Subproject 2:
Chemistry: Chemotaxonomic investigation of the fatty acid composition in different organs of various fish species/populations from Lake Victoria and evaluation of their industrial applications in Uganda.
Researchers and location of project
The researchers on this project will be Dr. Mpango G. B. and Mrs Sheila Adikini Mugabe of the Department of Chemistry, Makerere University, in collaboration with Professor Otto Grahl-Nielsen of Chemistry Department, University of Bergen, Norway.
Fieldwork, in connection with sample collection, will be carried out at selected landing sites, especially the islands and shores of L. Victoria. This is to be done with the help of a qualified Fisheries officer and local fishermen. The fundamental part of the chemical research will be done at the Department of Chemistry, Makerere University, while the analysis of the results will be done at both Makerere and the University of Bergen, Norway.
Abstract
Lake Victoria is the second largest fresh Water Lake in the world and contains a wide variety of fish. The introduction of large fish varieties (e.g. Lates niloticus, Oreochromis niloticus, O. leucostoctus, Tilapia melanopleura and Tilapia zilli) in the 1950's and early 1960’s led to a loss of genetic diversity. Consequently, L. niloticus and O. niloticus have virtually eliminated a number of indigenous species. This has led to a transformation of the fish community leaving the L. niloticus, O. niloticus and Rastrineobola argentea as the only abundant commercial species. It is therefore necessary to re-examine and take stock of the existing fish species in the lake. Morphological, genetic and biochemical analysis techniques have traditionally been used in the differentiation of plant and animal species and/or populations. None of these techniques consistently exceeds 80% resolution between species and/or populations, usually giving results in the range of 70 - 80%. In this project it is hoped that the large differences in the relative amounts of fatty acids (i.e. fatty acid profile) in various fish oils could be an indication of a possible use of this feature for taxonomic determination. Strong environmental and seasonal influences on the fatty acid composition will also be taken into account. It is assumed that in-born factors may be important in determining the composition of the fatty acids in the various organs of a given fish species and/ or population as indicated by several studies involving marine fish. This project initially aims at establishing the actual existing species and/or populations of fish by the application of a new rapid and simple chemometric method. This implies the use of, for example, the fatty acid profile of selected tissues of the fish as a taxonomic character or chemical marker) for the identification of various fish species and/or populations.
Heart, muscle and brain tissues of fishes, such as Nile perch, Nile Tilapia, Bagrus, cat fish, etc., will be subjected to methanolysis followed by gas chromatography of the resulting fatty acid methyl esters. Principal component data analysis of the chromatographic results will then be applied to the representative fatty acids to distinguish the different fish species and/or populations.
Secondly, in Uganda, despite the large stock and variety, the abundant fishes remain largely under-utilized as foodstuff, but especially as raw materials for the oil/fat-based industries. Fishes such as the Nile perch and Nile Tilapia, with exploitable amounts of oils, will be classified, their oils extracted, purified for local use as a substitute in the daily diet, and also modified by hydrogenation for industrial use as raw materials.
Introduction
With recent substantial increases in harvests from the Uganda sector of Lake Victoria, related mostly to the Nile perch (Lates niloticus) and Tilapia (Oreochromis species), several industrial ventures have been established and many others are planned. Operations are mostly geared to serve mainly export markets, although a small percentage is sold locally, especially the skeletal remains after filleting. It should be recalled that fisheries in Uganda have undergone successive disruptions since the early 1920's. The major contributing factors being the intensive non-selective fisheries, extreme modification of the drainage area, invasion of the introduced species (Ogutu-Ohwayo and Hecky, 1991), and the progressive change of physical and chemical factors in the aquatic environment, especially the recent arrival of the water hyacinth.
The growing numbers of merchant fish traders, with at present more than six operational fish processing plants, effectively exerts constant pressure on standing stocks. As a result, this excessive demand may lead to acute over-fishing that could automatically lead to a loss of genetic diversity of the affected species. Size composition of the stocks in terms of age groups, species, and/or populations is declining very fast due to the uncontrolled use of smaller meshed nets. This will inevitably lead to a decline or complete disappearance of stocks of the existing species (Acere, 1988). Consequently, only three species Nile perch, Nile Tilapia and R. argentea are currently abundant in L. Kyoga and apparently in most of L.Victoria. Thus the original multispecies fishery, based mostly on cichlids (haplochromines, tilapias), cyprinids (Barbus labeo, Rastrineobola) and siluroids (Bagrus, Clarias, Synodontis, Schilbe), has changed dramatically to one based on the named three species (Achieng, 1990 and Getabu, 1987). It is therefore necessary to re-examine and evaluate the status of fish species and their populations in the lake.
Overall Objective
The overall objective of the project is to formulate and establish a useful package of knowledge and research skills using a newly developed specific taxonomic technique (Chemotaxonomy) for fishes in Lake Victoria and to high-light the importance of fish oil to health and industrial development in Uganda.
Specific Objectives
(a) To investigate the existing fish species and/or populations in Lake Victoria by the chemometric evaluation of the fatty acid composition of the oils in various tissues of the fish. This is to be achieved via methanolysis of the oils in the various tissues, followed by gas chromatography. Multivariate data analysis will then be carried out to assess the similarities and differences between the different fishes in L.Victoria.
(b) To evaluate the possible contribution of a chemometric method, to further understanding of the taxonomic relationship between the different fishes, within L. Victoria.
(c) To extract, purify and compare the fresh-water fish oils to the commercially available cod-liver oil (or seven seas). The purified oils from the different fishes will then be modified by hydrogenation in order to determine their economic viability as tallow or lard substitutes.
d) To isolate and purify the free-fatty acids from the oil/fats, for use as industrial raw materials.
Justification
Based on the above information, there is a growing need to re-visit the lakes and rivers in Uganda to check the surviving native and introduced fish species in order to identify the endangered populations, if the ecosystem is to be preserved. Traditionally, this has been done using the common taxonomic techniques, e.g. morphological, genetic or biochemical analysis methods, which are tedious, time-consuming, and costly. The present, proposed method is rapid and definitely less costly.
Summary on previous work
Determination of fatty acids in marine fish is the subject of numerous publications (Jangaard, et. al 1976; Jeffries, 1972; Sarget, 1976; Clarke, 1977). Nonetheless, the methods used involved mostly the monovariate (individual fatty acid analysis) approaches with a few multivariate approaches (Grahl-Nielsen, et. al. 1983; Federle et. al. 1983). Recently, a new rapid and simple chemometric technique has been developed and successfully used to distinguish populations of marine fish (Grahl-Nielsen and Ulvund, 1990; Grahl-Nielsen and Viga, 1990), Gyrocotyle species (Grahl-Nielsen. Berland and Bristow, 1990) and Picea abies and Picea obovata, both plants of the Pinaceae family in Norway (Grahl-Nielsen et. al. 1991).
The method involves the methanolysis of the fatty acids in selected samples of the tissues of the organism, followed by gas chromatography of the fatty acid methyl esters, and finally multivariate data treatment (principal component analysis) of the gas chromatographic results. Work on the component fatty acids of fats of fresh water fish has been carried out using British, Indian, and South American fresh water fish (Reiser et. al. 1963; Brenner and Bottino, 1962). In all cases, the fatty acids were similar for the total intestinal, muscles and liver lipids. Only the proportion of the unsaturated C20 and C22 acids differed in the different fish species.
Work on fresh water fish in Lake Victoria has concentrated on the fisheries industrialization and the socio-economic impact of such developments in Uganda (Reynolds and Ssali, 1990). Numerous articles have also appeared concerning feeding experiments of tilapias and Nile perch from Lake Victoria (Ochumba, et. al. 1991; Mbuga, 1991; El-sayed and Teshima, 1991 and Olsen, et. al. 1990). Other reports have also been published on seasonal variations of the total lipids, free fatty acid, triglycerides, phospholipids and cholesterol contents of the fresh water Tilapia (El-sayed et. al. 1984; Howell and Matthews, 1991).
In all cases considered above, no work has been reported on the use of the fatty acid profile of fish various tissues, as a possible taxonomic character or chemical marker. A lot has been reported regarding the total lipid, contents in fresh-water fishes, but no effort has been made to high-light the economic importance of fish oils as raw materials for industrial development in Uganda. Similarly, no effort has been made to identify such oils as dietary substitutes or even to indicate the endangered fish species such as the Bagrus or cat-fish which are slowly becoming extinct from the waters of Lake Victoria and Lake Kyoga.
Preliminary studies in our laboratories at Makerere University, involving the isolation, characterization, purification, hydrogenation, and methanolysis followed by gas chromatography of Nile perch and Nile tilapia oils have shown the two oils to be quite different both in physical and chemical characteristics.
Thus although the fatty acid composition is the same there are differences in average percentage values between the oils of the two fishes. It was observed that the main difference occurs in the unsaturated fatty acids. Thus while C22:5n3 and C22:6n3 were 1.50 " 0.07% and 4.10 " 0.15% for Nile perch, they were 1.58 " 0.15% and 1.52 " 0.05% for Nile tilapia respectively. Multivariate data analysis of the gas chromatographic results showed the two fish to belong to different groupings as expected.
Hence there is need to develop further the new technique of chemometry of the fatty acid composition of the fresh water fishes for a fast check on the species and/or populations as they migrate from one place to another due to environmental stress. This will not only contribute to the understanding of the taxonomic relationship between different fishes, but will also highlight the quality of the fresh water fish oils. In addition, the results may serve to emphasize the need to use the fresh water fish oils as a supplement in the daily diet of the average Ugandan who cannot afford the price of other nutrients such as cod-liver oil. The realization will also serve to reduce the country's importation of tallow and lard substitutes and improve on the health of the local population through better nutrition.
Outline of the proposed research (methodology)
The project is to be divided into four major phases as outlined below. For chemometric analysis, both the crude and refined oils/fats, will be used in order to compare the constituents in each oil, after methanolysis and gas chromatography. Multivariate data analysis of the chromatographic results of the crude and refined oil methyl esters will be carried out.
The effects of refining the physical and chemical characteristics of the oils will be studied in order avoid unnecessary denaturation of the chemical constituents.
Sample collection and methanolysis
Fish samples of various fish species to be studied will be collected from selected landing sites on both L. Victoria and L. Kyoga and the selected fishes identified by a qualified fisheries officer. Tissues from specific organs such as muscles, heart, brain, and intestines will be carefully dissected out and frozen immediately until methanolysis and gas chromatography can be carried out on them. Each fish species and/or population will be treated separately until the results of gas chromatographic analysis of all species are ready for multivariate data analysis.
Isolation, purification, physical, and chemical data determination
In this phase, the total lipids will be isolated from a relatively large quantity of the selected fish species (Population) after collection. Physical and chemical data, such as refractive index, iodine value, saponification value, peroxide value, etc., will be determined on the oil before and after purification or refining to check on any changes that might occur during refining. Methanolysis and gas chromatography of the resulting methyl esters will be carried out on the oils from each species and/or population. Multivariate data analysis of the resulting methyl esters from all species will then be carried out to check on the identity of each species’ oil.
Modification of the oils by Catalytic Hydrogenation
This is to involve the use of Raney Nickel catalyst during the hydrogenation of the total lipids. The physical and chemical characteristics will be determined before methanolysis and gas chromatography of the hydrogenated oils. The process of hydrogenation is of major importance in the fats and oils industry since it accomplishes two main objectives:
(a) It allows the conversion of liquid oil into semi-solid or plastic fats more suitable for specific applications in industry.
(b) It improves the oxidation stability and hence the shelf life of the oil.
Isolation of the Fatty Acids
The composition of the fatty acids of industrial importance depends upon the fat or oil from which they are derived. Those most commonly used for fatty acid production include beef tallow, coconut, cottonseed, soybean and palm oils.
In this phase, attempt is to be made at the isolation and identification of fatty acids from fish oils in both natural and hydrogenated forms. Column chromatographic techniques will be used for the fractionation of the hydrolysate. Methanolysis and gas chromatography of the resulting methyl esters will be used to conclusively identify the individual separated component.