Comp. Biochem. PhysioL Vol. 83B, No. 1, pp. 51-55, 1986 0305-0491/86 $3.00+0.00 Printed in Great Britain © 1986 Pergamon Press Ltd LIPOLYSIS POST MORTEM IN NORTH ATLANTIC KRILL OLAV SAETHER, 1 TROND E. ELLINGSEN 2 and VIGGO MOHR 1 IDepartment of Biochemistry, The Norwegian Institute of Technology, The University of Trondheim, N-7034 Trondheim-Nth, Norway (Tel: (07)594071); and 2Biotechnology Research Unit, Division of Applied Chemistry, SINTEF, N-7034 Trondheim-Nth, Norway (Received 25 March 1985) A~tract--1. Three common species of North Atlantic krill, Meganyctiphanes norvegica (M. Sars), Thysanoessa inermis (Kroyer) and T. raschii (M. Sars), have been stored at 0°C post mortem, and the lipolytic activity followed by measuring changes in the lipid composition during storage. 2. Both phosphoglycerides and triacylglycerols were subjected to extensive hydrolysis with the formation of free fatty acids in all krill species examined, whereas wax esters, constituting a considerable proportion of the lipids in the Thysanoessa species, were not hydrolysed at all. 3. In M. norvegica the triacylglycerols and phosphoglycerides were hydrolysed at similar rates, whereas in T. inermis and T. raschii the phosphoglycerides were hydrolysed most rapidly. 4. For all krill species examined, the rate of production of free fatty acids was nearly constant during the initial phase of storage, and subsequently declined on prolonged storage. 5. At the end of the storage period of 16-24 days, the free fatty acids constituted about 35% of the total lipid in M. norvegica, and about 50% in the Thysanoessa species. 6. The rate of production of free fatty acids was about the same in all the three species of krill and seemed to be independent of the total lipid content. INTRODUCTION Recent studies on Antarctic krill have demonstrated that both phosphoglycerides and triacylglycerols serve as depot lipids in Euphausia superba, whereas E. crystallorophias deposits wax esters, triacylglycerols and phosphoglyceridcs (Ellingsen and Mohr, 1981; Ellingsen, 1982). Furthermore, Ellingsen (1982) has shown that storage of samples of krill post mortem resulted in extensive degradation of triacylglycerols and phosphoglycerides with the formation of free fatty acids, thus suggesting that these species of Antarctic krill contain significant levels of active lipases and phospholipases. The work by Ellingsen (1982) formed the basis of a program for examination of the chemical com- position and post mortem changes in three common species of North Atlantic krill: M. norvegica, T. inermis and T. raschii. The lipid composition of North Atlantic krill has been examined in detail by Sargent and Falk-Petersen (1981) and Saether et al. (1985). Saether et al. (1985) have provided clear evidence that triacylglycerols are the only depot lipids in M. norvegica, whereas phosphoglycerides, wax esters and triacylglycerols serve as depot lipids in T. inermis and T, raschii. The post mortem changes in the lipid of these species have not as yet been studied. The present communication provides data relating to the post mortem changes in the lipids of these species of North Atlantic krill. MATERIALS AND METHODS The krill used in the present studies were caught in Balstorden and Ullsfjorden in Northern Norway during the period 23 February-21 April 1982. Details of the catches are given in Table I. The catching and handling of the krill have 51 been described in detail by Saether et al. (1985). On board the vessel 20-50 ml samples of whole and homogenized krill were distributed into 100 ml plastic bottles with screw caps within 15-30min of catching, and stored as described below. Krill were also frozen in CO2-ice within 5-15 min of catching and stored at -80°C for no longer than I year before being used experimentally. A closer examination of the different Thysanoessa species was carried out in the laboratory. Blocks of krill were thawed and sorted accord- ing to species at 0°C within 4-6hr of thawing. Each specimen was subsequently refrozen on CO2-ice after sort- ing. Krill belonging to the same species and haul were pooled and homogenized in the frozen state, using an Ultra-Turrax TP 18-10 (Janke & Kunkel KG). Samples of 2-5 g of the homogenates were distributed into 10 ml glass tubes with screw caps. The samples of both fresh and frozen/thawed krill were stored in an ice-water bath, and individual samples removed as a function of time, and stored at -80°C until analysed. The entire content of each sample was homogenized and used in the analysis. The lipids were extracted with chloroform-methanol-water according to Hardy and Keay (1972). The main classes of lipid were separated by thin layer chromatography in accordance with Ellingsen (1982), as described by Saether et al. (1985). The dry weight of each sample was determined by drying I-2 g of homogenate for 24 hr at 105°C. RESULTS AND DISCUSSION The post mortem changes in the lipid constituents of three common species of North Atlantic krill, M. norvegica, T. inermis and T. raschii, were examined at 0°C. The studies were based on five catches of krill from fiords in northern Norway over a period of 2 months (February-April). Based on measurements of length, a majority of the krill examined seemed to be sexually mature (Saether et al., 1985).
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`52 OLAV SAETHER et al. Table 1. Data on the krill catches Total lipid Haul Krill content (% No. Date Locality species of dry weight) 21 23.02.82 Svartnes in T. inermis 35.3 Balsfjorden T. raschii 27.6 unsorted Thysanoessa* 33.1 23 23.03.82 Ulslorden M. norvegica 26.0 24 23.03.82 Svartnes in T. inermis Bals0orden T. raschii unsorted Thysanoessat 25.5 27 20.04.82 Ulsl~orden M. norvegica 22.7 28 21.04.82 Svartnes in T. inermis 14.9 Balstorden T. raschii 11.4 unsorted Thysanoessat 13.9 *Mixture of 70% T. inermis and 30% T. raschii. tMixture of 80% T. inermis and 20% T. raschii. In M. norvegica both triacylglycerols and phos- phoglycerides were hydrolysed with the formation of free fatty acids post mortem, suggesting that both lipases and phospholipases were active during stor- age. Judging from the data, the hydrolysis of tri- acylglycerols and phosphoglycerides proceeded at similar rates. The rate of release of fatty acids gradu- ally decreased during storage in both catches exam- ined, and at the end of the storage period, free fatty acids accounted for about 35% of the total lipid (Fig. 1). The rate of release of free fatty acids, on a dry weight basis, was also similar in the two catches (Fig. 2). In the Thysanoessa species the increase in the free fatty acid content post mortem was primarily due to extensive hydrolysis of phosphoglycerides, but the hydrolysis of triacylglycerols was also significant (Fig. 3). The rate of free fatty acid production was about the same in krill having widely different total lipid contents (13.9-33% dry wt), thus suggesting that the rate of lipid hydrolysis was independent of the total lipid content. (Table 1 and Fig. 2). At the end of the storage period free fatty acids constituted about 50% of the total lipid in the Thysanoessa species, which is significantly higher than in M. norvegica. A com- c~ 80 d- 64 o-.1 ~.j 48 el) b-- 'I10 ~ 32 ~l.I- 0 .-.-:- 16 I 0 ~'~ HAUL 23 ..A /- 6 12 18 i4 BO J -, HAUL 27 64 z~ .......... 48 _.~,dk 32 I yAf- j-, 0 6 12 IB i 24 30 TIME (DAYS) TIME (DAYS) Fig. 1. Content of main classes of lipid during storage ofM. norvegica (haul 23 and 27) at O°Cpost mortem. Wax esters: O, triacylglycerols: A, free fatty acids: A, cholesterol + diacylglycerols: IS, phospho- glycerides: m. 15 COI ~W >->- 9 F-r~ ~-~ 6 u-o 3 v 0 THYSANOESSA SP, _ .__+__~ /~_m t. 5 10 15 TIME (DAYS) io 25 10 M, NO,RVEGICA ././~/" /e 'lo 25 TIME (DAYS) Fig. 2. Content of free fatty acids in different hauls of Thysanoessa species (haul 21: •, 24: and 28: A) and in M. norvegica (haul 23: • and 27: ) stored at 0°C post mortem.
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`Post mortern lipolysis in krill 53 O-- ,ll F1 0 F-- V rl~ O- ,1J .--I Z 60 4B 36 24 12 0 HAUL 21 if / i 6 12 18 24 60 48 36 24 L2 0 HAUL 28 /~' e 6 12 18 TIME (DAYS) 30 30 4B, 36 24 12 0 HAUL 24 /~////_A 6 12 18 TIME (DAYS) 24 30 Fig. 3. Content of main classes of lipid during storage of Thysanoessa species (haul 21, 24 and 28) at 0°C post mortem. Wax esters: O, triacylglycerols: A, free fatty acids: A, cholesterol + diacylglycerols: IS, phosphoglycerides: 1. parison of the rates of hydrolysis in North Atlantic and Antarctic krilI reveals that the rates observed in M. norvegica, the Thysanoessa species and E. crys- tallorophias are nearly the same, and higher than that detected in E. superba (Fig. 4). The content of wax esters and cholesterol varied little during storage of the North Atlantic krill species post mortem, sug- gesting that there is no significant formation of cholesteryl esters during the first week of storage (Fig. 3). The contents of wax esters and cholesterol were likewise constant during the first week of storage of E. crystallorophias (Ellingsen, 1982). Esterification of cholesterol has been observed during storage of fish post rnortem (Lovern et al., 1959). i0 ¢.)UJ >->- 6 u.w 4 ~u_ u-o 2 0 f TIME (DAYS) 15 Fig. 4. Content of free fatty acids during post mortem storage at 0°C of M. norvegica (haul 27: A), Thysanoessa species (haul 28: A) and Antarctic krill species, examined by Ellingsen (1982) (E. crystallorophias: S, E. superba: 1). The studies of the post mortem changes in the Thysanoessa species on board the research vessel were based on catches containing 70-80% T. inermis and 20-30% T. raschii. A separation of T. inermis and T. raschii required microscopy of each individual, which was not possible on board the vessel, due to priority given to other experiments. However, frozen krill from haul 28 were thawed in the laboratory, sorted according to species, homogenized and stored at 0°C for a closer examination of lipolytic activity in T. inermis and T. raschii. The results indicate a similar pattern of lipid degradation in the two species (Fig. 5). Furthermore, the rate of release of free fatty acids was also nearly the same in the two species (Fig. 6). It is of interest to consider the lipolytic activity in relation to the lipid class composition of the different species of krill. In M. norvegica triacylglycerols are the only depot lipids, whereas in T. inermis and T. raschii phosphoglycerides, wax esters and tri- acylglycerols serve as depot lipids (Saether et al., 1985). E. crystallorophias also deposits phos- phoglycerides, wax esters and triacylglycerols, whilst E. superba uses only phosphoglycerides and tri- acylglycerols as depot lipids (Ellingsen, 1982). A systematic comparison of the different species of krill reveal no correlation between the relative proportion of the main classes of lipid, and the rate at which the lipids are hydrolysed. It appears, therefore, that the lipolytic activity in each species depends on other factors, such as the level of lipases and phos- pholipases in the krill, the extent of emulsification of the substrates and the degree of contact between enzymes and substrates. Studies of different species of fish have also failed to reveal any correlation between
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`54 OLAV SAETHER et al. '-;~ 40 a. <~- 20 _I ZI~ lO I ~ v 0 T, INERMIS TIME (DAYS) kº_ T, RASCH I I ~__ ~'-~--~- ........ .... i 2 3 4 5 TIME (DAYS) Fig. 5. Content of main classes of lipid during storage of homogenized, frozen and thawed T. inermis and T. raschii (haul 28) at 0°C. Wax esters: O, triacylglycerols: A, free fatty acids: A, cholesterol + diacylglycerols: F-l, phosphoglycerides: 1. the lipase- and phospholipase activity and the content of triacylglycerols and phosphoglycerides (Olley et al., 1962; Shono and Toyomizu, 1973; Nair et al., 1978). As pointed out above, phosphoglycerides were subjected to more extensive hydrolysis than tri- acylglycerols in the Thysanoessa species (Figs 3 and 5). A similar observation was made in the study of the lipid degradation in the Antarctic krill E. superba and E. crystallorophias (Ellingsen, 1982), whereas in M. 5 o.}-r ,", co 4 ~M >->- 3 I-- ,'-1 u.w 2 -r tall-- u.o i v 0 f , , 2 4 TIME (DAYS) B I0 Fig. 6. Content of free fatty acids during storage of homoge- nized, frozen and thawed T. inermis (haul 28: A) and T. raschii (haul 28: FI) at 0°C post mortem. norvegica the lipase activity was of the same order as the phospholipase activity (Fig. 1). It has been proposed that the more extensive degradation of phosphoglycerides than triacylglycerols in Antarctic krill species may be due to the more hydrophilic character of the phosphoglycerides, and hence a better enzyme to substrate contact (Ellingsen, 1982). The same may apply to North Atlantic Thysanoessa species. Triacylglycerols are more hydrophobic, and may as a result, require more effective emulsification for hydrolysis to take place. Wax esters are even less polar than triacylglycerols, and inadequate emulsification and/or a low enzyme level may explain why the wax esters were not subjected to post mortem hydrolysis neither in the Thysanoessa species nor in E. crystallorophias (present results and Ellingsen, 1982) The triacylglycerols in M. norvegica were subjected to much more extensive hydrolysis than those of the Thysanoessa species, which may be due to a higher lipase content, and/or better emulsification and, hence, a better enzyme-substrate contact. The lipids and lipolytic enzymes are presumably separated in the tissues of living krill. Thus, pro- teolytic activity is probably required to bring the lipids into contact with the lipolytic enzymes post rnortem, as suggested by Ellingsen (1982). An exten- sive proteolysis post mortem has been established BO - '-' 64 o-~ 48 0"1 ..-I O')F- ~ 32 0 M,NORVEG'ICA ____--.& 6 12 IB 24 30 TIME (DAYS) 6O 4B, 36 24 12 01 THYSANOESSA SP, /.~ J /// 6 12 IB 24 TIME (DAYS) 30 Fig. 7. Content of main classes of lipid during storage of homogenized M. norvegica (haul 23) and Thysanoessa species (haul 24) at 0°C post mortem. Wax esters: O, triacylglycerols: A, free fatty acids: A, cholesterol + diacylglyeerols: F-l, phosphoglycerides: 1.
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`Post mortem lipolysis in krill 55 both in Antarctic krill (Ellingsen, 1982) and in North Atlantic krill (unpublished results), which may ex- plain why lipolysis in all species of krill examined so far proceeded without any distinct, initial lag-phase (Figs 1 and 3; Ellingsen, 1982). Storage of homogen- ized samples of M. norvegica revealed a three-fold increase in the initial rate of hydrolysis of both phosphoglycerides and triacylglycerols as compared to whole krill, whereas in the Thysanoessa species, there were no significant differences in the rate of hydrolysis of whole- and homogenized animals (Figs 1-3, 7). Separate studies have revealed that the bacterial activity seems to be low during the first week of storage of M. norvegica and Thysanoessa species at 0°C (unpublished results). Thus, there is reason to believe that the post mortem changes observed during the first week of storage are primarily due to autolytic processes. Low bacterial activity during the first week of storage post mortem has also been observed in Antarctic krill (Ellingsen, 1982). In conclusion, the present work and the studies of Ellingsen (1982) have provided evidence that the hydrolysis of both phosphoglycerides and tri- acylglycerols is very rapid and extensive in both North Atlantic- and Antarctic krill, whereas the content of wax esters seem to be constant during post mortem storage of wax ester rich species. It is also concluded that the rate of production of free fatty acids, on a dry weight basis, is about the same in M. norvegica, T. inerrnis and T. raschii as in the Antarctic krill E. crystallorophias, examined by Ellingsen (1982). REFERENCES Ellingsen T. E. (1982) Biokjemiske studier over antarktisk krill Dr. ing. thesis, Department of Biochemistry, The Norwegian Institute of Technology. The University of Trondheim, N-7034 Trondheim-Nth, Norway (in Nor- wegian with summary in English). EUingsen T. E. and Mohr V. (I 981) Lipids in Antarctic krill. Composition and post mortem changes. Proc. l lth. Scan- dinavian Symposium on Lipids. (Edited by Marcuse R.), pp. 110-116. SIK, Gothenborg, Sweden. Hardy R and Keay J. N. (1972) Seasonal variations in the chemical composition of Cornish mackerel, Scomber scombrus (L), with detailed reference to the lipids. J. Fd TechnoL 1, 125. Lovern J. A., Olley J. and Watson H. A. 0959) Changes in the lipids of cod during storage in ice. J. Sci. Fd Agric. 10, 327. Nair P. G. V., Antony P. D., Gopakumar K. and Nair M. R. 0978) Lipid breakdown in oil sardine (Sardinella longiceps) during frozen storage. Fish. Technol. 15, 81-84. Olley J., Pirie R. and Watson H. (1962) Lipase and phos- pholipase activity in fish skeletal muscle and its re- lationship to protein denaturation. J. Sci. Fd Agric. 13, 501-516. Saether O., Mohr V. and Ellingsen T. E. 0985) Lipids of North Atlantic krill. (To be published.) Sargent J. R. and Falk-Petersen S. (1981) Ecological investigations on the zooplankton community in Balsfjorden, northern Norway: lipids and fatty acids in Meganyctiphanes norvegica, Thysanoessa raschii and T. inermis during mid-winter. Mar. Biol. 62, 131-137. Shono T. and Toyomizu M. (1973) Lipid alteration in fish muscle during cold storage--I. Expression of lipid hydro- lysis and oxidation in jack mackerel muscle based on decrease in C22:6 acid. Bull. Jpn. Soc. Sci. Fish. 39 411-416.
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