Sodium acetate 4. Succnic anhydride 5. Acetic acid 6. Sulphuric acid 7. Hydrochloric acid 8. p-nitroaniline 9. Sodium metaborate 1. 2. 3. 4. 5. 6. 7. 8. 9. Glasswares and equipments Spectrophotometer (475nm) Vacuum oven Refrigerator High speed Centrifuge machine High Speed Blender Polyethylene bottle pH meter Ice bath Water bath

(NaBO2) 10.Sodium carbonate 11.Sodium barbital 12.Dry histamine dihydrochloride 13.4-methyl-2pntanone(methyl isobutyl ketone) 14.Benzaldehyde (chlorine free) 15.Sodium borate 16.Absorbent Cotton 17.Ethyl alcohol absolute 18.NaOH 19.Methanol 20.Oxalic acid Methods

10. 11. 12. 13.

Erlenmeyer flask Oswald pipette Centrifuge tubes Volumetric flasks

14. Beakers 15. pipettes

Histamine content analysis: Histamine content will be analyzed according to AOAC, 2005. Microbiological analysis:
Total plate count was determined by pour plate method according to Harrigan and McCane (1979) using plate count agar and distilled water as diluents.

Proximate analysis Proximate constituents crude protein, crude fat, ash, calcium and phosphorous content will be done according to K.C and Rai, 2005. Budgetary Distribution Particulars Sample collection and Transportation Chemical and equipments Report writing Stationery and others Total Amount (NRs.) 50,000.00 100,000.00 10,000.00 10,000.00 1,70,000.00 Remarks -

References Bhusal D. (2011).Dried indigenous fish kept in the market for selling in Nepal. Available at http://fishconsult.org/?p=1694 Accessed on 4 April, 2013.

Niroula S. (2010). Fish Farming in Nepal, Available at http://www.scribd.com/doc/31497481/Fish-Farming-in-Nepal Accessed on 4 April, 2013. Kharel G.P., Acharya P.P, Rai, B.K, 2007. Handbook of Traditional Foods of Nepal. 2007. Timilsina A (2007). Comparative study on the hygienic quality of traditionally dried fish dried with lab dried fish. B. Tech dissertation presented to Central department of food technology, CCT, TU, Dharan, Nepal.