Journal of the Science of Food and Agriculture
J Sci Food Agric 83:1139–1142 (online: 2003) DOI: 10.1002/jsfa.1515
Lipase activity in different tissues of four species of fish: rohu (Labeo rohita Hamilton), oil sardine (Sardinella longiceps Linnaeus), mullet (Liza subviridis Valenciennes) and Indian mackerel (Rastrelliger kanagurta Cuvier) Jyotiranjan Nayak, PG Viswanathan Nair,∗ K Ammu and Suseela Mathew Department of Biochemistry and Nutrition, Central Institute of Fisheries Technology, Matsyapuri, Cochin, Kerala, India
Abstract: Lipase activity in stomach and pyloric caeca, liver, intestine and red muscle of rohu (Labeo rohita Hamilton), oil sardine (Sardinella longiceps Linnaeus), mullet (Liza subviridis Valenciennes) and Indian mackerel (Rastrelliger kanagurta Cuvier) was studied. Distinct differences in lipolytic activity in different tissues of these fish were observed. Rohu showed the highest activity in all tissues in comparison with the other three species of fish. Among the three size groups of mullet, medium-sized mullet showed higher activity than the other two groups in all tissues except intestine. Rohu hepatopancreatic lipase exhibited more hydrolytic activity on fish oil than rohu intestinal lipase. 2003 Society of Chemical Industry
Keywords: crude enzyme; lipase activity; fish oil; intestinal lipase; mullet; fish species
INTRODUCTION The widespread use of enzymes in industrial processes has necessitated the identification of costeffective and easily available sources of these enzymes. Lipases (triacylglycerol acylhydrolase; EC 3.1.1.3), which catalyse the hydrolysis of fatty acid ester bonds in triacylglycerols and related components, are widely used in the dairy industry,1 detergents,2 the oleochemical industry,3 the food industry4 and the production of biofuels.5 At present the main source of these enzymes is microorganisms. Taking into account the growing demand, it is essential that alternative sources are identified. Lipolytic activity has been detected in plants, higher vertebrates and micro-organisms, and lipases have been isolated and purified from various sources such as porcine pancreas,6 rat pancreas7 and microorganisms.8,9 The potential of fish and other marine organisms as a source of lipase is an area meriting detailed investigation. Studies on lipolytic activity in tissues of various fish are available.10 – 13 The objective of the present work was to compare digestive and muscle lipase activity in marine, brackish and freshwater fish and to determine whether they can be a possible source of lipase for industrial application.
MATERIALS AND METHODS Materials Fish employed in the experiments were Labeo rohita Hamilton (rohu), Sardinella longiceps Linnaeus (oil sardine), Liza subviridis Valenciennes (mullet) and Rastrelliger kanagurta Cuvier (Indian mackerel). They were collected from markets and farms in and around Cochin, India. Each time about 2–3 kg of fish were obtained fresh, transported to the laboratory in ice and analysed within 6–8 h of collection. Lipase activity in stomach and pyloric caeca, intestine, red muscle and liver/hepatopancreas of these fish was studied. In the case of mullet, three different size groups (see Table 2) were examined to evaluate any differences in enzyme activity associated with fish size. The chemicals polyvinyl alcohol (PVA), bovine serum albumin (BSA), tributyrin and sodium taurocholate were purchased from Sigma Chemical Company (St Louis, MO, USA). Fish oil from oil sardine (S longiceps) used in the experiment was obtained from a commercial source. The fatty acid composition of the oil is shown in Table 1. All other chemicals used were of analytical grade. Methods Preparation of crude enzyme extract All procedures in the preparation of the crude enzyme extracts were carried out at 0–4 ◦ C. Fish
∗
Correspondence to: PG Viswanathan Nair, Department of Biochemistry and Nutrition, CIFT, Willingdon Island, Matsyapuri-PO, Cochin 682 029, Kerala, India E-mail:
[email protected] (Received 14 May 2002; revised version received 12 March 2003; accepted 14 May 2003)
2003 Society of Chemical Industry. J Sci Food Agric 0022–5142/2003/$30.00
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J Nayak et al Table 1. Fatty acid composition of fish oil from oil sardine
Fatty acid
Content (% by weight of total fatty acids)
C14:0 C15:0 C16:0 C17:0 C18:0 C16:1 C18:1 C20:1 C22:1 C18:2 C18:3 C18:4 C20:4 C20:5 C22:4 C22:5 C22:6
8.1 0.3 27.0 1.0 3.8 6.8 15.4 2.3 3.7 4.3 0.8 1.7 2.6 10.6 1.2 0.8 9.6
were weighed individually and then incised ventrally. The digestive tract was dissected and ligated at specific points to collect stomach and pyloric caeca,∗ liver/hepatopancreas† and intestine. After skinning, red meat from the lateral line was collected from the fish. Tissues from each section were pooled, washed gently with a stream of cold distilled water, blotted dry with filter paper and weighed. The pooled tissues were homogenised in twice their weight of chilled McIlvaine buffer (0.1 M, pH 7.0). The homogenates were centrifuged at 10 000 × g for 30 min. The lipid cap formed was removed and the supernatant was recentrifuged at 15 000 × g for 1 h to obtain a clear supernatant. The clear supernatant was treated as the crude enzyme extract in subsequent assays. The protein concentration of crude enzyme extract was determined by the method of Lowry et al 14 using BSA as standard. Assay of lipase activity Lipase activity was determined by the titrimetric method of Mukundan et al 13 Crude enzyme extracts (3 ml) of the various tissue preparations were added separately to reaction mixtures consisting of 5 ml of
* True stomach is absent in rohu. The anterior part of the intestine, known as false stomach, was used for assay of stomach and pyloric caeca activity. † Diffused hepatopancreas is present in rohu and oil sardine.
tributyrin emulsified with PVA (0.1 M) and 5 ml of 0.05 M calcium chloride and incubated at 37 ◦ C for 1 h. The reaction was terminated by the addition of 20 ml of 80% ethanol. Blank determinations were conducted in a similar manner, except that the extracts were introduced after termination of the reaction. Lipase activity was estimated by titration with standard 0.1 M NaOH using thymolphthalein as indicator. Specific enzyme activity was expressed as µmol butyric acid liberated mg−1 protein min−1 . Hydrolysis of fish oil by intestinal and hepatopancreatic lipase of rohu The activity of the enzyme preparation on fish oil substrate was determined using a modified method of Ando et al 15 The assay system consisted of 2.5 ml of 25% fish oil emulsified in 1% PVA, 2.5 ml of 0.05% BSA in phosphate buffer (0.1 M, pH 7.0), 2.5 ml of 0.5% sodium taurocholate and 0.5 ml of 0.05 M calcium chloride, to which 1.5 ml of hepatopancreas or intestine extracts were added separately. Air in the reaction flask was replaced by nitrogen gas, and flasks were incubated at 30 ◦ C for 5 h with constant shaking. After 5 h, 20 ml of 80% ethanol was added to terminate the reaction. Controls were run in a similar way, but the crude enzyme extracts were added after termination of the reaction. Lipase activity was estimated by titration with standard 0.1 M NaOH using thymolphthalein as indicator. Specific enzyme activity was expressed as µmol oleic acid liberated mg−1 protein h−1 . Statistical analysis Treatment effects were analysed by one-way analysis of variance (ANOVA). Differences in means were evaluated for significance by Duncan’s multiple range test. The SPSS statistical package (version 7.5) was used for statistical analysis.
RESULTS AND DISCUSSION Lipase activity in different tissues of three size groups of mullet Data on lipase activity in stomach and pyloric caeca, liver, intestine and red muscle of mullet of three different size groups are presented in Table 2. ANOVA showed that there were significant differences in lipolytic activity in stomach and pyloric caeca, liver and intestine among groups 1, 2 and 3 (p < 0.05).
Table 2. Lipase activity (µmol butyric acid mg−1 protein min−1 ) in different tissues of three size groups of mullet (Liza subviridis)
Group 1 (114.55 ± 10.005 g) Group 2 (54.20 ± 6.066 g) Group 3 (23.02 ± 0.868 g)
Liver
Intestine
Stomach and pyloric caeca
Red muscle
0.22 ± 0.02a 0.24 ± 0.02a 0.18 ± 0.007b
0.19 ± 0.01a 0.17 ± 0.004a 0.31 ± 0.02b
0.24 ± 0.02a 0.30 ± 0.04b 0.28 ± 0.02b
0.15 ± 0.02 0.16 ± 0.006 0.16 ± 0.01
Values are mean of five determinations ± SD. Means followed by different letters within the same column differ significantly from each other at p < 0.05 level.
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Lipase activity in different tissues of four fish
Group 3 mullet were seen to have lower lipase activity in liver when compared with groups 1 and 2, whereas the latter two groups did not show any statistically significant difference in liver lipase activity (p < 0.05). In the case of intestine the highest lipase activity was noted in group 3 and the lowest in group 2. However, the difference between groups 1 and 2 was not significant (p < 0.05). Lipase activity in stomach and pyloric caeca was lower in group 1 (p < 0.05) than in groups 2 and 3. No significant difference in stomach and pyloric caeca lipase activity was observed between groups 2 and 3 (p < 0.05). A report on rainbow trout indicates that, in general, esterase activity increased with growth, but the highest activity was found in medium-sized fish.16 The differences in activity among the three size groups of mullet may be due to differences in feeding habits,17 food composition18 and/or physiological status of the fish with respect to their growth. Lipase activity in red muscle of all groups was almost the same. From the life history of mullet it is known that mature mullet migrate to the sea for spawning.19 More activity could be expected owing to spawning migration of the fish, but this was not reflected in the lipase activity of red muscle in this study. Lipase activity in different tissues of four species of fish Data on lipase activity in different tissues of rohu, oil sardine, mackerel and mullet are presented in Table 3. ANOVA showed that there were significant differences in lipolytic activity in stomach and pyloric caeca, liver/hepatopancreas, intestine and red muscle among rohu, oil sardine, mackerel and mullet (p < 0.05). Lipase activity of intestine was found to be significantly higher in rohu and oil sardine than in mackerel and mullet. No significant difference in intestinal lipase activity was noted between rohu and oil sardine or between mackerel and mullet. Rohu and oil sardine do not have a well-developed compact pancreas, and this may be the reason for higher intestinal lipase activity in these species. The lipolytic activity is compensated by the secretion of lipase in the intestinal mucosa in addition to the secretory activity of the diffused pancreas.20 Lipase activity bears a definite correlation with the feeding habits of fish.21 This may be another reason for such variations among the species. Lipase activity was found to vary significantly in stomach and pyloric caeca of the four species. This variation may be due to differences in feeding habits,21
composition of diet,22 substrate specificity of lipase23 and/or volume of pyloric caeca.16 Significant differences in red muscle lipase activity were found among the four species of fish studied. The weakest lipase activity was exhibited by red muscle of oil sardine, mullet and mackerel. However, among these three marine fish, the highest activity was noted in mackerel. Many factors, eg active swimming,24 specificity of lipase for fatty acids,25 etc, influence lipase activity in tissues. Hepatopancreas of rohu and oil sardine exhibited more lipase activity than that from the other two species of fish. Lipase activity of hepatopancreas was significantly higher in rohu than in the other three species. In the present study it was observed that diffused hepatopancreas had higher activity than discrete liver. However, it may be noted in this context that the assay here was carried out using tributyrin as substrate, and the true lipase activity associated with discrete liver26 hydrolyses esters of long-chain aliphatic acids from glycerides at oil/water interfaces.27 Comparison of fish oil hydrolysis by crude hepatopancreatic and intestinal lipase preparations of rohu As the intestine and hepatopancreas of rohu showed the highest lipolytic activity with tributyrin (compared with organs of the other three species of fish), these two organs were selected for fish oil hydrolysis to study their action on long-chain fatty acid-rich glycerides. Fish oil hydrolysis by rohu intestinal and hepatopancreatic lipase is illustrated in Table 4. It was noted that the hepatopancreatic lipase released more fatty acids from the fish oil glycerides than the intestinal lipase. In general, fish oil is rich in longchain and polyunsaturated fatty acids. This result indicated that the enzyme in the hepatopancreas was more efficient at hydrolysing glycerides of fish oil than that in the intestine. A similar observation was made in carp (Cyprinus carpio).28
CONCLUSION The result of the study comparing activities of lipases in different tissues of mullet, rohu, oil sardine and mackerel indicated that distinct differences exist in the lipolytic activity in different tissues of these fish. It was also observed that the lipase activity was related to the different stages of growth. Fish can be considered as a
Table 3. Lipase activity (µmol butyric acid mg−1 protein min−1 ) in different tissues of rohu, oil sardine, mackerel and mullet
Fish Rohu Oil sardine Mackerel Mullet
Intestine
Stomach and pyloric caeca
Red muscle
Liver/hepato-pancreas
0.540 ± 0.05aA 0.494 ± 0.02aA 0.135 ± 0.02bA 0.193 ± 0.01bA
0.527 ± 0.023aB 0.428 ± 0.027bB 0.173 ± 0.027cB 0.231 ± 0.021dB
0.282 ± .04aC 0.114 ± .01bC 0.231 ± .02cC 0.158 ± .02dC
0.587 ± 0.02aD 0.243 ± 0.01bD 0.228 ± 0.02bD 0.226 ± 0.02bD
Values are mean of five determinations ± SD. Means followed by different lowercase letters within the same column and by different uppercase letters within the same row differ significantly from each other at p < 0.05 level.
J Sci Food Agric 83:1139–1142 (online: 2003)
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J Nayak et al Table 4. Fish oil hydrolysis by rohu hepatopancreatic and intestinal lipase
10
Tissue Hepatopancreas Intestine
Specific activity (µmol oleic acid liberated mg−1 protein h−1 ) 2.76 ± 0.08 2.54 ± 0.09
Values are mean of triplicate determinations ± SD.
potential source for industrial lipases, especially where enzymes optimal for long-chain and unsaturated fatty acids are required. The enzymatic activity of the crude extract of extracellular lipase from Pseudomonas fragi, which is a commercial source of the enzyme, was estimated to be 0.62 µmol mg−1 protein min−1 ,29 the assay conditions being 35 ◦ C, pH 9.0 and tributyrin as substrate. The values obtained here for lipase activity in different organs of fish are comparable to this value, suggesting that fish organs might also be a good source for industrial lipase production.
11
12
13
14
15
16
17 18
ACKNOWLEDGEMENT The authors are grateful to Dr MR Raghunath, Senior Scientist, Bangalore Research Center of Central Institute of Freshwater Aquaculture, for checking the manuscript.
19 20
21
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J Sci Food Agric 83:1139–1142 (online: 2003)
