"INDIARAMESH et al.: SEASONAL VARIATIONS IN FATTY ACID PROFILES OF THREADFIN REAMSIOL"INDIAN

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1 "INDIARAMESH et al.: SEASONAL VARIATIONS IN FATTY ACID PROFILES OF THREADFIN REAMSIOL"INDIAN Indian Journal of Geo-Marine Sciences Vol.45(5), May 2016, pp Seasonal variation in fatty acid profiles of threadfin breams, Nemipterus mesoprion (Bleeker, 1853) and Nemipterus japonicas (Bloch, 1791) with respect to their size R. Ramesh, S.K. Chakraborty & G. Venkateshwarlu* Central Institute of Fisheries Education, Panch Marg, Off Yari Road, Versova, Andheri (West), Mumbai , India. *[ address: Received 07 April 2013; revised 13 June 2013 In order to evaluate its nutritive value, seasonal variation in total lipid and fatty acid profiles of different sizes of threadfin breams (Nempiterus mesoprion and N. japonicus) were studied. Total lipid content was significantly (P<0.05) high in large sizes of N. mesoprion and N. japonicus captured during winter and lowest in small groups during post-monsoon season. A total of 37 fatty acids were identified by GC-MS encompassing saturated, mono unsaturated, n-6, n-3 poly unsaturated and odd and branched chain fatty acids. High levels of n-3 PUFA ( %) and favourable n-3/n-6 PUFA ratio of 2.25 to 3.3 revealed nutritional significance of threadfin breams in human diet. Difference between small, medium and large groups of N. mesoprion and N. japonicus of respective seasons based on fatty acid composition were clearly established by principal component analysis. [Key words: threadfin breams, fatty acids, principal components, seasons and size] Introduction Fish has been reported to possess low cholesterol, high protein content and valuable polyunsaturated fatty acids such as docosahexaenoic acid and eicosapentaenoic acid 1. These fatty acids have several health benefits such as reduced risk of coronary heart disease and control of arterial hypertension 2, 3. They prevent human breast cancer growth, asthma and inflammatory disorders of the immune system 4, 5. Lipid content and fatty acid composition of fish also varies in response to changes in water temperature, salinity and depth through adaptive mechanisms 6. It is known that the quality of seafood products is dependent on the genetic basis, size and reproductive period of fish, as well as on the characteristics of the environment, e.g., ph, salinity and temperature of the water, composition of phyto- and zooplankton during the year 7. Threadfin breams (Nemipterus mesoprion and N. japonicus) are one of the major demersal finfish resources exploited along the East and West coast of India. Annual catch for was about 1, 74, 079 tonnes which formed 5% of the total marine catch of India (CMFRI, 2012). Earlier, studies were conducted on threadfin breams regarding food and feeding habits, chemical and physical status of the fishes 8, 9 and lipid profiles of N. japonicus of Royapuram coast of Bay of Bengal, Tamilnadu 10. Although the lipid content and fatty acid profiles have been reported, there is no comprehensive information available on seasonal variations on lipid content and fatty acid profiles of threadfin breams with respect to size. Due to their commercial importance and availability throughout the year, the present study was carried out to discern the seasonal variations in the fatty acid profiles of threadfin breams with respect to different size groups by using multivariate technique i.e. Principal Component Analysis (PCA). Materials and Methods Samples were collected randomly from landing centres along Mumbai coast during post-monsoon (August), winter (December) and summer (April). The fish were measured for fork length and body weight, and classified as large (fork length > 20 cm), medium (15 20 cm) and small (< 15 cm) (Table 1). Table 1- Morphometric data of different size groups of threadfin breams (N. mesoprion & N. japonicas) sampled for the study Size Body Length(cm) Body weight(g) Small < Medium Large >

2 RAMESH et al.: SEASONAL VARIATIONS IN FATTY ACID PROFILES OF THREADFIN BREAMS 697 N FATTY ACID PROFILES OF THREADFIN REAMSIOL"INDIAN J MAR SCI VO Total lipid was extracted from muscle tissue of threadfin breams by Folch method 11 and the fatty acid methyl ester (FAME) was prepared from the isolated lipids by heating with methanolic NaOH and then with BF 3 methanol for esterification. An aliquot of 5 ml n-heptane was added to recover the methyl esters in organic phase. The mixture was washed with saturated NaCl solution and two phases were separated using a separating funnel. The upper n-heptane phase was pipetted out and stored in 10 ml all glass vials until further analysis 12. Fatty acid methyl esters were separated by using a Shimadzu QP2010 quadruple Gas Chromatography Mass Spectrometer (GC-MS) Instrument equipped with a Carbowax (30 m x 0.25 mm ID; 0.25-µm film thickness) capillary column (Cromlab S.A.). Helium was used as the carrier gas. Injector and detector temperatures were set at 250 C. Injection was performed in split mode (1:15). The column temperature was programmed initially at 50 C for 2 min and then increased at a rate of 10 C per min to a final temperature of 230 C. FAMEs were separated at constant pressure (23.1 kpa) and peaks were identified by comparing standard mass spectra with the relative abundances of m/z ranging from to 550. The values of fatty acids are presented in area percentage of total identified fatty acids. Statistical analysis was performed on total lipid content data and significant difference at P<0.05 in mean values was evaluated using Duncan test with multiple comparison by employing one way ANOVA with SPSS statistical package. Multivariate data of fatty acid composition subjected to different size and seasons were analyzed PCA using software Unscrambler (Version 9.5, CAMO, Norway). PCA is a data compression method based on the correlation among variables. The purpose of carrying out PCA was to express the main information in the variables by a lower number of variables, the so-called principal components (PC 1, PC 2, ). The first principal component, PC 1, is defined as the direction of maximum variance of the whole data set. PC 2 is the direction that describes the maximum variance in the orthogonal subspace to PC 1. Subsequent components are taken orthogonally to the ones previously chosen and describe the maximum of the remaining variance. When redundancy is removed, only the first few principal components are required to describe most of the information contained in the original data set. A high positive or negative loading reveals a significant variable in the actual PCA model. Score plots from the PCA explore the main trends in the data and their respective loadings reveal the significant fatty acids. Both the scores and loadings were explained by the bi-plot. Results and Discussion Table 2 shows the total lipid content extracted from muscle tissue and it was found in the range of %. Significant differences (P<0.05) were observed during different seasons with respect to different sizes. Generally, fish can be grouped into four categories based on their fat content: lean fish (<2%), low fat (2 4%), medium fat (4 8%), and high fat (>8%) 13. In the present study, the threadfin breams fall under category of lean and low fat fishes. Total lipid content varies from species to species depending upon the season, location, availability of food 14. High lipid content (3.13±0.02%) was observed during the winter season compared to other seasons. It indicates that fish may depend on reserve energy during winter season due to unavailability of food in aquatic environment. The differences in these values could be due to many factors as fat content in fish vary according to seasons, species and geographical variations. Age variation and maturity in the same species may also contribute to the significant differences in the total lipid 15. In the present study, use of capillary column for resolution of components and MS (Mass spectrometer) for identification of fatty acids enabled us to characterize 37 fatty acids encompassing n-3, n-5, n-6, n-7, n-9 and odd and branched chain fatty acids (Table 3 & 4). Table 2 - Total lipid content (%) in the muscle tissue of different size groups of threadfin breams observed during different seasons Nemipterus mesoprion Nemipterus japonicus Seasons Small Medium Large Small Medium Large Postmonsoon 0.36±0.01 aa 0.47±0.04 aa 0.52±0.02 aa 0.49±0.02 aa 1.43±0.03 ab 1.63±0.06 ab Winter 1.92±0.44 ba 2.26±0.35 bb 3.13±0.02 bc 1.62±0.34 ba 2.11±0.55 bb 3.06±0.52 bc Summer 1.19±0.44 ca 1.58±0.35 cb 2.53±0.02 cc 0.50±0.04 aa 2.01±0.05 bb 2.47±0.52 cb Values are (Mean± SD)Superscripts a, b and c represents significant differences (P< 0.05) among same sizes in different seasons (Mean±SD). A, B and C represent significant differences (P< 0.05) among different sizes in each season.

3 698 INDIAN J MAR SCI VOL 45, NO. 5 MAY 2016 Table 3 - Fatty acid composition (% of total fatty acids by peak area) of muscle of different size groups of Nemipterus mesoprion caught during three seasons Post-monsoon Winter Summer Fatty acids small medium large small medium large small medium large 6: : : : : i-15: : i-16: : i-17: : i-18: : : : : Total SAFA :1n :1n :1n :1n :1n :1n :1n :1n Total MUFA :2n :2n :3n :4n :4n :5n Total n-6 PUFA :3n :4n :3n :4n :5n :5n :6n Total n-3 PUFA n-3/n-6 ratio

4 RAMESH et al.: SEASONAL VARIATIONS IN FATTY ACID PROFILES OF THREADFIN BREAMS 699 Table 4 - Fatty acid composition (% of total fatty acids by peak area) of muscle of different size groups of Nemipterus japonicus caught during three seasons Post-monsoon Winter Summer Fatty acids small medium large small medium large small medium Large 6: : : : : i-15: : i-16: : i-17: : i-18: : : : : Total SAFA :1n :1n :1n :1n :1n :1n :1n :1n Total MUFA :2n :2n :3n :4n :4n :5n Total n-6 PUFA :3n :4n :3n :4n :5n :5n :6n Total n-3 PUFA n-3/n-6 ratio

5 700 INDIAN J MAR SCI VOL 45, NO. 5 MAY 2016 In earlier studies on fatty acid profiles of threadfin breams, only eight fatty acids were identified by Nazeer et al 10 and thirty fatty acids were reported by Chedoloh et al 16. In both N. mesoprion and N. japonicas, palmitic acid (16:0) was found to be the most dominant fatty acid accounting % followed by stearic acid ( %). Arachidionic acid (AA, 20:4, n-6) was the principal n-6 PUFA at a level of % of total fatty acids. Eicosapentaenoic acid (EPA, 20:5, n- 3), docosapentaenoic acid (DPA, 22:5, n-3) and docosahexaenoic acid (DHA, 22:6, n-3) have contributed as major n-3 PUFA accounting %, %, % respectively. Levels of n-6 and n-3 fatty acids were high during post-monsoon and winter season, where as saturated fatty acids (SAFA) and monounsaturated fatty acids (MUFA) were abundant in summer season apparently in all the size groups. The presence of high levels of saturated fatty acids indicates the presence of active enzymes responsible for de novo synthesis. These SAFA and MUFA are generally abundant in fish from warm or temperate regions, whereas PUFA show high levels in fish from cold regions 17, 18. It is in agreement to earlier study that threadfin breams contain higher levels of saturated fatty acids. Edible portion of the threadfin breams muscle tissue contained higher levels of PUFA which may be due to their feeding mechanism, as they depend entirely on marine food chain, which are rich sources of long chain PUFA. The n-3 PUFA levels were found higher than those of n-6 PUFA, as typical for marine fish 19. The n-3/n-6 ratio is a better index in comparing relative nutritional value of fish 20. In the light of the diversified roles of n-3 and n-6 PUFA, right balance between these fatty acid groups is recommended. In this background, the observed high ratio of n-3/n-6 PUFA in the present study (2.25 to 3.3) emphasises the excellent quality of threadfin breams meat. Within the general patterns, the fatty acid composition is known to be affected by diet food deprivation and non-dietary factors including season and environmental temperature 21, 22. Environmental temperature is known to affect the fatty acid composition of the tissue of the poikilothermic fish as the degree of unsaturation increases by decreasing temperature. In the present study, the PUFA content was increased in post monsoon and decreased in summer of threadfin breams. Small sized threadfin breams have lower lipid contents compared to medium and large sized groups captured during all the seasons. However, they possessed relatively high proportion of nutritionally important PUFA compared to the larger fishes and have significant n-3/n-6 ratio. Besides linear fatty acids, branched-chain saturated fatty acids (iso fatty acids such as iso- 15:0, iso-16:0, iso-17:0 and iso-18:0) found in the range of %. These fatty acids may occur in small amounts and have been detected in the range of 0.1-1% each 13, 23. Vlaeminck et al 24 suggested that the polyunsaturated and odd and branched chain fatty acids (OBCFA) of marine food chain received considerable attention because of their various biological activities in health and diseases. Other reasons for interest in odd and branched chain fatty acids are their reported anti-carcinogenic effects. Marine fishes do not have capacity to synthesise odd and branched chain fatty acids. So they derive entirely from marine food chain 25. In the present study, OBCFAs did not show any significant difference in both the species with respect to size and season. Multivariate statistical methods were successfully applied in lipid research in fishes like tilapia, rays and Bombay duck 21, 26. PCA has been reported to reduce the dimensionality of multivariate data while preserving most of the variance within it 27. In view of the large data comprising of two species N.mesoprion and N. japonicus with 37 variables (fatty acids), the principal component analysis was applied in the present study to observe the relationships among different seasons and sizes with respect to fatty acids. A graphic representation of the projection of variables and samples on to the first two principal components is given in Figures 1, 2 and 3 in the form of bi-plots. Based on PCA, the distribution of different fatty acids with respect to different seasons and sizes was mentioned in the bi-plots (Fig. 1, 2 & 3). About 69% (average) of total information in the data explained by the PC 1 and PC 2. The PC 1 separated N. mesoprion from N. japonicus based on fatty acid composition (Fig 2 and 3).

6 RAMESH et al.: SEASONAL VARIATIONS IN FATTY ACID PROFILES OF THREADFIN BREAMS 701 Fig. 1 - Muscle fatty acid distribution in different size groups of N. mesoprion and N. japonicus caught during postmonsoon season Fig. 2 - Muscle fatty acid distribution in different size groups of N. mesoprion and N. japonicus caught during winter season Fig. 3 - Muscle fatty acid distribution in different size groups of N. mesoprion and N. japonicus caught during summer season In all the three seasons, both the threadfin breams were segregated into clusters demonstrating the influence of species nature over body size on the fatty acid composition of fish. Segregation of each species into a cluster was observed more distinct in winter and summer seasons compared to post-monsoon season. Further, the cluster of N. japonicus was found dominated by highly unsaturated fatty acids such as DHA and DPA. In the present study, odd and branched-chain saturated fatty acids such as iso-15:0, iso-16:0, iso-17:0 and iso-18:0 were found randomly without associating to any particular species or size. PCA also revealed that the fishes can be distinguished based on their muscle fatty acid profile. Conclusions Present study revealed that the threadfin breams possess higher levels of n-3 PUFA, especially EPA and DHA which are known for their human health promoting cardio protective role. The fish caught during winter season has significantly (p<0.05) higher lipid content in muscle compared to other seasons irrespective of the body size. PCA carried out in the present study revealed that it is an important tool to investigate the relationship among fish species of different size groups caught during different seasons based on fatty acid compositions. References 1 Ersoy, B. & Çelik, M., Essential elements and contaminants in tissues of commercial pelagic fish from the Eastern Mediterranean Sea, J. Sci. Food Agric., 89(2009) Burr, M.L., Fish food, fish oil and cardiovascular disease, Clin. Exp. Hypertens. A14 (1992) Millar, J.A. & Waal-Manning, H.J., Fish oil in treatment of hypertension, N. Z. Med. J., 105(1992) 155 pp. 4 Dry, J. & Vincent, D., Effects of fish oil diet on asthma: results of a 1-year double-blind study, Int. Arch. Allergy Appl. Immunol. 95(1991) Rose, D.P. & Connoll, J.M., Effects of dietary omega-3 fatty acids on human breast cancer growth and metastases in nude mice. J. Natl. Cancer Inst. 85(21) (1993) Refik Tanakol, Zeliha Yazıci, Erdal Sener & Ergin Sencer, Fatty acid composition of 19 species of fish from the Black Sea and the Marmara Sea, Lipids, 34(3) (1999)

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