A Comparison of Proximate Composition And Fatty Acid Profile of Indus River Fish Species

Abstract

Eight species of freshwater fish from the Indus River were analyzed for their proximate composition and fatty acid (FA) profile. Differences were observed (P < 0.05) for moisture (59.95–79.45%), ash (0.05–4.95%), crude protein (17–20.09%) and lipid (0.85–18.32%) contents. The changes in FA profiles of fish species were significant (P < 0.05). The monounsaturated (MUFA) fatty acid content (24.55–48.35 g/100 g) was higher than the saturated fatty acid (SFA) (25.04–41.02 g/100 g) and polyunsaturated fatty acid (PUFA) (15.72–35.34 g/100g). The predominant PUFAs were eicosapentaenoic acid (C20:5 n-3) and docosahexaenoic acid (C22:6 n-3). All of the fish species had n-3/n-6 within the recommended values (1–4), which confirms he importance of River Indus fish as a significant dietary source of n-3 PUFA.

Keywords:

• Indus River fishes
• Proximate composition
• Fatty acid profile
• Gas chromatography

INTRODUCTION

Fish lipids play an important role in human nutrition by supplying both energy and essential fatty acids (FA) requirement to satisfy the various physiological needs of the organisms.[1] Fish lipids have been intensely investigated since their protective effect on cardiovascular diseases was first studied. Fish lipids are well known to be rich in long chain n-3 polyunsaturated fatty acid (PUFA), especially eicosapentaenoic acid (EPA),[2] and docosahexaenoic acid (DHA).[3],[4] These fatty acids play a vital role in human nutrition, disease prevention and health promotion. [5],[6] Long chain n-3 PUFAs cannot be synthesized by humans and must be obtained through the diet.[7] Scientific data indicate that the consumption of fish oil containing n-3 PUFAs reduces the risk of coronary heart disease[8],[9],[10], decreases mild hypertension, prevents certain cardiac arrhythmias and sudden death, lowers the incidence of diabetes, and appears to alleviate symptoms of rheumatoid arthritis. Epidemiological data indicated that there was a decrease in the number of deaths caused by coronary heart diseases in people who consumed fish or fish oil containing small amounts (0.4 g) of n-3 series fatty acid on a regularly daily basis.[11]

Besides, it appears that n-3 PUFAs play a vital role in the development and function of the nervous system (brain), photoreception (vision), and the reproductive system.[7] Considering all these facts and the lower the risk of cardiovascular diseases in particular, the importance of consuming fish rich in n-3 PUFAs can hardly be neglected.[12] It has been reported that the FA profiles of fish are affected by various factors, including genetic factors,[13] environmental factors,[14],[15] and the nutritional quality of each dietary component.[16] Researchers have shown that freshwater fish generally contain lower proportions of n-3 PUFA than marine. [17],[18] Fish need PUFAs to provide tolerance to low water temperatures. Decreases in PUFA concentrations in lipids would therefore be expected in warmer waters (nearer the equator) like Pakistan. Although it is generally recognized that PUFA composition may vary among species of fish, little attention has been paid to the PUFA composition of different species when selecting fish for diets.[19]

According to Agriculture Statistics of Pakistan, the total fish production in country was reported as 604.9 tons in 2006 (425.0 ton of marine and 179.9 ton of freshwater fish).[20] River Indus is one of the longest rivers in the world with an astonishing length of 2900 km.[21] However, there are limited research works carried out on the Indus River fish and no report was available in the literature concerning the fatty acid profile of fish species. Therefore, an attempt was made to study proximate composition and FA profiles of 8 selected Indus river fish species and compared, among those species with special reference to n-3 PUFA contents, a potential quality parameter for health food source.

Materials And Methods

Sampling

Eight river fish species were analyzed: the Wallago attu (WA) (Jarko), Oreochromis mossambicus (OM) (Dayo), Labeo calbasu (LC) (Daahi), Bagarius bagarius (BB) (Fouji khago), Labeo gonius (LG) (sariyo), Clupisoma garua (CG) (Doghno), Aorichthys aor (AA) (Singhari), Eutropiichthys vacha (EV) (shali). The selection criterion was the volume of sales for the species.[20]. The fishes were caught from river Indus at Jamshoro point and were identified by the Fisheries Department, Sindh University Jamshoro. The experimental fishes were obtained in natural conditions between March and April, 2007. Prior to analysis the scale was removed and the fishes were dressed and washed. Fish sampling, cleaning and evisceration were done according to Standard Operating Procedures [22]. Ten fish samples of each species were selected individually for the study, all samples were immediately frozen at −20°C until analyzed within the two following weeks.

Determination of Proximate Composition

The fish samples were analyzed for proximate composition according to the official methods of the Association of Official Analytical Chemists,[23] moisture by the sample drying technique in an oven at a temperature of 105°C for 16–18 h and ash by direct analysis in a furnace at 550°C for 12 h while Nitrogen was determined by Kjeldahl method and protein was calculated from the nitrogen content.

Extraction of Total Lipids

Total lipids were extracted from fish muscle samples according to Folch et al. 1957.[24] A chloroform - methanol solvent mixture (2:1, v/v) was added to frozen samples in the ratio of 20:1 (v/w) of solvent – tissue. The samples were homogenized with solvent mixture three times for 10 min at 3000–4000 rpm. Each homogenization step was followed by cooling of the sample for 1 h at 4°C. Four milliliters of 0.034% MgCl₂ was added to the extract for each 1 g of tissue. The chloroform - methanol extracts were incubated overnight at 4°C for complete separation between organic (containing the extract of total lipids) and aqueous layers. The top (aqueous) layer was removed, and the bottom (organic) layer was washed with 2:1 (v/v) chloroform– methanol and placed into a glass tube. The total lipid fraction was obtained by evaporating the lower phase. The solvent was removed in a rotary evaporator (Heidolph, W. Germany) under reduced pressure (10–50 mm Hg) at 40°C. These extracts, representing total lipids, were weighed, and results were noted for each fish species. Total lipid contents were determined gravimetrically. Further, each extract was dissolved in 2 mL 2:1 (v/v) of chloroform–methanol and the resulting extract of total lipids was stored at 4°C until further analysis.

Fatty Acid Analysis

The lipids were saponified and esterified for fatty acid analysis by standard IUPAC method.[25] The fatty acid methyl esters (FAMEs) were analyzed on a Perkin Elmer gas chromatograph model 8700 (Perkin-Elmer Ltd, Buckinghamshire, England) fitted with biscynopropyl siloxane stationary-phase, SP-2340 [(60m × 0.25 mm) (Supelco, PA, USA] and an FID. Oxygen-free nitrogen was used as a carrier gas at a flow rate of 3.5 mL/min. The initial oven temperature was 130°C which was raised to 150° C at a rate of 4°C/min and further to 220° C at a rate of 2°C/min and held for 7 min. The injector and detector temperature were set at 260 and 270°C, respectively. A sample volume of 2.0 μL was injected. FAMEs were identified by comparing their relative and absolute retention times to those of authentic standards of FAME obtained from Sigma Chemical Co. All of the quantification was done by a built-in data-handling program provided by the manufacturer of the gas chromatograph (PerkinElmer) as reported earlier by Talpur et al.[26]

Statistical Analysis

The results presented for fish of particular species are means ± standard deviation obtained in the analysis of eight fish. The difference between the mean values of parameters examined (proximate composition and fatty acid composition) were submitted to one-way analysis of variance (ANOVA) using SPSS 10.0 (SPSS 1990, SPSS Inc., IL, USA), statistically significant differences were reported at (P < 0.05).

RESULTS AND DISCUSSION

Proximate Composition

The proximate composition of Indus River fish species differs significantly (p < 0.05). The most pronounced is the difference in the level of fat among the fish species (Table 1). Eutropiichthys vacha (EV) contained significantly higher (P < 0.001) fat content than other species (18.32 vs. 0.85–8.25%). The fat content in fish varies according to seasons, species and geographical region. Age variation and sex maturity in the same species also contribute to the significant differences (P < 0.05) in the total lipid contents.[27],[28] Based on the classification of Bennion[29] most of the fish studied were lean fish with the fat content of less than 5% by weight. This includes Wallago attu (WA) (0.85%), Labeo calbasu (LC) (0.98%), Labeo gonius (LG) (1.50%), Clupisoma garua (CG) (3.75%), Aorichthys aor (AA) (1.78%). The medium fat fish with 5–10% fat includes Oreochromis mossambicus (OM) (7.89%), Bagarius bagarius (BB) (8.25%) whereas the Eutropiichthys vacha EV (18.32%) falls in the category of fatty fish having fat content more than 10% by weight. Similarly Rahman et al.[19] have reported wide variation in fat content (1.17–34.00%) among 20 species of freshwater Malaysian fish. An inverse relationship was found between the fat and moisture contents in this study, which is already reported for other fish species.[30],[31] The lower moisture content was found in EV 59.95% (fat content 18.32%), while LG fish species possessed the higher moisture content 79.45% (fat content 1.50%).

Table 1 Proximate composition of fish muscle (%, wet basis)

	Component (%)
Species	Moisture	Ash	Fat	Protein
WA	78.80^a ± 0.43	2.90^b ± 0.14	0.85^d ± 0.25	17.00^c ± 0.12
OM	71.58^b ± 0.30	1.65^b ± 0.02	7.89^b ± 0.37	18.50^b ± 0.28
LC	77.00^a ± 0.20	2.30^b ± 0.09	0.98^d ± 0.20	18.83^b ± 0.29
BB	73.20^b ± 0.26	0.50^c ± 0.05	8.25^b ± 0.36	18.05^b ± 0.09
LG	79.45^a ± 0.50	0.05^c ± 0.02	1.50^c ± 0.21	19.00^a ± 0.15
CG	72.30^b ± 0.30	4.95^a ± 0.54	3.75^c ± 0.34	18.40^b ± 0.14
AA	77.03^a ± 0.20	2.02^b ± 0.12	1.78^c ± 0.21	19.05^a ± 0.28
EV	59.95^c ± 0.45	1.45^b ± 0.06	18.32^a ± 1.34	20.09^a ± 0.16
WA: Wallago attu; OM: Oreochromis mossambicus; LC: Labeo calbasu; BB: Bagarius bagarius; LG: Labeo gonius; CG: Clupisomagarua;AA: Aorichthys aor; and EV: Eutropiichthys vacha. Note: Values are given as mean ± S.D. from triplicate determinations. Different superscripts in the same column indicate significant differences (p < 0.05).

The ash content showed significant differences (P < 0.05) among eight fish species investigated with lowest content in LG (0.05%) while CG fish had the highest (4.95%) ash content. The reason could be minimum skeleton in small indigenous fish species (LG) compared to other species. This fact is duly supported by Mazumder. et al.[32] where authors have found less ash contents for small indigenous fish species over other fishes. The protein content was significantly higher (P < 0.05) in EV (20.9%) and LG (20.5%) fishes, followed by AA (19.50%) while OM, LC, and BB fishes had similar protein values ranging from 18.50% to 18.33%. The lowest% protein values were from CG (17.40%) and WA (17.00%) fishes. These results are comparable with the results published for protein (6.7–19.4%) and ash (0.6–4.0%) for some north-eastern Pacific forage fish species by Susan et al.[33].

Fatty Acid Analysis

The FA contents in the eight Indus River fish species are presented in Table 2. The changes in FA profiles of fish species in terms of total and individual saturated and unsaturated fatty acids were significant (P < 0.05). Overall, the monounsaturated (MUFA) fatty acid content (24.55–48.35 g/100 g) of River Indus fishes was higher than the saturated fatty acid (SFA) content (25.04–41.17 g/100 g) and polyunsaturated fatty acid (PUFA) content (15.72–35.34 g/100 g). This is similar to the study carried out by Ackman & Mcleod[34] for five Indian carp species i.e. Labio bata, C. catla and C. mrigala, L. rohita and L. calbasu. The palmitic acid (C16:0) was the predominant fatty acids accounted for 52–68% of total SFA. The maximum palmitic acid content was 25.44 ± 0.51 g/100 g in WA, while the minimum value was 16.45 ± 0.12 g/100 g in CG. Rahman et al,[19] found comparable values of palmitic acid (12.7 – 26.6 g/100 g) for freshwater fishes from Malaysia. MUFA accounts 72.10 g/100g of the total fatty acids in all fish samples, and the major monoenic FA's were palmitoleic acid (C16:1 n-7) and oleic acid (C18:1 n-9). The maximum C16:1n-7 content was 9.56 ± 0.06 in OM while the minimum value 3.75 ± 0.02 was observed for WA, and the maximum C18:1n-9 content was 32.32 ± 0.04 in BB while the minimum value was 8.88 ± 0.00 g/100 g in LG. Similarly Kaneniwaa et al,[35] reported C16:1 n-7 and C18:1 n-9 as the major MUFA in muscle of carp and other fish species (Chinese sea bass and Bluntnose black bream) living in Chinese freshwater. Later, Osman et al.[36] reported the high levels of oleic and palmitoleic acids as a characteristic property of freshwater fish oils.

Table 2 Fatty Acid Composition of Total Lipids from Muscle of Indus River fishes (g/100 g Fatty acids)

Fatty acids	Species
	WA	OM	LC	BB	LG	CG	AA	EV
(a) Saturated fatty acid (SFA) composition (%)
C14:0	1.98^c ± 0.01	5.00^a ± 0.00	4.10^a ± 0.12	2.29^b ± 0.06	2.77^b ± 0.08	1.64^c ± 0.00	1.67^c ± 0.01	1.68^c ± 0.05
C15:0	0.81^c ± 0.03	2.74^a ± 0.02	1.92^a,b ± 0.04	0.64^c ± 0.05	1.32^b ± 0.01	0.98^c ± 0.06	0.83^c ± 0.00	0.79^c ± 0.06
C16:0	25.44^a ± 0.51	22.22^a ± 0.40	20.94^a ± 0.78	21.46^a ± 0.38	20.94^a ± 0.36	16.45^b ± 0.12	16.97^b ± 0.05	22.90^a ± 0.08
C17:0	0.98^d ± 0.10	2.16^b ± 0.09	3.24^a ± 0.12	0.88^d ± 0.04	2.41^b ± 0.01	2.48^b ± 0.13	2.03^b ± 0.04	1.63^c ± 0.01
C18:0	9.22^a ± 0.19	4.62^d ± 0.24	7.90^b ± 0.37	6.93^b ± 0.01	7.39^b ± 0.41	6.30^b ± 0.12	5.41^c ± 0.03	7.36 ^b ± 0.07
C20:0	0.23^b ± 0.00	0.77^b ± 0.00	0.23^b ± 0.00	0.81^b ± 0.01	0.08^c ± 0.00	1.59^a ± 0.01	1.77^a ± 0.01	0.68^b ± 0.01
C24:0	1.51^b ± 0.05	3.47^a ± 0.12	1.79^b ± 0.00	0.79^c ± 0.00	2.47^b ± 0.00	3.28^a ± 0.02	0.57^c ± 0.01	0.69^c ± 0.00
∑SFA	40.17	41.02	40.19	25.04	37.38	32.72	29.25	35.73
(b) Monounsaturated fatty acids (MUFA) composition (%)
C14:1	0.23^b ± 0.01	0.18^b ± 0.04	1.65^a ± 0.03	0.57^b ± 0.02	1.32^a ± 0.02	0.87^b ± 0.00	0.29^b ± 0.00	0.36^b ± 0.03
C15:1	0.12^c ± 0.21	2.37^a ± 0.20	1.45^b ± 0.01	ND	0.13^c ± 0.01	ND	1.57 ^b ± 0.02	0.42^c ± 0.02
C16:1n-6	0.96^b ± 0.07	2.03^a ± 0.03	2.78^a ± 0.02	0.68^c ± 0.01	2.85^a ± 0.01	0.89^c ± 0.01	0.54^c ± 0.02	1.02^b ± 0.01
C16:1n-7	3.75^d ± 0.02	9.56^a ± 0.06	6.08^c ± 0.01	8.26^a ± 0.01	7.95^b ± 0.01	5.42^c ± 0.01	7.68^b ± 0.00	7.09^b ± 0.01
C17:1	0.41^c ± 0.01	1.04^a ± 0.00	0.37^c ± 0.01	0.68^b ± 0.00	0.16^c ± 0.03	1.83^a ± 0.00	1.81^a ± 0.01	0.96^b ± 0.00
C18:1n-9	15.24^b ± 0.01	11.09^b ± 0.02	11.31^b ± 0.12	32.32^a ± 0.04	8.88^c ± 0.00	17.01^b ± 0.06	12.95 ^b ± 0.09	25.66^a ± 0.08
C18:1n-7	3.29^b ± 0.04	4.32^a ± 0.00	3.88^b ± 0.00	4.76^a ± 0.01	4.60^a ± 0.02	3.55^b ± 0.01	3.80^b ± 0.00	4.93^a ± 0.04
C20:1	0.12^c ± 0.07	ND	ND	1.08^b ± 0.01	ND	1.42^b ± 0.00	2.18^a ± 0.00	0.88^c ± 0.02
C22:1	0.43^d ± 0.02	1.39^c ± 0.15	ND	ND	0.13^d ± 0.00	2.15^b ± 0.03	6.11^a ± 0.01	0.49^d ± 0.00
∑MUFA	24.55	31.98	27.52	48.35	26.02	33.14	36.93	41.81
(c) n-6 Polyunsaturated fatty acids (PUFA) composition (%)
C18:2	5.38^a ± 0.00	1.20^d ± 0.26	4.47^b ± 0.01	3.82^c ± 0.00	5.84^a ± 0.01	3.93^c ± 0.01	3.89^c ± 0.01	4.92^b ± 0.01
C20:2	0.82^c ± 0.01	2.95^b ± 0.01	0.37^c ± 0.01	0.54^c ± 0.02	ND	4.03^a ± 0.02	4.24^a ± 0.03	0.72^c ± 0.01
C20:4	7.25^a ± 0.01	2.28^c ± 0.12	1.91^d ± 0.03	1.93^d ± 0.01	7.13^a ± 0.03	5.84^b ± 0.01	1.93^d ± 0.06	1.54^d ± 0.03
∑PUFA n-6 1	3.45	6.43	6.75	6.29	12.97	13.8	10.06	7.18
(d) n-3 (PUFA) composition (%)
C18:3	2.48^b ± 0.01	2.27^b ± 0.00	2.89^b ± 0.07	1.69^c ± 0.01	4.37 ^a ± 0.04	2.57^b ± 0.01	2.65^b ± 0.01	2.35^b ± 0.01
C20:3	0.96^e ± 0.04	2.03^c ± 0.01	0.73^e ± 0.04	1.10^d ± 0.03	3.78^b ± 0.01	7.39^a ± 0.00	2.16^c ± 0.01	0.71^e ± 0.00
C20:5	2.77^d ± 0.02	5.25^b ± 0.01	18.53^a ± 0.02	2.80^d ± 0.00	4.56^c ± 0.01	1.16^e ± 0.01	2.45^d ± 0.01	1.66^e ± 0.00
C22:5	3.06 ^b ± 0.01	5.71^a ± 0.07	1.56^d ± 0.01	2.07^c ± 0.00	3.15^b ± 0.00	1.22^d ± 0.03	6.38^a ± 0.04	1.78^d ± 0.01
C22:6	10.95^a ± 0.02	5.09^c ± 0.00	1.33^f ± 0.00	3.11^d ± 0.01	6.51^b ± 0.02	3.50^d ± 0.05	5.60^c ± 0.03	2.04^e ± 0.01
∑PUFA n-3 2	0.22	20.35	25.04	10.77	22.37	15.84	19.24	8.54
Note: Each value is an average of ten samples, with its standard deviations. ND: not detected Values in the same line with different letters are significantly different at p < 0.05.

Table 3 The polyunsaturated/saturated fatty acids (PUFA/SFA) and n-3/n-6 fatty acids ratios of eight different Indus River fishes

Species	Ratio of PUFA/SFA	Ratio of n-3/n-6
Jarko	0.84	1.50
(Wallago attu)
Dayo	0.65	3.16
(Oreochromis mossambicus)
Daahi	0.79	3.71
(Labeo calbasu)
Fouji khago	0.68	1.71
(Bagarius bagarius)
sariyo	0.94	1.72
(Labeo gonius)
Doghno	0.91	1.15
(Clupisoma garua)
Singhari	1.00	1.91
(Aorichthys aor)
shali	0.44	1.19
(Eutropiichthys vacha)

The total unsaturated fatty acid n-3 (8.54–25.04 g/100 g) was found to be higher than that of n-6 (6.29–13.8 g/100 g) in the eight fish species. In the present study, DHA ranged from 1.33–10.95% of total FA^,s. Sargent[37] reported that n-3 PUFA, principally DHA, has a role specific and important role in neural cell membranes, i.e. the brain and eyes. Moreover, it is considered a desirable property in fish for human nutrition and health. Similarly Ackman & Mcleod[34] have reported significant levels (P < 0.05) of n-3 fatty acids in Freshwater fish from cold northern waters. However, Nair & Gopakumar[38] have reported higher n-6 (2.43–26.2 g/100g) than n-3 fatty acids (14 -11%) in freshwater fishes. Freshwater fish are capable of performing the conversion of linolenic acid to the longer n-3 PUFA by chain elongation and desaturation.[39]

The content of Arachidonic acid (AA) (C20:4 n-6) ranging from 1.54–7.25 g/100 g, was comparable with Chinese fresh water fish species.[34] Bowman[40] reported that AA is a precursor for prostaglandin and thromboxan, which will influence the blood clot and its attachment to the endothelial tissue during wound healing. Bessonart et al.[41] have pointed out the importance of AA in fish metabolism since it is known to be the main predecessor fatty acid of eicosanoids in fish. Fish that have higher contents of AA were OM (2.28 g/100 g), CG (5.84 g/100 g), LG (7.13 g/100 g), and WA (7.25 g/100 g). Based on earlier study Rahman et al.[19] in case, the contents of AA in freshwater fishes were high than marine fishes.[36] The present study n-3/n-6 calculated in the range of 1.15–3.71. The n-3/n-6 ratio has been suggested to be the best index when comparing relative nutritional values of fish oils from different species.

Previous studies have shown that the n-3/n-6 PUFA ratio ranged from 1 to 4 for freshwater fish species.[1] The UK Department of HMSO,[42] recommends an ideal relationship of n-3/n-6 of 4.0, at maximum. In this study the n-3/n-6 ratios ranked in the following order: Labeo calbasu (Daahi), Oreochromis mossambicus (Dayo), Aorichthys aor (Singhari), Labeo gonius (sariyo), Bagarius bagarius (Fouji khago), Wallago attu (Jarko), Eutropiichthys vacha (shali), Clupisoma garua(Doghno).

The PUFA/SFA ratio of the eight fish species were examined in the range of (0.44–1.00). Previously Saglik & Imre[43] have reported comparable values for PUFA/SFA for different fish species from Turkey. A minimum value of PUFA/SFA ratio recommended is 0.45,[42] which was lower than those obtained from all freshwater fish species investigated in present work (0.65–1.00), except for EV fish species (0.44); indicating that the Indus river fish species studied are a good source of PUFA, particularly AA, EPA and DHA, hence suitable for inclusion in highly unsaturated low-fat diets.

CONCLUSIONS

The river fish species investigated in present study reveals that Indus river fishes are good sources of n-3 fatty acids particularly EPA and DHA, and should be recommended for dietary inclusion to reduce risks of cardiovascular disease. The Labeo calbasu (LC) proved to be species most suited to a fish based diet due to higher n-3/n-6 ratio particularly higher EPA content, despite the fact it contains small amount of fat. Moreover, the FA characteristics of these fish species indicated a great potential for culturing this river fish species for future nutrition research and marketing. In addition, the information presented in this study may be valuable for the pharmaceutical and food industries in the selection of fresh water fish and fish oil for chemical studies.

Notes

42. HMSO, UK. 1994. Nutritional aspects of cardiovascular disease (report on health and social subjects' No. 46. London: HMSO.