Enzymic processing of marine raw materials

Process

Biochemistry

28

(1993)

I-15

Review Enzymic Processing of Marine Raw Materials Asbjmn Norwegian (Received

Gildberg Institute 27 February

of

Fisheries and Aquaculture, PO Box 677, N-9001 1992;

accepted

6 April

Tromss,

Norway

1992)

The literature on processing and modt$cation of marine raw materials by enzyme technology is reviewed. The intention has been to survey all processes developed, and particularly industrialised processes, where marine raw materials incIuding$sh, shell&h, or algae are treated with emymes. The utilisation oj’endogenous enzymes in autolytic processes is briefly discussed, but the major goal has been to survey methods where enzymes are added to the raw materials. At present, preparation of,fish protein hydrolysates is the most important commercial process, but enzyjmic methods for accelerated$sh ripening and selective tissue degradation are being recognised as interesting alternatives to conventional processing.

from heavily salted small fish or fish waste. and during a storage period of several months the digestive enzymes hydrolyse most of the protein yielding an aqueous solution rich in small peptides and free amino acids.3 Today, fish sauce is mainly used as a condiment on rice dishes, and the annual production in South-East Asia is about 250000 tons.’ The addition of enzymes in fish processing probably first took place in Vietnam where one of the traditional fish sauce products is made by mixing enzyme-rich pineapple juice with eviscerated fish and salt.5 Fish silage is a more recent invention which has many characteristics in common with fish sauce. Fish waste or by-catch fish is preserved with acid, and the digestive enzymes active at low pH quickly hydrolyse most of the protein yielding an aqueous solution that is suitable as a protein supply in feed products.6, ’ Fish silage was first made in Scandinavia during the second world war.8m10The silage method can be considered as an alternative to fish meal production where oil separation is

INTRODUCTION Since ancient times enzymes have been used in cheesemaking, brewing and leather tanning. In the treatment of marine raw materials like fish, shellfish and algae, exogenous enzymes have been used only to a minor extent. The effect of endogenous enzymes, however, is utilized in the traditional processes of fish fermentation and curing. In the curing process the proteolytic enzymes in the digestive tract are of vital importance. When uneviscerated or gibbed fish are stored in brine, digestive enzymes leak from the digestive tract into the surrounding tissue and cause partial hydrolysis and softening of the muscle tissue. Fermented fish products have long traditions in South-East Asia,’ and fish sauce, which is the major fermented fish product, was known in ancient Greece and Rome.2 Endogenous enzymes play an essential role in hsh sauce preparation. It is made Corresponding author : Dr 29 000: Fax: (083) 29 100.

A. Gildberg.

Telephone

: (083) 1

Proc~~.~

Biochemi.~try

0032.9592/92/$05.00

@

1992

Elscvicr

Science

Publishers

Ltd, England

2

Asbjern

achieved by enzymatic hydrolysis instead of by heating and pressing. Commercial silage production has been very limited but renewed interest in this technology has arisen due to problems with waste handling in the aquaculture industry. Methods for fish protein hydrolysate production by enzyme addition have been developed in America, Asia and Europe.‘1m’4 Commercial production of fish protein hydrolysates is still limited on a world basis, but it has reached a significant level in a few countries. In France, which is probably the biggest producer, fish protein hydrolysates are used as milk replacers and flavour compounds.‘“,“j Enzymatic treatment of stickwater in the fish meal industry is a related process. By digestion of the stickwater proteins, the evaporation process is enhanced and energy is saved.‘7.1” Some Scandinavian fish meal factories apply this method in commercial production. In Norway and Iceland much work has been done to develop methods for selective tissue degradation as an alternative to mechanical methods in the fish processing industry. Industrial-scale equipment for enzymatic rinsing of cod swimming bladder, roe rinsing and deskinning of squid has been constructed.4.L9.20 Active application of enzymes in the treatment or processing of marine raw materials is still not very widespread, but recent literature reveals an increasing interest in the development of gentle enzymatic methods as alternatives to mechanical or chemical treatments which often may damage the product and reduce product recovery.4*1g*“1 This paper reviews the literature on modification and processing of marine raw materials by the use of enzymes. The whole application range from basic research to commercial production is covered. It should be emphasised, however, that the flow of information about commercial processes is very restricted. For this reason some applications may be missing in this survey.

FISH SAUCE

AND FISH

SILAGE

During the preparation of fish sauce or fish silage a protein hydrolysate is formed by the action of endogenous enzymes. In fish silage the enzymes also promote oil separation and the silage process may to some extent be considered as an alternative to fish meal production. The production of fish sauce is very simple. Small fish, like sardines, or minced fish material is mixed

Gildberg

with 2@40 % sea salt and stored at ambient tropical temperatures for 612 months. The amber protein hydrolysate liquid is tapped, filtered and bottled. Usually about half of the amino nitrogen is recovered in the sauce fraction. The remaining, partly digested fraction is usually extracted several times with brine to obtain lower grade fish sauce fractions until most of the proteinous material is recovered. Normally, high-quality fish sauce contains about 10 % peptides and amino acids and about 25 % salt, whereas low-grade leaching extracts may contain less than 3 % proteinous material.’ The nutritive value of low-grade fish sauce is low, but since fish sauce is principally used as a condiment, it may still be sold. Protein hydrolysis is caused mainly by trypsinlike enzymes.3 Although the bacterial count of fish sauce is very low, halophilic bacteria may play an important role in flavour development together with digestive peptidases from the fish.22,Z3 The high salt concentration in fish sauce partly inhibits the activity of trypsin-like enzymes and the rate of autolysis is 10w.~.24Autolysis is also inhibited by impurities in the sahz5 Due to the slow rate of autolysis, storage periods of more than 6 months are necessary before the sauce can be harvested. From an economic point of view it is desirable to reduce the production time. This can be achieved by several methods. If the storage temperature is raised to about 45 “C for a couple of weeks in the initial storage phase, the total production time may be reduced from 1 year to 2 months.2”.26 A similar reduction can be achieved if the initial pH is raised to 11 by addition of sodium hydroxide at a moderate salt concentration.24 Under such conditions the trypsin-type enzymes will be very active, whereas endogenous trypsin inhibitors will be denatured.3s2* Due to limited storage stability at these conditions, the material should be neutralised by hydrochloric acid and salt added to a final concentration of 25 % within 1 day. Initial acidification to promote rapid autolysis has also been attempted,27*28 but this is not convenient because some endogenous enzymes which are important for flavour development during storage at neutral conditions are denatured at low pH. As mentioned previously, the addition of enzymerich fruit components has been used to accelerate the protein hydrolysis in traditional fish fermentation. In Vietnam a special fish sauce product is made by the addition of pineapple juice to fish meat,5 whereas chopped pineapple or papaya is used in some fermented fish products in Thailand.2g

Enzymic processing

The unripe papaya is rich in papain, whereas pineapple, particularly the stem, is rich in bromelain. Both these enzymes are cysteine proteases most active under weak acid conditions. During the last few decades several experiments with enzyme addition to accelerate the fermentation process have been performed. Enzymes from mainly plant sources including papain, bromelain and ficin have been tested30e3” and high sauce recovery was obtained after only 2-3 weeks of storage. Although the flavour development was not satisfactory with either of the enzymes, the best results were obtained by application of bromelain preparations. There are reasons to believe that enzyme addition is used today in commercial fish sauce production in Thailand (Thongthai, C., 1991, pers. comm.). Fish silage has many similarities with fish sauce. During autolysis an aqueous phase rich in small peptides and free amino acids is formed. The silage is normally acidified to pH 3-4, which is optimal for protein digestion by the fish pepsins. Since no salt is added in silage, the autolysis is much faster than in fish sauce, and a high recovery of aqueous phase and an oil-rich fraction is normally obtained after a few days of storage. The aqueous phase has a bitter taste and is not suitable for human consumption. Hence, the silage method may be considered as an alternative to fish meal production where the separation of oil- and protein-rich fractions is promoted by the action of endogenous enzymes.7a lo The fish silage protein, however, is much more degraded than the fish meal protein, and it is most suitable as a feed to immature animals with a poorly developed digestive system, or in limited amounts to mature animals.“” 36 Various acids or acid mixtures have been used as preservatives in fish silage, but today most of the commercially produced silage is preserved by formic acid. If 2.5% formic acid is used, the silage will be preserved at about pH 4. If the pepsin rich stomach is included, a rapid autolysis is usually achieved when the silage is stored at 20-40 oC.6.31.37,38 To obtain rapid hydrolysis in fish silage with low protease activity it may be necessary to add enzymes or enzyme-rich raw materiallo In contrast, if silage is made from fish viscera, which are very rich in proteolytic enzymes, the autolysis will not be accelerated if more enzymes are added.12 After 3-5 days of storage at 25-30 “C the protein hydrolysate yields about 60 % of the total silage vo1ume.3y The amber hydrolysate liquid contains about 12% peptides and amino acids and little lipid. It can be

of marine raw materials

3

concentrated to about 50% dry matter by evaporation. Such a concentrate has a good storage stability and is suitable as a protein source in feed for domestic animals like calves, piglets and poultry.104” The upper phase of the silage is a lipidrich emulsion, and the oil can be recovered from this phase by separation after heating to 90 0C.34.43 Fish viscera are very rich in pepsins, which are stable at low pH and can be recovered from the aqueous phase of viscera silage by ultrafiltration.40s44 If mineral acids are used to preserve the silage, the permeate may be neutralised and applied as a nitrogen source in microbial growth media.44m47 Industrial production of pepsins and peptones from cod viscera has been established in Norway.”

PROTEIN

HYDROLYSATES

This section deals with the use of added enzymes to prepare protein hydrolysates from fish and shellfish. Shortly after the Second World War Canadian researchers developed methods for the enzymatic preparation of protein hydrolysates from fish meat.ll White fish viscera were also used as a raw material and pepsin, papain and other commercial protease preparations were added.‘* Enzyme addition did not improve the hydrolysate yield because the concentration of endogenous enzymes was high. After 24 days at 25-50 “C more than 80 % of the viscera tissue was solubilised in all the samples, including the control where no enzyme was added. Hydrolysates were dried by spray-, vacuum- or drum-drier. The spray-drier gave the finest product. In India, research on fish protein hydrolysates started early.13 Equal amounts of minced fish meat and water were mixed and papain (O-1 % of the fish weight) added. The hydrolysis was performed at pH 5 and 55 “C, and stopped by boiling after 2 and 17 h, respectively. After filtration, the two hydrolysates contained equal amounts of total nitrogen but the amount of amino nitrogen was much higher in the 17 h hydrolysate. Feeding trials with rats showed that the net protein utilisation was best with this hydrolysate and the reason was supposed to be higher content of tryptophan in this preparation. Aromatic amino acids and cysteine are slowly released during hydrolysis38,48 and tryptophan may be the growth-limiting amino acid in hydrolysates made by short-time incubation. In Japan, research on enzymatic fish protein hydrolysis probably started around 1960. Hydro-

4

Asbjwtz

lysates made from mackerel waste contained an unknown growth factor which gave significant growth improvements with rats if 10 % of a caseinbased protein diet was substituted by protein from fish hydrolysate.48 Other experiments with mackerel showed that the nutritive value was dependent on the kind of protease used. Whereas hydrolysates made by applying proteases from a Streptomyces species had a higher nutritive value for rats than casein, hydrolysates made by proteases from Bacillus subtilis were inferior to casein4’ This was probably due to an imbalance of the essential amino acid composition in the latter preparation. Proteinases from plants or microorganisms have usually been used to prepare fish protein hydrolysates. ‘as” Hale showed that the hydrolysate yield varied little when various proteinase preparations were used to hydrolyse heat-denatured protein from haddock fillet.“’ About 60 % solubles were obtained after 1 day of hydrolysis with the plant enzymes papain, bromelain and ficin, with two microbial proteinase preparations or with mammalian pepsin or trypsin. A similar yield was obtained after 4 h hydrolysis of fresh cod muscle by ficin.5”.53 With the use of high enzyme addition (1 % of the fish dry weight) the yield was 70%, but this was achieved in a rather dilute suspension. If only one part of water was added to the fish mince, the yield of solubles was reduced to 60% at otherwise identical conditions. This was probably due to product inhibition. In industrial production a compromise must be made between the hydrolysate yield and the amount of water that must be removed to obtain a dry product. A suspension of equal amounts of fish and water appears to be convenient from an economic point of view. Since fish tissue is a very complex substrate and large also contains amounts of proteinase inhibitors,j4 it is impossible to explain the hydrolysis in detail. Langmyhr studied the hydrolysis of cod muscle protein by trypsin and found that the best yield was obtained if the enzyme was added at low temperature and the temperature was increased slowly to the optimum for the enzyme.“” By this procedure the fish protein was partly digested before it started to aggregate, and the aggregates formed during heating were relatively small allowing good access to the hydrolysing enzyme. In industrialscale production, this advantage is achieved automatically if the enzyme is added before the material is heated, since it will always take some time to heat large volumes. The functional properties of the hydrolysates are

Gil&erg

important, particularly if they are used as ingredients in food products. The solubility of fish protein increases with the degree of hydrolysis, and the degree of hydrolysis increases with the incubation time and amount of hydrolytic enzyme added.50,56 Experiments where sardines were hydrolysed by the bacterial proteinase Alcalase showed that emulsifying properties and surface hydrophobicity were reduced when the degree of hydrolysis increased.57 Fish protein derivatives with good emulsifying properties can be prepared by acetylation. 58 The best results were obtained when the myofibrillar protein fraction was acetylated and partly hydrolysed by bromelain. This preparation was tested as an additive in various foods. Tilapia muscle hydrolysed by Alcalase has been tested as an ingredient in biscuits to improve the nutritive value.54 Acceptable products were obtained by a 10% addition of dried hydrolysate. Mackie found that bromelain hydrolysed both fish muscle and fish waste more efficiently than trypsin.” Many results indicate that bromelain gives hydrolysates with better organoleptic quality than other proteinases.30~31~e0 Although fish protein hydrolysates are not used for human consumption in the USA,” there are several US patents concerning such use. Two of these describe methods for the preparation of fish protein hydrolysates with a neutral non-bitter flavour.““.62 In one of the methods lean fish is gutted, deskinned and run through a bone separator.” The meat mince is washed and centrifuged before stabilised fat is added during homogenising to yield an emulsion. Bromelain is then added, and the temperature raised to 55 “C for 15 min before the preparation is heated to 80 “C to pasteurise the product and inactivate the enzyme. After a second homogenising, the product is spray-dried. The final product contains about 70 % protein and 25 % fat, is easy to suspend in water and forms a stable emulsion which does not separate during storage for several days. The second patent describes a two-step hydrolysis in which various raw materials including sardine, mackerel, pollack and krill can be used.62 To whole fish is added an equal amount of water, and the temperature is raised to above 60 “C to inactivate endogenous enzymes. After 15 min the temperature is reduced to 60 “C, pH adjusted to 9 and a thermostable proteinase active at high pH added. After 1 h incubation the pH is adjusted to 5.5, and a proteinase active at low pH is added. After another 1 h digestion, the hydrolysate is separated

Enzymic processing FISH

I

HYDROLYSIS HEATING AND

(MODERATE

ENZYME (HEATING

STIRRING)

INACTIVATION TO ABOVE So'=C)

SOLIDS

V

SOLUBLE FISH PROTEIN HYDROLYSATE Fig.

1. Outline

of

a

common

hydrolysateproduction.

procedure for fish protein

on a three-layer separator centrifuge where oil and sludge are removed. The hydrolysate is then filtered and vacuum evaporated to 60-70 % dry matter, and it is claimed to have no bitter taste or fish smell. A short-time hydrolysis to avoid the formation of bitter peptides is a characteristic of both methods. For the same reason initial inactivation of the endogenous enzymes is applied in one of the methods. If bitter peptides are already present debittering may be achieved by application of In addition to bitter specific peptidases.lG.“” peptides, rancid fat is the major flavour problem in fish protein hydrolysates. If the product contains more than 1% fish fat, the fat must either be removed by extraction or stabilised by antioxidants.“’ Although various methods have been developed for fish protein hydrolysate production, the industrialised processes seem to be rather uniform. A common procedure for fish protein hydrolysate production is illustrated in Fig. 1.

of marine raw materials

5

It is difficult to survey the extent of industrial fish protein hydrolysate production although it is well known that much of the fish flavour, fish soup and fish paste products available on the market are prepared by enzymic hydrolysis.‘6*64 In commercial feed production the major applications of fish protein hydrolysates are as protein and flavour supply in fish feeds5*@ and pet fooda and as a milk replacer to calves and piglets.15,65,86 In the US the main application of fish protein hydrolysate is as weaning feed for piglets, and the market price of the spray-dried product is about US $2/kg.66 The pet food market is rapidly growing in Europe and the US. In the US the annual turnover amounts to about US $8 M.’ Fish protein hydrolysates are mainly used in cat feed as a pellet coating to improve the palatability. About 3 % hydrolysate is used, and this would correspond to almost 20000 tons dry hydrolysate per year if all dry cat feed were to be coated. One company in Japan makes a product called ‘Bio-fish flour’.65 This is made by enzymic digestion of sardines and used in fish feed and as a milk replacer for calves and piglets. The production capacity is 150 tons/day. At present, Japanese and French companies seem to be in the forefront in the field of fish protein hydrolysate production. In France process development has been in progress since 1963,68 and some basic research has also been done regarding special qualities of fish protein hydrolysates. Vinot et al. studied the effect of commercial fish protein hydrolysate on mouse lymphocytes and concluded that certain compounds hydrolysate can promote immunoin the stimulation.69 This may explain observations made in Norway where the disease frequency of domestic animals was reduced when a fish silage autolysate was a part of the feed (Hoydal, S., 1992, pers. comm.). Fish protein hydrolysates have proved to be excellent as nitrogen sources in microbial growth media *‘* 7om72 and today fish peptones are produced commercially in Norway and Japan.44v65 Antarctic krill is an enormous protein resource which is poorly utilised. The krill is very rich in proteases and autolyses very quickly. Ellingsen and Mohr showed that most of the krill protein was hydrolysed to free amino acids after 2 days of storage at 20 0C.73 Based on these results they described an industrial process for production of free amino acids. Feeding trials with rats have shown that fish protein hydrolysate made from fresh raw material

6

Asbjmn

has a nutritive value similar to casein.4a*74 However, a high degree of hydrolysis may reduce the protein utilisation, particularly if used in large amounts for mature animals.7”x ” This is probably due to increased deamination of some amino acids during digestion. In premature animals like fish larvae, calves and piglets, the digestive system is not fully developed and feeding heavy digested protein may be advantageous. In feed for mature animals a small addition of fish protein hydrolysate may be more valuable as a flavourant and appetiser, and possibly also as an immunostimulant.67*6g Enzymic treatment of stickwater is a process developed to reduce the cost of water removal in the fish meal industry.17*1s*77 Since removal of water by evaporator is cheaper than by dryer, it is convenient to evaporate the stickwater as much as possible before drying. By applying Alcalase digestion in the second evaporation step at a temperature of 5&55 “C, the final dry matter concentration can be increased from about 60 % to about 70 %. Also cheap plant enzyme preparations can be used for this purpose.” There are indications, however, that enzyme digestion reduces the nutritive value of the stickwater protein,” and this is in accordance with the results discussed in the previous paragraph. In coastal regions with agriculture, some fish waste has always been used as fertiliser. It may be looked upon as a waste of valuable resources to use fish protein hydrolysate as a fertiliser, but some interesting results obtained in cultivation of vegetables should be mentioned. General growth stimulation has been observed by supplying fish protein hydrolysate, and it has been shown that it retards the ageing in lettuce and peas and delays flowering and fruiting in tomatos.80 The latter may be an advantage in areas where frost in early spring is frequent. Spraying hydrolysate on young tomato plants also reduces stress and improves survival during transplanting. Thus, fish protein hydrolysates have a wide spectrum of applications ranging from high value peptones and food ingredients with special functional properties to feed and fertiliser. The choice of raw material, hydrolytic enzyme and processing parameters must be decided from the type of application involved. Extensive hydrolysis gives a low-molecularweight product rich in essential amino acids, but with little functional properties. Such hydrolysates are suitable as peptones in microbial growth media and as milk replacers. but inferior as food ingredients. Hydrolysates for food applications must

Gildherg

be prepared by controlled hydrolysis to secure good flavour and functional properties. The choice of enzyme is also very important. It seems to be generally accepted that the plant enzyme bromelain produces hydrolysates with a pleasant flavour and therefore is very useful in the preparation of hydrolysates for food application. If efficient hydrolysis and high recovery are the most important factors, thermostable microbial enzymes such as Alcalase will be appropriate. Plastein is a protein substance which may be prepared by reversing hydrolysis in a concentrated protein hydrolysate. El Plastein has other functional properties and often a higher content of essential amino acids than the protein of the raw material.” The method can be described briefly as follows. An enzymatic protein hydrolysate is concentrated to about 40 % dry matter before the plastein synthesis is initiated by addition of a suitable proteinase. The hydrolysate is usually prepared by pepsin digestion at pH 2, whereas the plastein reaction runs optimally at pH 4-5 with either pepsin, papain or certain microbial proteinases as catalysts. In research laboratories plastein has been made from various marine raw materials like fish waste,“-*” sardines’“*” and marine algae.” The interest in plastein is mainly due to special functional properties, low lipid content and favourable amino acid composition. However, the preparation process is quite expensive and it is unknown whether commercial production of plastein from marine raw materials exists.

RIPENING

BY ENZYME

ADDITION

The annual world production of cured fish products amounts to several hundred thousand tons. Most of these are herring products and have been produced for more than 1000 years in the North Sea countries.4.“’ Traditionally, whole herring, with or without head, are mixed with salt and stored cool in barrels for a few months before they are washed, filleted and packed. During storage, enzymes from the digestive tract leak out and cause partial digestion of the muscle proteins. The fish meat becomes smooth and pliable and attains a pleasant rich flavour. The high salt concentration (12-15 %) and low temperature (S-IO “C) slow down digestion and the maturation process usually takes from 2 to 7 months.as~ag Experiments with the addition of enzymes have

Enzymic

processing

of marine

been in progress since the early 1970~.~~*~“~~~ Trypsin-type enzymes from the pyloric caeca are the main contributors in the ripening process. The concentrations of these enzymes vary with the season. Regarding North Sea herring, which is the major raw material, it is only fish caught in the period from May to August that contains sufficient enzyme to promote satisfactory ripening.RRs”’ If enzymes are added, good products can be obtained also with herring caught in early spring and autumn. Ritskes showed that good quality matjes herring could be made from herring caught in early April if pancreatic enzymes from pig or cattle were added.s1 One gram of commercial enzyme preparations was added per kilogram of herring. If the enzyme preparations contained high lipase activity, however, the product attained an inferior flavour. Ruiter showed that spent herring caught in October was well maturated if minced pyloric caeca from herring caught in mid-summer was added with the salt.87 The traditional maturation procedure is time consuming and demands large storage capacity. When whole fish is salted the filleting waste becomes too salty to be used as animal feed. By filleting before salting, two advantages are obtained: the storage capacity can be reduced and the filleting waste can be used as animal feed. However, enzymes must be added to obtain maturation. Several methods involving enzyme addition have been patented. A British patent describes the preparation of matjes from herring where head and viscera are removed.” Five per cent salt and 2-5 % sugar are added together with an enzyme preparation mainly consisting of trypsin and chymotrypsin. Satisfactory ripening was obtained after 3 weeks of storage at 5 “C while the ordinary maturation time for such products is 2-3 months. It was emphasised that an unsatisfactory flavour was developed if pepsin or significant amount of lipase were present in the enzyme preparation. Opshaug has patented a maturation procedure in which herring fillets are ripened in a brine containing digestive enzymes from herring.“’ One part herring fillets is immersed in 1.3 parts brine containing 12 % salt, 6 % sugar, a spice mixture and 5 % minced herring pyloric caeca. After only 5 days at 3 “C the fillets attain the smooth consistency and flavour which characterise traditionally matured products. The final salt concentration is only 6%, and extensive washing before consumption is not necessary. The amount of pyloric caeca used in this method corresponds to the natural content in

raw materials

7

summer-caught herring. A Canadian patent describes a simular procedure where fish enzymes are added to obtain herring ripening.“O A German company (Kordts KG, Heide, Wennemannswisch, manufactures Germany) enzymes and microbial preparations for use in accelerated ripening of herring fillets and it is claimed that good quality matjes herring can be produced within 1 week of storage by the application of these products. In Norway, research on accelerated ripening of acidified herring fillets by enzyme addition has been in progress since about 1980.g2 An acceptable flavour has been obtained but the stability of the final product was poor due to residual enzyme activities. The main advantage with the traditional longtime storage method is that the enzyme activities gradually decline during the storage giving a quite stable final product with very little residual enzyme activity. To obtain stable products with rapid methods, it is necessary to reduce the residual activities as much as possible. Simpson and Haard used pure trypsins from cattle or cod in ripening experiments with herring and squid.s3 They obtained ripening reactions by addition of 20 mg trypsin to 1 kg raw material. At 10 “C the cod enzyme was more efficient than the cattle enzyme, but the activity of cod trypsin declined more rapidly than the activity of cattle trypsin. Both observations accorded with the lowtemperature characteristics of cod trypsins and show that the fish enzymes probably are more suitable tools in accelerated ripening than mammalian enzymes. 94 Although the new rapid methods imply obvious advantages like a reduced storage capacity requirement and improved utilisation of the raw material, the commercial application of such methods still appears to be limited. The main reason for this is probably the problems with stabilizing the final product. In Norway the annual production of herring fillets matured by enzyme addition is about 700 tons.” The extent of such production in other countries is unknown.

SELECTIVE

TISSUE

DEGRADATION

Hydrolytic enzymes digest certain tissue structures leaving others intact. Some tissue structures which are normally resistant to certain enzymes, may become susceptible to digestion when chemical

Asbjarn Gildberg

Fig. 2. Processing

lint for enzymatic

deskinning

of squid

(developed

by Biotec-Mackzymal

in Norway

and produced

by Carnitech

in Denmark).

conditions such as PI-I, temperature or the salt concentration are changed. Due to these factors enzymes can be used as specific tools in processing where the aim is selective removal or modification of certain tissue structures. The oldest example of such enzyme application on marine raw materials is probably in the tanning of fish skin. In this process pancreatic enzymes are used to digest elastin and keratin structures leaving the major skin protein, collagen, unchanged. This is the same method as used in tanning of mammalian skin. Today, tanning of fish skin has little commercial significance, but the interest is increasing, and small-scale industrial production exists in Korea, Alaska and Nova Scotia.86 Since the 1970s research and development on fish processing by biotechnological methods have been in Norway and Icein progress, particularly land.4. lg. 97-y9It has been recognised that mechanical processes have obvious limitations regarding selectivity and gentle treatment of the raw materials. In many situations mechanical processing implies low yield and quality reduction. In some cases it may even be impossible to solve a problem by purely technical means. This is the situation with the production of salted cod swim bladder in Iceland. The swim bladder is enclosed in a thin black membrane, but the market demands an almost white product. It is impossible to remove the membrane efficiently by mechanical means but the problem has been solved by subjecting the raw material to enzyme hydrolysis for 20 min. The annual production is about 40 tons, and the major market is Italy.4 An enzymic method for the

removal of the connective tissue membrane enveloping cod liver has also been developed in Iceland, but this method has not yet been commercialised.4 A method for the enzymic deskinning of herring has been developed in Norway.” The whole herring is treated in 5 % acetic acid at 10 “C, to denature the skin collagen, before it is transferred to the enzyme bath containing fish pepsin and 0.5 % acetic acid. After 1-2 h at 20 “C the skin is partly solubilised and can be washed off. Due to differences in skin structure and thickness on different parts of the body, it is difficult to achieve uniform deskinning, and this is probably the main reason why this method has not been commercialised. A method for the deskinning of squid has been more successful. The squid skin consists of two layers. The conventional mechanical deskinning method removes only the outer skin leaving the rubbery inner membrane intact. The enzymic deskinning process involves soaking the squid tubes in a solution containing a plant protease which selectively attacks the rubbery membrane without degrading the muscle tissue.‘O.gs After washing and blanching the squid tubes are vacuum packed and frozen. A complete production line for enzymic deskinning of squid has been developed (Fig. 2). Deskinned skate wings is a commercial product in Iceland. The conventional deskinning method leaves residual skin fragments which must be removed manually. An enzymatic method has been developed to reduce the need for manual treatment.4,gx The skin collagen is denatured by a rapid heat treatment before the skate wings are soaked in

Enzymic

processing

Fig. 3. Process equipment for enzymatic rinsing of roe in caviar production (developed by Biotec-Mackzymal and produced by Trio Industries in Norway).

an enzyme solution for a few hours at 10 “C. The enzyme preparation used contains both proteinases and carbohydrases. The enzymic method is not yet commercialised. A simular method for the deskinning of tuna has been described in a US patent.‘“” A method for the enzymatic rinsing of shellfish is also given. Experiments with enzymatic rinsing of shellfish in Norway and Iceland have revealed that it is difficult to achieve release and separation of the muscle tissue without undesirable muscle softening.* Japanese researchers have shown that the very resistant byssus threads of mussels can be degraded by a bacterial serine proteinase.‘O’ The practical significance of this discovery is unknown. One problem in conventional caviar production is that it is difficult to release the roe particles from the connective tissue of the roe sac without destroying a large amount of the roe. Sometimes the yield of intact roe may be as low as 50 %,g7 A US patent describes a method where proteinases can be used to release salmon roe from the connective tissue.“’ It is claimed that various proteinases can be used at acid, neutral and alkaline conditions.

of marine

raw materials

9

Research iq. Norway has shown that enzymic roe rinsing is a very delicate process demanding strict control with all the condition parameters to avoid damage of the roe cell wall. Several enzymes have been tried, but satisfactory results have only been obtained by using a special enzyme preparation of fish origing7 After the enzyme treatment the roe may be separated from the connective tissue by sedimentation in a floatation tank. By accurate adjustment of salt concentration and floatation conditions it is also possible to remove damaged roe together with the connective tissue. The caviar yield by this procedure is about 90 %97*99 and the process has been developed to full industrial scale. Figure 3 shows a picture of the process equipment. Industrial production by this enzymic method has been initiated in Canada, the US, Australia and several Nordic countries. So far mainly salmon and trout caviar have been produced, and the annual production is about 50 tons. Newly spawned eggs from species like walleye and catfish have an external adhesive layer of mucoprotein which causes clumping and reduced survival during hatching.l”-lo5 A gentle proteinase treatment may remove the mucoprotein layer without damaging the chorion. Experiments with walleye eggs have shown that a treatment with trypsin-type enzymes (O-5 mg/ml) immediately after fertilisation improved both the hatching yield and survival of larvae.‘““~iU4 The latter is probably due to reduced mould growth on eggs where the mucoproteins have been removed. Alcalase has been used successfully to reduce adhesiveness of fertilised catfish eggs.‘“” Enzymic removal of the chorion from fish eggs is used in research experiments to facilitate observation of embryogenesis and microinjection of naked genes. A large number of enzymes were tested on eggs from various species. Goldfish eggs were efficiently dechorionated by trypsin treatment (2.5 mg/ml) and showed excellent viability.‘O” So far selective tissue degradation by enzyme treatment has gained little commercial importance in the processing of marine raw materials. To the author’s knowledge only three of the methods mentioned in this review have been industrialised. These are enzymatic rinsing of cod swim bladder,” enzymic deskinning of squidZOand enzymic rinsing of roe in caviar production.lg Alteration from mechanical to biotechnological processes is a challenging task that demands a competence which is scarce in the conventional processing industry. However, this situation is gradually changing and

10

Asbjtwn Gildberg

the use of enzymes as gentle knives will certainly be an interesting alternative in the near future.

MISCELLANEOUS

METHODS

This section deals with various enzymatic methods which fall outside or on the border line of the application categories already discussed. Shellfish are rich in the polysaccharide chitin, and it is estimated that the annual world production of shellfish waste contains about 120000 tons of chitin.lo7 Chitin and its derivatives have numerous applications, but enzymes have scarcely been used in the recovery and processing of chitin. In the US a method for enzymatic hydrolysis of chitin to its monomer N-acetylglucosamine has been developed.lo8 Ilo Protein and minerals are removed from the shellfish waste by chemical extraction to obtain a fairly poor chitin fraction. A small part of the chitin is used as a growth substrate for a bacterium (Serratia marcescens) with a high chitinase production. The chitinase is secreted and the culture filtrate is used as an enzyme preparation for hydrolysis of the bulk of the chitin. If chitin from shrimp waste is used, the yield during hydrolysis for 24 h at 30 “C was 80%.“” The product, IVacetylglucosamine, was used as a growth substrate for yeast to produce single-cell protein for animal feed. More than 40 different strains of yeast were tested and Pichia kudriavzevii was most suitable because it could grow well on a simple medium at low pH (45) and high temperature (37 “C) reducing the risk of competition by contaminating microorganisms. Yeast is suitable as an ingredient in fish feed both because it is a good vitamin and protein source, and because the yeast cells are rich in proteoglucans which stimulate the non-specific immune defence system.“’ A commercial evaluation of this process, however, concluded that it was not feasible in the US.11o In China research and development on enzymatic chitin digestion is in progress, but little detailed information is available.l12 Chitosan is prepared by chemical deacetylation of chitin. Chitosan has a wide spectrum of applications ranging from medical and biotechnological use to clarification of beer and waste-water treatment.lo7 One future perspective is to use this polymer to make biodegradable packaging material.“’ During chemical deacetylation, the 160 kDa chitin molecule is degraded to units smaller than 50 kDa. For certain applications this may be

inconvenient. By enzymic deacetylation it is possible to obtain full length chitosan molecules.‘13 Some fungus species are rich in chitin deacetylase.r14 Astaxanthin is the carotenoid giving the attractive pinkish-red colour to salmon meat. This is also the major carotenoid in shrimp and lobster. It can be efficiently extracted from the waste fraction of shrimp and lobster if trypsin is added to the extraction buffer.l15,r16 In the extraction of shrimp waste at low temperature, cod trypsin gave a higher recovery of both astaxanthin and protein than bovine trypsin. 11’ Experiments with crawfish waste have shown that autolysis at acid conditions improves the astaxanthin recovery by subsequent extraction.‘l’ In France there is a market for nucleic acids from cod and herring milt. DNA preparations are usually obtained by a chemical extraction procedure including alkali and heat treatment to remove the protein. During this process the DNA is partly degraded. For certain applications, highly polymeric DNA is needed. For this purpose a more gentle process was developed where the protein fraction is removed by papain digestion (Sandsdalen, E., 1991, pers. comm.). In Japan, enzymic hydrolysis is used to make a food product from skipjack milt.ll’ Several commercial enzyme preparations were tried, but the best result was obtained when the milt was digested by a crude enzyme extracted from skipjack liver. The application of polyunsaturated fatty acids to prevent cardiovascular diseases is well established. Marine oils have a high content of polyunsaturated fatty acids, and there is a large commercial interest in preparing oils where such acids are enriched to very high concentrations. This can be carried out by various methods and also by the use of enzymes. Haraldsson surveyed the application of lipases to modify marine oils for this purpose.‘2o The main principle utilised is that some of the most valuable polyunsaturated fatty acids are poor lipase substrates. This is most pronounced with docosahexaenoic acid (DHA). Hoshino et al. showed that the total amount of DHA and eicosapentaenoic acid (EPA) could be doubled in the triglyceride fraction of cod liver oil if the oils were treated by lipases from Cundidue cylindracea or Aspergillus niger.“l Hills et al. obtained a five times enrichment of DHA in the free fatty acid fraction after selective esterification of unsaturated fatty acids in cod liver oil by a lipase from Mucor miehei.‘“’ The same lipase has been used in the enrichment of DHA in sandeel oi1.‘23

Enzymic

processing

Free fatty acids or fatty acid methylesters were used as substrates. In both cases DHA was enriched in the unmodified fraction. The Mucor miehei lipase has also been used to enrich EPA and DHA in cod liver oil by interesterification reactions.lz4 Even if EPA and DHA are normally poor lipase substrates, it has been shown that triglycerides from sardine oil with a high content of these acids were efficiently hydrolysed by a lipase from Chromobacterium

viscosum.‘25

Some raw materials have an exceptionally high content of polyunsaturated fatty acids. About 10 % of the dry matter in cod roe are phospholipids, and about one-half of these are polyunsaturated fatty acids. Hydrolysis of the phospholipid fraction from cod roe by a pancreatic lipase gave 24% EPA and 40% DHA in the free fatty acid fraction.‘26 In laboratory experiments the recovery was 60%, corresponding to a yield of 6 g polyunsaturated fatty acids per kilogram cod roe. The commercial production of marine oils enriched with EPA and DHA is considerable. The extent of enzyme application in such production is uncertain. Although such products are popular at present, the health implications of using enriched oils instead of native oils are being disputed. Marine algae are important raw materials for the production of polysaccharides utilised in food, fish feed and cosmetics as well as in biotechnological separation methods and cell cultivation.127 Enzymes are probably not used in commercial production of marine polysaccharides. For research purposes protoplasts are produced from algal cell by enzyme treatment. Depolymerisation enzymes are used together with pectinase, sulphatase and macerase to remove the cell wa11.12R~‘2” The gene for an elastase-like enzyme from abalone has been cloned, and utilisation of this enzyme in the processing of marine algae has been indicated. 13”

CONCLUSlON The present use of enzymes in processing marine raw materials is limited. The utility of endogenous enzymes in autolytic processes, however, is considerable. Herring maturation and fish sauce production are the most important processes where the effects of endogenous enzymes are utilised. Processing by enzyme addition has gained most importance in the production of fish protein hydrolysates. Such products are generally made 3

of marine raw malrrials

II

from any cheap fish raw material and applied either for food or feed purposes. Hydrolysates used for consumption must have good functional and organoleptic properties. This is normally obtained by a short hydrolysis at gentle conditions, and the choice of enzyme is very important. The pineapple enzyme bromelain is often preferred because it gives a favourable taste to the products. In feed production efficient hydrolysis and high recovery is most important, and thermostable microbial enzymes like Alcalase may be recommended. Selective enzymic hydrolysis as an alternative to mechanical separation has many interesting perspectives, but remains in its initial development phase. So far only a few commercial processes have been established. Alteration from purely mechanical processes to enzyme technology is very demanding, particularly in the fish processing industry where biotechnological competence is limited. This situation, however, is gradually improving, and enzyme technology certainly will gain more importance in the future, both as an alternative to conventional processes and as a tool in the development of new products.

ACKNOWLEDGEMENTS Financial support from the Norwegian Council of Fisheries Research (NFFR) is acknowledged.

REFERENCES 1. Amano, K., The influence on nutritive value of fish with special reference to fermented fish products of South-East Asia. In Fish in Nutrition, ed. E. Heen & R. Kreuzer. Fishmg News Books Ltd, London, UK, 1962, pp. 189-200. 2. Corcoran, T. H., Roman fish sauces. Clawicol .I., 58 (1963) 204-10. 3. Orejana, F.M. & Liston. J., Agents of proteolysis and its inhibition in patis (fish sauce) fermentation. J. Food Sci.. 47 (1982) 198-203. G. & Steingrimsdottir, V., Applicatton of 4. Stcfansson. enzymes for fish processing in Iceland~Present and future aspects. In .4dvances in Fisheries Technology and Biotechnolozv ._. for Increased Profitnhilitv. ed. M.N. Voigt & J.R. Botta. Technomic Publ. Co.. Inc.. Lanc&ter, Philadelphia. USA, 1990. DD. 237-50. 5. Owens, J.D. & Mendoza, i’.S., Enzymatically hydrolysed and bacterially fermented fishery products. J. Food Tech&., 20 (1985) 273393. I.N. & Windsor, M.L., Fish silage. J. Sci. Food 6. Tatterson, Agric., 25 (1974) 369-79. 7. Raa, J. & Gildberg, A., Fish silage: A review. CRC’ C‘rif. Rev. Food Sci., Nutr., 16 (1982) 383419. 8. Edin, H., Undersiikningar angaande importavstegningens aggviteproblem. Nerd. Jordbr. Forsk, 22 (1940) 142-58. ”

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Asbjm-n Gildberg

9. Petersen, H.. Acid preservation of fish and fish offal. FAO Fish. Bull., 6, (1953) 18-26. 10. Gilberg, Y., Framgangsmate til 1 utvinne olje og proteinmel av fett-og oljeholdige hvirveldyr av marin opprinnelse. Norwegian Patent No. 81705, 1953. Il. Tarr, H.L.A.. Possibilities in developing fisheries byproducts. Food Techno/., 2 (1948) 268 77. 12. Freeman H.C. & Hoogland, P.L., Processing of cod and haddock viscera. I Laboratory experiments. J. Fish. Res. Bd Can., 13 (1956) 869-77. 13. Sripathy, N.V., Kadkol, S.B., Sen, D.P., Swaminathan. M. & Lahiry, N.L.. Fish hydrosates. III. Influence of degree of hydrolysis on nutritive value. J. Food Sri., 25 (1963) 36559. 14. Mackie, I.M., Proteolytic enzymes in recovery of proteins from fish waste. Process. Biochem., 9(10) (1974) 12-14. 15. Pigott, G.M., Enzyme modification of fishery by-products. In Chemistry and Biochemisfry of Marine Food Products, ed. R.E. Martin, G.J. Flick, C.E. Hebard & D.R. Ward, AVI Publ., CT, USA, 1982, pp. 447-52. 16. In, T., Seafood flavourants produced by enzymatic hydrolysis. In Making Projits out of Seafood Wustrs, ed. S. Keller, Alaska Sea Grant College Program. Report No. 9&07, Fairbanks, AK, USA, 1990, pp. 1977201. 17. Jakobsen. F. & Lykke-Rasmussen, O., Energy-savings through enzymatic treatment of stickwater in the fish meal industry. Process Biochem., 19(5) (1984) 165-9. 18. Jakobsen, F., Effect of enzymatic treatment of stickwater on evaporator capacity and fouling. Process. Biochrm., 20(6) (1985) 103-8. 19. Raa J., Biotechnology in aquaculture and the fish processing industry: A success story in Norway. In Advnnws in Fisheries Technology and Biotechnology for Increased Profitability, ed. M.N. Voigt & J.R. Botta. Technomic Pub]. Co., Inc., Lancaster, Philadelphia USA, 1990. pp. 509924. 20. Nilsen K., Viana, M.T. & Raa, J., Biotechnology in squid processing: Removing skins enzymatically. Irzfofish Irrternat., 2189 (1989) 27-8. 21. Martin, A.M. & Patel, T.R., Bioconversion of wastes from marine organisms. In Bioconvrrsion of Waste Materials to Industriul Producrs, ed. A.M. Martin. Else&r Sci. Pub]., London, UK, 1991, pp. 417-40. 22. Dougan, J. & Howard, G.E., Some Auvouring constituents of fermented fish sauce. J. Sci. Food Agric., 26 (1975) 887-94. 23. Beddows, C.G., Ardeshir, A.G. & bin Daud, W.J., Development and origin of the volatile fatty acids in budu. J. Sri. Food Agric., 31 (1980) 8692. 24. Gildberg. A., Accelerated fish sauce fermentation by initial alkalification at low salt concentration. In Current Topics ir2 Marine Biotechnology, ed. S. Miyachi, I. Karube & Y. Ishida, Fuji Technology Press Ltd, Tokyo, 1989, pp. 1014. 25. Hamm, W.S. & Clague, J.A. Temperature and salt purity effects on the manufacture of fish paste and sauce. Research Report 24, Fish and Wildlife Service, US Dept. Interior, 1950. 26. Mabesa, R.C., Carpio, E.V. & Mabesa, L.B., An accclcrated process for fish sauce (patis) production. In Post-harvest technology. preservation and Quality of Fish in South-East Asiu, ed. P.J.A. Reilly, R.W.H. Parry & L.E. Barile. Echanis Press, Manila, The Philippines, 1990, pp. 4559. 27. Beddows, C.G. dt Ardeshir, A.G.. The production of soluble fish protein solution for use in fish sauce manufacture. IT. The use of acids at ambient temperature. J. Food Technol., 14 (1979) 613-23. 28. Gildberg, A., Hermes, J.E. & Orejana, F.M., Acceleration of autolysis during fish sauce fermentation by adding acid

29.

30.

31.

32.

33.

34. 35.

36. 37. 38. 39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

and reducing the salt content. J. Sci. Food Agric. 35 (1984) 136339. Phithakpol, B., Fish fermentation technology in Thailand. In Fish Fermentation Technology, ed. C.E. Lee, K.H. Steinkraus & P.J.A. Reilly. Yurim Publish. Co., Seoul, The Republic of Korea, 1989, pp. 143-52. Beddows, C.G., Ismail, M. & Steinkraus, K.H., The use of bromelain in the hydrolysis of mackerel and the investigation of fermented fish aroma. 1. Food Technol., 11 (I 976) 379-88. Beddows, CC. & Ardcshir, A.G.. The production of soluble fish protein solution for use in fish sauce manufacture. I. The use of added enzymes. J. Food Technol.. 14 (1979) 603-12. Kumalaningshi, S., Accelerated method of fish sauce production from lemuru fish (Scardinella sp.) In Postharvest Technology, Presersution and Quality of Fish in South-Eust Asia, ed. P.J.A. Reilly, R.W.H. Parry & L.E. Barile. Echanis Press, Manila, The Philippines, 1990, p. 21. Lisac, H., The liquid ensilage of Mediteranean sardines for animal feeding. Proc. Gen. Fish. Coun. Medit., 6 (1961) 111-19. Wignall, J. & Tatterson, I., Fish silage. Process Biochem., ll(10) (1976) 17-19. Hardy, R.W., Shearer, K.D. & Spinclly, J., The nutritional properties of co-dried fish silage in rainbow trout (Sulmo gairdneri) diets. Aquaculture, 38 (1984) 3544. Stone, F.E. & Hardy, R.W., Nutritional value of acid stabilized silage. J. Sci. Food Agric., 37 (1986) 797-803. Backhoff, H.P., Some chemical changes in fish silage. J. Food Technol., 11 (1976) 353-63. Raa, J. & Gildberg, A., Autolysis and protcolytic activity of cod vicera. J. Food Technol., 11 (1976) 619-28. Gildberg, A. & Raa, J., Properties of a propionic acid/formic acid preserved silage of cod viscera. J. Sci. Food Agric., 28 (1977) 647-53. Gildberg, A. & Almas, K.A., Utilization of fish viscera. In Food Engineering and Process Applications-Vol. 2, Unit Operations, ed. M. 1eMaguer & P. Jelen. Elsevier Sci. Publ., Londpn, UK, 1986, pp. 383 93. Krogdahl. A., Fish viscera silage as a protein source for poultry. I. Experiments with layer-type chicks and hens. Acta Agric._Scand., 35 (1985) 3-23. Krogdahl, A., Fish viscera silage as a protein source for poultry. II. Experiments with meat-type chickens and ducks. Acta Agric. Scund., 35 (1985) 24-32. Raa, J., A. & Strom, T., Gildberg, Silage production--theory and practice. In Upgrading Waste for Feeds and Food, ed. D.A. Ledward, A.J. Taylor & R.A. Lawrie. Butterworths, London, UK, 1983, pp. 117-32. Almds, K.A., Utilization of marine biomass for production of microbial growth media and biochemicals. In Advunces in Fisheries Technology und Biotechnology for Increased Pr&ability. ed. M.N. Voigt & J.R. Botta. Technomic Publ. Co., Inc., Lancaster, Philadelphia. USA 1990, pp. 361-72. Clausen, E., Gildberg, A. & Raa, J., Preparation and testing of an autolysate of fish viscera as a growth substrate for bacteria. Appl. Environ. Microbial., 50 (1985) 1556-7. Almas, K.A. & Sandsdalen, E., Utilization of by-products from the fishing industry for industrial production of media for microbial growth. In Current Topics in Marine Biotechnology, ed. S. Miyachi, 1. Karube & Y. Ishida. Fuji Technology Press Ltd, Tokyo, Japan, 1989, pp. 365-8. Vecht-Lifshitz. SE., AlmHs, K.A. & Zomer, E., Microbial growth on peptones from fish industrial wastes. Lett. Appl. Microbial., 10 (1990) 183-6. Yanase, M. & Murayama, S., Studies on fish solubles. III.

Enzymic

processing

Effect of unidentified growth factor in fish solubles on growth of rats. Bull. Tokai Reg. Fish. Res. Lab., 43 (1965) 73-6.

H., Murayama, S., Onishi, T., Iseki, S. & 49. Higashi, fish protein”. I. Watanabe, T.: Studies on “liquefied Nutritive value of “liquefied fish protein “. BUN. Tokai Reg. Fish. Res. Lab., 43 (1965) 77-86. 50. Mackie. I.M., Fish protein hydrolysates. Process Biochem., 17(l) (1982) 268, 31. available 51. Hale, M.B., Relative activities of commercially enzymes on the hydrolysis of fish protein. Food Technol., 23 (1969) 107-10. 52. Mohr, V., Fish protein concentrate production by enzymatic hydrolysis. In Biochemical Aspects of New Protein Food, ed. J. Adler-N&en, B.O. Eggum, L. Munck & H.S. Olsen. Pergamon Press, Oxford, UK, 1978, pp. 53362. V., Enzymes technology in the meat and fish 53. Mohr, industries. Process Biochem., 15(6) (1980) 18-21, 32. 54. Hjelmcland, K., Protcinasc inhibitors in the muscle and Isolation and cod (Gadus morhua). serum of characterization. Comp. Biochem. Physciol., 76B (1983) 365-72. 55. Langmyhr, E., Enzymatisk Hydrolyse av Fiskeprotein. Dr ing. thesis, Norges Tekniske Hogskole, University of Trondheim, Norway, 198 1. 56. Quagly, G.B. & Orban, E., Influence of the degree of hydrolysis on the solubility of the protein hydrolysates from sardine (Sardina pilchardus). J. Sci., Food Agric., 38 (1987) 271-6. 51. Quagly, G.G. & Orban, E., Influence of enzymatic hydrolysis on structure and emulsifying properties of sardine (Sardina pilchardus) protein hydrolysates. J. Food Sci., 55 (1990) 1571-3, 1619. 58. Spinelli, J.. Groninger, H., Koury, B. & Miller, R., Functional protein isolates and derivatives from fish muscle. Process Biochem., lO(l0) (1975) 31-7. S.U. & Tan, L.K., Acceptability of crackers 59. Yu, (“keropok”) with fish protein hydrolysaie. ht. J. Food Sci. Technol., 25 (1990) 20118. 60. Rutman, M., Process for preparing high-energy fish protein concentrate. US Patent No. 3561973, 1971. 61. Pcpplcr, H.J. & Reed, G., Enzymes in food and feed Enzyme Biotechnology-Vol. 7a, processing. In Technology, cd. J.F. Kennedy. VCH Publishers, Weinheim, Germany,- 1987, pp. 546603. T., Process for preparation of fish 62. Ikeda, I. & Takasaki, meat extracts. US Patent No. 4036993: 1977. K., Egawa, M., Onruka, H. & Oba, K., 63. Sugiyama, Characteristics of sardine muscle hydrolysates prepared by various enzymatic treatment. Nippon Suixm Gakkaishi, 57 (1991) 475-9. 64. Shoji, Y., Creamy fish protein. In Making Profits out of Seafbod Wastes, ed. S. Keller. Alaska Sea Grant Program. Report No. 9&07, Fairbanks, AK, USA, 1990, pp. 87-93. H., Shirakawa. Y. & Shoii. Y.. 65. Uchida, Y., Hukuhara, Bio-fish flour. In Making Profits out ofseafood Was&, ed. S. Keller. Alaska Sea Grant Program, Report No. 9&07, Fairbanks, AK, USA, 1990. pp. 95-9. 66. Goldhor, S.H., Curren, R.A., Solstad, O., Levin, R.E. & Nichols, D., Hydrolysis and fermentation of fishcry byproducts : Costs and benefits of some processing variables. In Making Profits out of Seafood Wastes, ed. S. Keller. Alaska Sea Grant Program, Report No. 90-07, Fairbanks, AK, USA, 1990, pp. 203-S. 61. Willard, T., Utilization of marine by-products in pet foods. In Making Projits out of Seafood Wastes, ed. S. Keller. Alaska Sea Grant Program, Report No. 9&07, Fairbanks, AK, USA, 1990. pp. 121-5.

of marine

raw materials

13

68. Noel, H.S., By-catches for protein. World Fishing, March (1974) 77. and 69. Vinot, C., Bouchez, P. & Durand, P., Extraction purification of peptides from fish protein hvdrolysates. In &rent Topics &Marine Biotechn&ogy, ed: S. Miyachi, I. Karube & Y. Ishida. Fuji Technology Press Ltd, Tokyo, Japan, 1989, pp. 3614. 70. Beuchat, L.R., Preparation and evaluation of a microbial growth medium formulated from catfish waste peptone. J. Milk Food Technol., 37 (1974) 277-81. 71. Jassim, S., Salt, W.G. & Stretton, R.J., The preparation and use of media based on a simple fish waste extract. Left. Appl. Microbial., 6 (1988) 13943. 72. Gildberg, A., Batista, I. & Strom, E., Preparation and characterization of peptones obtained by a two-step enzymatic hydrolysis of whole fish. Biotechnol. Appl. Biochem., 11 (1989) 413-23. 73. Ellingsen, T. & Mohr, V., A new process for utilization of Antarctic krill. Process. Biochem., 14(10) (1979) 1419. 74. Rebeca, B.D., Pena-Vera, M.T. & Diaz-Castaneda, M., Production of fish protein hydrolysates with bacterial proteases; yield and nutritional value. J. Food Sci., 56 (1991) 30914. 75. Stone, F.E., Hardy, R.W., Shearer, K.D. & Scott, T.M., Utilization of fish silage by rainbow trout (Salmo gairdneri). Aquaculture, 76 (1989) 109-18. 76. Hardy, R.W. & Masumoto, T., Specifications for marine by-products for aquaculture. In Making Profits out of Seafood Wastes, ed. S. Keller. Alaska Sea Grant Program. Report No. 9&07, Fairbanks, AK, USA, 1990, pp. 109-20. 77. Christensen, F.M., Enzyme technology versus engineering technology in the food industry. Biotechnol. Appl. Biochem.. 11 (1989) 249-65. 78. Schaffel, G., Bruzzone, P., Illanes, A., Curoffo, M. & Aguirre, C., Enzymatic treatment of stickwater from fishmeal industry with the protease from Curcurhita ficifolia. Biotechnol. Lett., 11 (1989) 521-2. F.G.; The effect of adding 79. Hulan, H.W. & Proudfoot, white fish meal containing enzyme digested or untreated stickwater solids to diets for broiler chickens. Animal Feed Sci. Technol., 16 (1987) 253-9. 80. Wyatt, B. & McGourty, G., Use of marine by-products on agricultural crops. In Making Profits out of Seafood Wastes, ed. S. Keller. Alaska Sea Grant Program, Report No. 9&07, Fairbanks, AK, USA. 1990, pp. 187-95. 81. Montecalvo, J., Constantinides, SM. & Yang, C.S.T., Enzymatic modification of fish frame protein isolate. J. Food. Sci., 49 (1984) 1305-9. 82. Onoue, Y. & Riddle, V.M., Use of plastein reaction in recovery protein from fish waste. J. Fish Rex Bd Can., 30 (1973) 1745-7. 83. Ragunath, M.R. & McCurdy, A.R., Synthesis of plasteins from fish silage. J. Sci. Food Agric., 54 (1991) 655-8. 84. Ooshiro, Z., Won, M.P., Makagawa, S., Itakura, T. & Hayashi, S., Approaches to the use of plastein reaction in oily fish. Preparation and characterization of plastein products. Mem. Fat. Fish. Kagoshima Univ., 30 (1981) 369-82. 85. Kim, SK. & Lee, E.H., Enzymatic modification of sardine protein concentrate. J. Korean Agric. Chem. Sac., 30 (1987) 234-41. 86. Yamashita, M., Arai, S. & Fujimaki, M., Plastein reaction for food protein improvement. J. Agric. Food Chem., 24 (1976) 1100-4. of proteases in the enzymatic 87. Ruiter, A., Substitution ripening of hcning. Ann. Technol. Agric., 21 (1972) 597-605.

14 88. 89. 90. 91.

92.

93.

94.

95.

96.

97

98 99 100 101

102.

103.

104.

105.

106.

107.

108.

109.

Asbjwn 4non., Production process for salted herrings (matjes). British Patenl No. 1403221, 1975. Dpshaug, K., Fremgangsmite for enzymatisk hurtiginodning av sild. Norwegian Patent no. 148207, 1983. Eriksson, C., Method of controlling the ripening process af herring. Canadian Patent No. 969419, 1975. Ritskes, T.M., Artificial ripening of maatjes-cured herring with the aid of proteolytic enzyme preparations. FL&. Bull., 69 (1971) 647. Slinning, K.E., Hurtigmodning av Sild. Report to the Res. Lab. of the Norwegian Canning Industry, Stavanger, Norway, 1985. Simpson, B.K. & Haard, N.F., Trypsin from Greenland cod as a food-processing aid. J. Appl. Biochem., 6 (I 984) 135-43. Asgeirsson, B., Fox, J.W. & Bjarnason, J.B.. Purification and characterization of trypsin from the poikilotherm Gadus morhua. Eur. J. Biochem., 180 (1989) 85-94. Kvdle, H., Bioteknologisk virksomhet i Norge. Report No. A88001 lo Bergen Foundation of Science, Bergen, Norway, 1988. Lewis, R.V., The aquatic leather industry: Opportunities for small manufactures in Alaska. In Making Profits auf of Seafood Wastes, ed. S. Keller. Alaska Sea Grant Program, Report No. 90-07, Fairbanks, AK, USA, 1990, pp. 101-3. Raa, J., Modern biotechnology: Impact on aquaculture and the fish processing industry. Paper presented at the 5th World Productivity Congress, Jakarta, Indonesia, 13-16 April 1986. Stefansson, G., Enzymes in the fishing industry. Food Technol., 42(3) (1988) 64-5. Wray, T., Fish processing: New uses for enzymes. Food Manufacture, 63(7) (1988) 48-9. Fehmerling, G.B., Separation of edible tissue from edible flesh of marine creatures. US Patent No. 3729324, 1973. Dohmoto, N. & Miyachi. S., Degradation of marine mussel-thread by bacterial proteinases. Paper presented at the 2nd International Marine Biotechnology Conferencc, Baltimore, Maryland, USA 13-16 October 1991. Sugihara, T., Yashima, C., Tamura, H., Kawasaki, M. & Shimizu, S., Process for preparation of Ikura (salmon egg). US Patent No. 3759718, 1973. Krise, W.F., Cummings, L.B., Shellman, A.D., Kraus. K.A. & Gould, R.W., Increased walleye egg hatch and larval survival after protease treatment of eggs. Prog. Fish-Culturist, 48 (1986) 95-100. Krise. W.F., Optimum protease exposure time for removing adhesiveness of walleye eggs. Prog. Fi.rh Culturist, 50 (1988) 126--7. Horvlith, L., Use of a proteolytic enzyme to improve incubation of eggs of the European catfish. Prog. FishCulturisf, 42 (1980) 110-l 1. Hallerman, E.M., Schneider, J.F., Gross, M.L., Faras, A.J., Hackett, P.B., Guise, K.S. & Kapuscinski, A.R., Enzymatic dechorionation of goldfish, walleye, and northern pike eggs. Transac. Amer. Fish. Sot., 117 (1988) 45&60. Knorr, D., Recovery and utilization ofchitin and chitosan in food processing waste management. Food Technol. 45(l) (1991) 114-120, 122. Carroad, P.A. & Tom, R.A., Bioconversion of shellfish chitin wastes: Process conception and selection of microorganisms. J. Food SCL, 43 (1978) 1158-l 157. 1161. Revah-Moiseev, S. & Carroad, P.A., Conversion of the enzymatic hydrolysate of shellfish waste chitin to singlecell protein. BiotechnoL Bioeng., 23 (1981) 1067-78.

Gildberg 110. 1“osio, LG., Fisher, R.A. & Carroad, P.A., Bioconversion sf shellfish chitin waste: Waste pretreatment, enzyme production, process design and economic analysis. J. Food Sci., 47 (1982) 901-5. 111. Raa, J., Rlarstad, G., Engstad, R. & Robertsen, B., The use of immunostimulants to increase resistance of aquatic srganisms to microbial infections. Paper presented at Conference on Diseases in Asian Aquaculture, Bali, Indonesia, 2629 November 1990. 112. Xiuli, H., Danmei, A. & Qian, R., Study on chitinase in cultures of Beauveria hassiana. In Chitin and Chitosan, ed. G. SkjPk-Braek. T. Anthonsen & P. Sandford, Elsevier Sci. Publ. London, UK, 1989, pp. 309-17. 113. Nestaas, E., Biochemicals and industrial raw materials from marine biomass the competitive situation and perspectives. Paper presented at the 1 lth International Fisheries Fair, Trondheim, Norway, 1 l-16 August, 1986. 114. Kafetzopoulos, D., Martinou, A. & Bouritis, V., Isolation and characterisation of chitin deacetylase from Mucor rouxii. Paper presented at the 5th International Conference on Chitin and Chitosan, Princeton, USA, 17-20 October 1991. 115. Simpson, B.K. & Haard, N.F., The use of proteolytic enzymes to extract carotenoproteins from shrimp waste. J. Appl. Biochem., 7 (1985) 212-22. 116. Ya, T., Simpson, B.K., Ramaswamy, H., Yaylayan, V., Smith, J.P. & Hudon, C., Carotenoproteins from lobster waste as a potential feed supplement for cultured salmonids. Food Biotechnol., 5 (1991) 87-93. A., Simpson, B.K. & Haard, N.H., Ex117. Cano-Lopez, traction of carotenoprotein from shrimp process wastes with the aid of trypsin from Atlantic cod. J. Food Sci., 52 (1987) 503-504, 506. 118 Chen, H.M. & Meyers, S.P., Ensilage treatment of crawfish waste for improvement of astaxanthin pigment extraction. J. Food Sci., 48 (1983) 151&20, 1555. 119 Murata, Y.. Hayashi, T., Watanabe, E. & Toyama, K., Preparation of skipjack spermary extract by enzymolysis. Nippon Suisan Gakkaishi, 57 (1991) 1127-32. G.G., The application of lipases for 120. Haraldsson, modification of fats and oils, including marine oils. In Advances in Fisheries Technology and Biotechnology for Increased Pro$tability, ed. M.N. Voigt & J.R. Bottd. Technomic Publ. Co. Inc., Lancaster, Philadelphia, USA, 1990, pp. 337-57. I., Yamane, T. & Shimizu, S., Selective 121. Hoshino. hydrolysis of fish oil by lipase to concentrate n-3 polyunsaturated fatty acids. Agric. Biol. Chem., 54 (1990) 1459-67. 122. Hills, M.J., Kiewitt, I. & Mukherjee, K.D., Enzymatic fraction of fatty acids: Enrichment of y-linoleic acid and docosahexaenoic acid by selective esterification catalysed by lipases. J. Amer. Oil Sot., 67 (1990) 561-4. 123. Langholz, P., Andersen, P., Forskov, T. & Schmidtsdorff, W., Application of a specificity of Mucor miehei lipase to concentrate docosahexaenoic acid (DHA). J. Amer. Oil Sot., 66 (1983) 1120-3. 124. Haraldsson, G.G., HSiskuldsson, P.A., Sigurdsson, S.T., Thorsteinsson, F. & Gudbjarnason, S., The preparation of triglycerides highly enriched with R-3 polyunsaturated fatty acids via lipase catalyzed interesterification. Tetrahed. Left., 30 (1989) 16714. 125. Osada, K., Takahashi, K. & Hatano, M., Hydrolysis and synthesis of icosapentaenoic acidaocosahexaenoic acid rich oil by lipase TOYO. J. Jpn Oil Chem. Sot., 39 (1990) 5c2. 126. Tocher, D.R., Webster, A. % Sargent, J.R., Utilization of porcine pancreatic phospholipase A, for the preparation

Enzymic processing of marine fish oil enriched in (n-3) polyunsaturated fatty acids. Biotechnol. A&. Biochem., 8 (1986) 83-95. 127. Weiner, R.M., Colwell, R.R., Jarmann, R.N., Stein, D. C., Somerville, CC. & Bonar, D.B., Application of biotechnology to the production, recovery and use of marine poly&charides. Bio/Technof., 3 (1985) 899-902. 128. Polne-Fuller, M., Biniminov, M. & Gibor, A.. Vegetative propagation. of Porphyra perforata. Hydrobiologio. 116/117 (1984) 308-13.

of marine raw materials

15

129. Knutsen, S.H., Large scale production of bacterial enzymes for depolymerization of matrix polysaccharides in seaweeds. Paper presented at Cost-48 Workshop, Las Palmas, de Gran Canaria, 8-17 February 1991. 130. Groppe, J.C. & Morse, D.E., Molecular cloning of novel serine protease cDNAs from abalone. In Current Topics in Marine Biotechnology, ed. S. Miyachi, 1. Karube & Y. Ishida. Fuji Technol. Press Ltd, Tokyo, Japan, 1989, pp. 285-8.