Fisheries Research, 4 (1986) 259-269

Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

259

A R e v i e w on A u t o l y s i s in F i s h
M.K. MUKUNDAN, P.D. ANTONY and M.R. NAIR

Central Institute of Fisheries Technology, Cochin-29 (India)

(Accepted for publication 3 June 1986)

ABSTRACT Mukundan, M.K., Antony, P.D. and Nair, M.R.,1986. A review on autolysisin fish. Fish. Res.,4: 259-269. Autolysis plays an important role in spoilage of fish. The enzymes involved in autolysis are many. However phosphorylases, lipases, cathepsins and gut enzymes predominate. The distribution and properties of these enzymes in some important fishes and their action on major tissue components such as carbohydrates, fat and proteins are discussed to establish a possible mechanism for autolysis in fish. In the light of this possible mechanism, some suggestions are given for limiting autolysis to improve the quality of fresh as well as processed fish.

INTRODUCTION

The spoilage of fish can be defined as the unacceptable changes occuring in fish muscle post-mortem. Such changes are enhanced as a result of careless handling and faulty processing or storage. Spoilage can be broadly classified into two types; bacterial spoilage and autolytic spoilage. Bacterial spoilage results from growth and multiplication of microorganisms at the expense of muscle constituents. Even though bacterial growth is the major cause of spoilage of fish, it can be effectively controlled by proper methods of sterilisation and use of bactericidal agents. The rate and extent of autolytic spoilage in fish is considerably less than bacterial spoilage, but autolysis plays an important role in flavour development and the onset of bacterial spoilage. Absolutely fresh and healthy fish is impermeable to bacteria due to the intact skin. Further, the absence of simple and easily available nutrients in absolutely fresh fish makes it difficult for bacteria to grow and multiply. However, after the death of the fish, autolysis sets in, making the fish skin permeable to bacteria and at the same time releasing simple sugars, free amino acids, free fatty acids, etc., which provide a nutrient-rich medium for bacteria to thrive. In simple terms, autolysis is defined as the degradation of muscle and skin
0165-7836/86/$03.50 1986 Elsevier Science Publishers B.V.

260 constituents by endogenous enzymes. Since the enzymes causing autolysis arise from within the fish muscle, the prevention and control of autolysis is very difficult unless drastic treatments are used. However, a clear understanding of autolysis would be useful in devising suitable methods to curb the process and effectively reduce bacterial spoilage, thereby preserving the delicate flavour. A lot of work has been done on post-mortem biochemical changes in fish muscle with a view to understanding the p h e n o m e n o n of autolysis. The present paper is an attempt to consolidate the results of research on autolysis done elsewhere, in the light of the work done by the authors. ROLE OF ENZYMES IN AUTOLYSIS

The nature of enzymes and their distribution

Live fish contain numerous enzyme systems required for the complex metabolic reactions taking place. These enzymes are distributed both in the intracellular and extracellular compartments throughout the fish muscle, but their individual concentrations vary with the nature and function of the tissue. In live fish, all these enzyme systems are put to use only as and when they are actually required. Consequently, most of the enzymes occur as some sort of inactive precursors as in phosphorylase b, chymotrypsinogen, etc., whose conversion to active forms requires the presence of certain cofactors, while in certain other cases the enzymes are kept isolated from their substrates, as in the case of a group of hydrolases kept inside lysosomes. However, there are also certain enzyme systems such as adenylate cyclase, ATP-ase, etc., which are present in the active form in tissues, but in such cases their concentration will be very low and their distribution will be limited to specialised tissues. Once the fish is dead, the ability of its body to regulate the enzymes will be lost. The absence of blood circulation, and hence the absence of oxyen and nutrient supply to the tissue, depletion of energy sources such as CTP ( creatine triphosphate) and ATP ( adenosine triphosphate ), and the breakdown of the body's scavenging system will bring an end to all anabolic or biosynthetic processes. In effect, in post-mortem fish muscle, only the catabolic or the degenerative and degrading reactions are active, leading to the accumulation of catabolic products.

Glycolysis and decrease in pH precedes autolysis

The major catabolic reactions occuring in post-mortem fish muscle relate to three groups of compounds, viz. carbohydrate, fat and protein. Even though the amount of carbohydrate in fish muscle is very low compared to protein and fat, its metabolism in live and post-mortem fish muscle is of paramount importance in deciding the extent of rigor mortis and autolytic spoilage in fish. Most

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fish are caught after vigorous struggles, during which period the fish utilises almost all its blood glucose and a substantial quantity of tissue and liver glycogen for deriving energy through the glycolytic pathway ( Tarr, 1965 ). Phosphorylase, the prime enzyme responsible for initiating glycogenolysis ( Harrow and Mazoor, 1962), is distributed widely in almost all tissues in substantial quantities (Crabtree and Newsholme, 1972; Mukundan and Antony, 1977), as is evident from Table I. The almost neutral pH optimum and the high activity of phosphorylase at ambient as well as near sub-zero temperatures (Nowlan and Dyer, 1974; Mukundan and Nair, 1977) considerably augment glycolysis/glycogenolysis in fish muscle leading to accumulation of lactic acid. The temperature and pH optimum values in Table II and the extent of lactic acid formation shown in Table III confirm this conclusion. In live fish, the lactic acid will be reconverted to glycogen if the fish is allowed to rest. However, this does not occur in a fish catch, as the fish struggle until they are dead. The fish are then usually frozen and stored until required. During this storage, breakdown of residual glycogen/glucose also occurs. Nowlan and Dyer (1974) in cod ( Gadus morhua), Sharp (1935) in haddock (Melanogrammus aeglefinus ) and Partmann (1963) in carp ( Cyprinus carpio) and rainbow trout ( Salmo gairdneri) have shown that glycolysis occurs at an enhanced rate at temperatures
TABLEI Distribution of key enzymes of carbohydrate and fat metabolism in the muscle of some fishes and shell fishes Fish Phosphorylase activity I (units g- ~) Lipase activity 2 (units g ~)

Oil sardine

( Sardinella longiceps )
Mackerel

68.0 63.0 75.2 60.05 26.4 13.95 6.97 4.1 5.2

5.68 4.8 3.0 1.5 4.1 0.8 0.81 0.65 0.60

( Rastrelliger kanagurtha )
Jew fish

( Jhonius dussumeri )
Threadfin bream

( Nemepterus japonicus )
Tilapia

( Tilapia mosambica)
Sea Naran

( Penaeus indicus )
Karikkadi

( Metapenaeus stylefera )
Poovalan

( Metapenaeus dobsoni)
Crab

( Scilla serrata )
1Mukundan and Antony, 1977 2M.K. Mukundan, unpublished data, 1984.

262 TABLE II The temperature and pH optima of the three major enzymes. Figures in parentheses indicate the range of temperature and pH over which the enzymes are active Enzyme Phosphorylase~ Lipase 2 Cathepsin 3 Optimum temperature ( C) 36 (up to 50) 37 (up to 45) 37 (up to 50) Optimum pH 6.1 (4-7) 8 (5-9.5) (3-4.4)

1Mukundan and Nair, 1977. 2Mukundan et al., 1985. 3P.D. Antony, unpublished data, 1982. TAt~bE III Pattern of formation of lactic acid, free fatty acid and free amino acid, and pH in the first 12 h of post-mortem mackerel Item Lactic acid (mg/100 g) Free fatty acid (rag/100 g) 1 Muscle pH Free amino acid (mg/100 g) 1Nair et al., 1976. 2Extrapolated value. b e t w e e n - 4 a n d - 1 C. As a r e s u l t of t h e glycogenolysis/glycolysis, a considerable a m o u n t o f lactic acid a c c u m u l a t e s in fish muscle, w h i c h is n e i t h e r neutralised n o r r e m o v e d f r o m t h e site of f o r m a t i o n a n d t h e r e f o r e reduces t h e p H of t h e tissue. Fresh fish 40.0 75.0 6.6 170.0 12 h post-mortem 210.0 85.02 5.9 330.0

Contribution of lipolysis to muscle pH

A n o t h e r set o f e n z y m e s t h a t are active d u r i n g struggling as well as postm o r t e m are lipases ( M u k u n d a n et al., 1985 ), including p h o s p h o l i p a s e s . T h e s e e n z y m e s are widely d i s t r i b u t e d in a l m o s t all fishes, especially t h e f a t t y fishes a n d t h e ones w i t h red m e a t . T h e lipase c o n t e n t of some i m p o r t a n t fishes are s h o w n in T a b l e I. M o s t lipases f r o m fish a n d shell fish p a n c r e a s a n d muscle s h o w e d lipolysis in t h e p H r a n g e 6 - 1 0 a n d at all t e m p e r a t u r e s f r o m - 2 0 to 4 0 C ( B r o c k e r h o f f a n d H o y l e , 1965; Leger, 1972; P a t t o n a n d Quinn, 1972).

263 Purified sardine lipase is shown to be active at pH values 5.5-10 (optimum pH 8) and temperatures between - 1 0 and 40C (Mukundan et al., 1982, 1985) (Table II). The properties of lipases, as well as their environment in fish free of inhibitQrs (Brockerhoff and Jensen, 1974a), are extremely favourable for lipid breakdown in fish muscle at ambient as well as low temperatures, as has been observed by several workers (Gopakumar, 1972; Nair et al., 1976; Mai and Kinsella, 1979; Shenouda, 1980). The primary products of lipolysis are free fatty acids and glycerol. Further degradation of fatty acids via fl-oxidation does not occur in sterile tissue due to the absence of respiratory oxygen. Hence in dead fish the products of lipolysis also accumulate at the site of formation. Of these products, free fatty acids, although weak, will make a significant contribution towards increasing the hydrogen ion concentration or lowering the pH of the tissue. The lower free fatty acids formed as a result of lipolysis will also impart offflavour to fish muscle and is known as hydroloytic rancidity (Deuel, 1951 ). The higher unsaturated fatty acids released will easily become vulnerable to oxidation by atmospheric oxygen, resulting in oxidative rancidity (Bailey, 1952 ). However, the water-soluble nature of lipase protein and the insolubility of the triglycerides, its substrate, considerably reduce the rate of lypolysis and resultant rancidity, as the enzyme can act only at the interphase of lipid-water-emulsion {Brockerhoff and Jensen, 1974b; Mukudan et al., 1985 ). The above conclusions are also evident from Table III, which shows the amount of lactic acid, free fatty acid and free amino acid, along with muscle pH, of ice-stored mackerel at 0 and 12 h post-mortem. It is seen that in the first 12 h the rate of formation of lactic acid is very fast. The rate of formation of free fatty acids is comparatively much lower, even though the initial content of lactic acid and free fatty acids are comparable. As most fatty acids are waterinsoluble and less ionic than lactic acid, their contribution to acidity will be much less. However, the combined effect of lactic acid and free fatty acids is to bring down the pH of post-mortem fish muscle to between 5 and 6 within a few hours after death (Gould, 1965; Mukundan and Nair, 1980; P.D. Antony, unpublished data, 1982). Acidic p H activates many autolytic enzymes The cell cytoplasm of fish muscle contains small circular organelles called Iysosomes. Lysosomes contain about 36 hydrolytic enzyme systems capable of degrading almost all of the tissue components such as carbohydrate, fat, protein, nucleic acids, etc. The normal function of lysosomal enzymes in live fish or any other living organism is to digest dead cells, as in pinocytosis/phagocytosis, for further processes of synthesis or oxidation for energy generation ( White et al., 1973 ). This function has led to lysosomes also being called "sui-

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cide bags" (Almas, 1981 ), as the breakage of lysosomal membrane will result in self-digestion. Fish-muscle lysosomes are rich in proteolytic enzymes, especially the cathepsins and proteases. The cathepsin content of some important fishes are listed in Table IV, along with those of certain mammals. The distribution of cathepsins was found not to be uniform in any given fish. Liver, kidney and spleen are rich in cathepsins, while skin, lung and heart-muscle tissues contain only very small amounts of these enzymes (Karmas, 1978). However, from Table IV it can be seen that fish-muscle content of cathepsins is at least 10 times higher than that of mammalian muscle. In live fish, appreciable amounts of these cathepsins are retained or reserved in an insoluble and inactive form by combining them with insoluble proteins, but they are easily broken down by acid hydrolysis at pH values between 5 and 6, liberating soluble and active cathepsins (Karmas, 1978). In live fish also, cathepsins are released only when they are required for degradation of tissue proteins for energy requirements at times such as spawning or in periods of starvation, and for removal of dead cells. However, in postmortem fish muscle the lowering of pH due to glycogenolysis/glycolysis and lipolysis will upset the regulation of proteolytic enzymes. The increased acidic environment of lysosomes causes rupture of the membrane, resulting in the release of cathepsins, proteases and a number of other hydrolases. Drastic changes in tissue temperature, such as in incorrect chilling and freezing, make the condition worse, as such treatments break open almost all the lysosomal packets releasing the entire store of cathepsins and other hydrolases into the tissue (Almas, 1981).
TABLE IV Catheptic activity in certain fish and mammalian muscle ~ Source Catheptic activity Tyrosine Units

Fish Carp ( Cyprinus carpio ) Mackerel ( Rastrelliger kanagurta ) 2 Cod ( Gadus morhua ) Herring ( Clupea harengus) Trout ( Salmo gairdneri) Mammals Pigs Cattle Rabbits tSiebert and Schmidt, 1964. ='P.D. Antony, unpublished data, 1982.

35 140 30 41 50 7 8 5

265 Most cathepsins and other proteolytic enzymes are highly active at acidic pH (Siebert and Schmidt, 1964; Hjelmeland and Raa, 1979; P.D. Antony, unpublished data, 1982; Samuel and Reynold, 1983). They are also active at a wide range of temperatures from - 1 0 to 60 C (Table II). Thus, of the lysosomal enzymes released into fish muscle, cathepsins have the most favourable conditions for vigorous activity in post-mortem fish muscle. Fish muscle is shown to contain at least four distinct groups of cathepsins, viz. cathepsins A, B, C and D (Siebert and Schmidt, 1964; Hjelmeland and Raa, 1982; Hjelmeland, 1983). All of them except cathepsin-c are endopeptidases, with activity over a variety of peptide bonds (Greenbaum, 1961; Alan and Krischke, 1981). Moreover, they are active at much lower temperatures than mammalian cathepsins. As a result, under optimal conditions of pH and temperature, the cathepsins and proteases present in fish muscle are observed to digest the entire fish protein in less than 1 day (Karmas, 1978). The very high proteolytic activity of fish muscle cathepsins can easily be explained from the observations of Siebert and Schmidt (1964). These authors studied the specificity of purified cod spleen cathepsin by permitting it to act on the fl chain of insulin, and were able to locate three major and five minor cleaving sites. Some of these sites are inaccessible to most of the proteases and the potent proteolytic enzyme pepsin. The same authors also observed 4-SH groups in their enzyme at the same time, but almost all sulphydril inhibitors had no effect on the enzyme activity. Further, the action of the enzyme was found to be independent of redox potential. P.D. Antony (unpublished data, 1982), working with purified mackerel cathepsins, also came to similar conclusions, suggesting possible complete digestion of fish-muscle proteins by cathepsins and proteases present in fish muscle.

Role of gut enzymes

Another group of enzymes activily engaged in autolysis of whole fish is the digestive enzymes of the gut. Fish gut contains a wide spectrum of enzymes capable of hydrolysing protein, fat and carbohydrate. Bramstedt and Auerbach (1961), Brockerhoff and Jensen (1974c) and Love (1970) have given brief descriptions about the variety and content of some of these enzymes. The role of gut enzymes in autolysis of capelin (Mallotus villosus) (Gildberg and Raa, 1978, 1979; Gildberg, 1978), cod (Gadus morhua) viscera (Raa and Gildberg, 1976), carp ( Cyprinus carpio) ( Onishi et al., 1976) and Antarctic krill ( Kubota and Sakai, 1978) were studied in detail. The major enzymes of fish gut are proteases, including pepsin, trypsin and chymotrypsin, followed by lipases and carbohydrases. A typical analysis of fresh oil-sardine guts showed the presence of 120 units of protease, 60 units of lipase and 12 units of carabohydrase g- 1min - 1 ( M.K. Mukundan, unpublished data, 1984).

266 Even though proteases are the major enzymes present in viscera, their optimum pH is in most cases around 3; a condition seldom found in fresh fish. However, the neutral pH of fresh fish very much favours carbohydrase and lipase activity (Mukundan et al., 1985), producing acidic pH favourable for protease activity. The enzymes hydrolyse and perforate the stomach and intestinal walls and leak out into the surrounding tissue, causing general hydrolysis (Gildberg, 1978), a mechanism evidently seen in capelin and oil-sardine. However, potent autolytic enzymes of the gut are effectively contained by the stomach and intestinal walls for about 4-7 days in fish stored at 0 C. Moreover, for processing or frozen/dried storage, gut enzymes can be effectively removed by evisceration. Consequently, gut enzymes are not given much attention except in cases such as silage and fermented fish products. The autolytic changes described above relate to the spoilage of fish muscle. There are also some autolytic changes which are considered beneficial for fish as a food material. An important group of enzymes responsible for such beneficial changes in post-mortem fish are the nucleodepolymerases. They are enzymes responsible for hydrolysing nucleic acids to mononucleotides. Two important enzymes of this category are acid ribonuclease and acid deoxyribonuclease (Karmas, 1978). As the name suggests, these enzymes are also active in acidic pH and are of lysosomal origin (White et al., 1973 ). The nucleotides produced as a result of the activity of these enzymes greatly enhance the flayour of fish and meat in general (Hall, 1978). Disodium salts of inosine 5'monophosphate and guanosine 5' -monophosphate are the most important flayour-generating nucleotides produced by these enzymes. Inosine monophosphate is also formed in small amounts by the degradation of ATP (Almas, 1981 ). Thus, as a result of lowering of pH due to glycogenolysis/glycolysis and lipolysis, cathepsins and other proteolytic enzymes are activated and released into the tissue. These enzymes start degrading the tissue proteins, at first tenderising the muscle (6-10 h after death). Up to this stage the fish will be in prime condition for cooking and consumption. As time passes, the autolytic process proceeds further and results in gradual loss of texture, taste and flavour due to the formation of polypeptides, peptides, free amino acids, free fatty acids, hypoxanthine, etc. Due to these changes, the fish skin will become easily permeable to bacteria, which also enter through the gills. The products of autolysis provide a nutrient-rich medium for bacteria to feed and proliferate. Once bacterial access to tissue is established, spoilage becomes rapid, resulting in total breakdown of fish tissue and producing a multitude of metabolites, including the foul-smelling amines, characteristic of spoiled fish. PREVENTION OF AUTOLYSISIS DIFFICULT Prevention of autolysis in fresh fish is very difficult. The reasons for this difficulty are many, but some important ones are listed below.

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(1) The enzymes responsible for autolysis are dispersed throughout the muscle, making their removal impossible. However, removal of viscera, including liver, spleen, kidney and red meat, which are rich sources of proteolytic enzymes, immediately after catch can considerably reduce autolysis of the musculature. (2) The use of chemical enzyme inhibitors to prevent autolysis is not possible, as such inhibitors may inhibit the enzyme systems of the consumer also. (3) If any suitable inhibitor which is harmless to the consumer were ever found, its quick distribution in intact fish muscle is almost impossible. However, such inhibitors would be very useful in reducing autolysis in fish mince, where the inhibitors can be effectively dispersed. Under these conditions, the only alternative available for control of autolysis is temperature manipulation. Blanching of fish in steam at 100 C or reducing the water activity of muscle by drying will denature and inactivate all enzymes, preventing autolysis; it will also partially or wholly coagulate all the fish proteins, depending upon the processing time. Salting, chilling, icing, freezing and irradiation will also reduce the activity of all enzymes, including cathepsins and other proteases, thereby considerably reducing autolysis. Storage of fresh fish immediately at sub-zero temperatures ( i.e. between - 1 and - 4 C, where enhanced glycolysis occurs) should be avoided. Another aspect to be considered in the possible control of autolysis is the pH of the fish muscle. If it is possible to keep the muscle pH around 7 with the help of suitable buffer systems, the activation of cathepsins and other proteases can be prevented, thus reducing autolysis. In this case also, uniform dispersion of the buffer in intact fish muscle would be a difficult problem except in fish minces. However, the possibility of dipping or spraying fish with suitable buffers followed by icing/chilling/freezing is bound to reduce autolysis and in turn bring in improvement in quality and an extension of shelf life.

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