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Bioactive compounds in fish and shellfish hydrolysates.

Objective

Two projects involving six laboratories were designed for developing methods for upgrading fish and crustacean wastes. The first one (FAR UP.3.654) was mainly directed on the potential use of enzymes in various processes of economical significance. The second one (FAIR CT 97 3097) aims to detect biologically active substances (immunostimulants, gastro-intestinal peptides, etc.) in hydrolysates of marine fish or invertebrates and to use them for enhancing growth and disease resistance of animals or as growth factors in bacteria or cell cultures.
Proteolytic enzymes (FRCC,FRIF)

Enzyme activities subjected to this study belong to the group of esterases : endoproteases (trypsin, chymotrypsin, elastase collagenase), exoproteases (aminopeptidases, carboxy-peptidases) ( Hajjou and Le Gal, 1993). Chitinases and lipases present in the same sources of material have also been studied as co-products. A pepsin activity was purified from blue shark stomach with the main objective of using methods manageable from pilot to industrial stage. Elastase activity was detected in pancreatic extracts of tropical tuna (Thunnus albacora) (Smine and Le Gal, 1995). The purification yielded an enzyme suitable for enzymatic hydrolysis of insoluble elastine. It is worth to indicate that this enzyme presents a practical interest for the preparation of elastine hydrolysates used in cosmetics.
A new crab serine collagenase (CSC) was isolated and its specificity towards several synthetic and natural substrates was investigated. The CSC hydrolyzes native bovine collagens types I and III at neutral pH, and is therefore a true collagenase. Beta-casein is strongly hydrolyzed by CSC, but not beta-lactoglobulin or elastin. (Roy et al., 1996). CSC has been used for the hydrolysis of a natural sponge collagen and could therefore be a convenient tool for collagen structural studies.
Scaling up the purification procedures for pepsin and trypsin from tuna and blue shark have been undertaken and samples of the resulting products have been, in turn, used for the preparation of fish hydrolysates. Following this research, several enzyme systems are now tested for practical use in three different domains: scale removing in the process of fish skin tanning, elastin and elastin hydrolysates in cosmetics and upgrading of waste waters from frozen crustacean treatments.

Biological activites in hydrolysates ( NOTR, UKPL)

The research investigates the effects of a muscle protein hydrolysates from Atlantic cod on the non-specific and specific immune responses of rainbow trout (Bogwald et al., 1996). Small acid peptides from cod muscle and cod stomach hydrolysates stimulates superoxide anion production ("oxidative burst") in head kidney leukocytes from Atlantic salmon (Salmo salar) ( Gildberg et al.,1996). Such stimulation has been obtained both after intraperitoneal injection in vivo, and after inoculating cell cultures in vitro. The stimulation has relatively short duration, but the level is similar to stimulation obtained by lipopolysacharides (LPS) from bacterial cell wall. New enzymatic hydrolysates will be prepared from the head of Atlantic cod and from shrimp waste. Fractions with immuno stimulating activity in vitro assay systems will be tested in vivo challenge experiments with fish fry to verify if the immuno stimulating peptides can provide protection against infection after oral administration.
Opioid activities (FRLR)
Evidence for the identity between opioid substances and peptides isolated and purified from enzymatic digests was obtained from the opioid activity elicited by synthetic peptides with the same sequences. Hydrolysates are constituted of very complex mixtures and essentially composed of peptides. A great care was taken to optimize the separation conditions in order to obtain resolved chromatograms allowing the collection of individualized peptidic fractions. Several fractions were able to inhibit the electrically induced muscle contractions. Naloxone (opiate antagonist) when added reversed all these induced inhibitions.
Gastrin-CCK( FRCC)
Gastrin and CCK-like peptides are present in cod viscera and somach extracts. However the levels of gastrin-like equivalents measured by RIA are low. Purified fractions corresponded largely to oxidized and hydrolysed forms of authentic cod gastrins or CCK.

Fish peptones (PTLI,FRQU)
A series of hydrolysates was selected for the preparation of culture media for bacteria and tested as growth promoters or enhancers. Growing media prepared with fish hydrolysates were used for the identification of different Salmonella, Vibrio, and Aeromonas strains. The results were similar to those obtained with commercial substrates. (Batista and Nunes,1997). The results of experiments performed on an E.coli strain showed that the rate of hydrolysis related to the quality of fish protein hydrolysates affect the final biomass in cultures.

CONCLUSIONS

Methods for extracting enzymes from fish and crustacean species of commercial importance, testing them in the preparation of different products and taking into account as priorities the economic constraints, the disposability and competitiveness of the process have been developed. The main source of biological material has been the digestive tract wastes of industrially commercialised fishes and crustaceans. Fish hydrolysates are more particularly investigated. The presence of biologically active compounds in fish hydrolysates is under investigation. Preliminary results indicates that mild hydrolysis of fish or crustacean wastes yield hormone-like (calcitonine, gastrine) material, immunoactive substances, peptones, etc..

REFERENCES
Batista I. and Nunes M.C; In: Seafood; Eds: J.B.Luten,T.Boerresen and J. Oehlenschlager; (1997);.Elsevier Amsterdam; pp.59-70
Boegwald J., Dalmo R.A., Leifsson R.M., Stenberg E. and Gildberg A.; Fish Shellfish Immunol..; 6 (1996) 3-16
De La Broise D., Dauer G. and Guerard F.; J. Marine Biotechnol. (1998)(In press)
Gildberg A., Batista I. and Strom E.; Biotechnol.Appl. Biochem.; 11 (1989) 413-424
Gildberg A., Boegwald J., Johansen A. and Stenberg E.; Comp.Biochem.Physiol. 114B (1996) 97-101
Gildberg A., Olsen R.L.;and Bjarnason J.B.; Comp.Biochem.Physiol.; 90B (1990) 323-330
Guerard F. and LeGal Y.; Comp.Biochem.Physiol. 88 (1987) 823-827
Hajjou M. and Le Gal Y. Biochim.Biophys.Acta; 1204 (1993) 1-13
Myrnes B. and Johansen; Prep.Biochem.; 24 (1994) 69-80
Piot J.M.,Zhao Q, GuillochonD. Ricart G. and Thomas D.; Biochem.Biophys.Res.Communs; 189 (1992)101-110
Roy P. ColasB.and Durand P.; Comp.Biochem.Physiol.; 115B ( 1996) 87-95
Smine K. and Le Gal Y.; J.MarineBiotechnol.; 2 ( 1995) 143-147
Van Wormhoudt A. and Dirksen H.; In: Frontiers in Crustacean Hemobiology. (1990) Birkhauser Verlag; pp 483-484
Van Wormhoudt A., Favrel P. and Guillaume J.; J.Comp.Physiol.; 159 ( 1989) 269-173
Zhao Q., Sannier F., Garreau I. and Piot J.M.; J.Liq.Chromatogr.; 18 (1995) 1077-1092
Zhao Q.,Sannier F.and Piot J.M.; Biochim.Biophys.Acta.; 1295 ( 1996) 73-80
Only 50% of the total catches of the fish industry in the EU are used for human consumption. The remains are either discarded at sea or processed to animal feed. These wastes constitute an important source of biologically active molecules possessing peculiar properties and practical application promises in various areas (agriculture, medicine, chemistry, biotechnology). Extracts, hydrolysates and enzymes from seawater fish species and marine invertebrates have revealed interesting characteristics. Many marine enzymes behave differently from their mammalian counterparts (Gildberg et al.1990, Guerard and Le Gal, 1987; Myrnes and Johansen, 1994). For example, high activities are obtained at 5-12°C for enzymes of marine origin, instead of 30-35°C for classical systems. Hence, these enzymes are suitable for gentle processing at low temperature.
On the other hand, hydrolysates and extracts, from fish or marine invertebrates viscera have been shown to contain biologically active factors that should be further investigated. Feeding with various extracts from marine invertebrates or fish eliminates the reduction of feed intake normally occurring when the fish is given feed containing antibiotics. This observation may reveal a possibility of both economical and environmentally compatible improvements in aquaculture. Another field of special interest is the characterization, in fish hydrolysates, of small gastro-intestinal peptides like gastrins and cholecystokinins (CCK) ( Van Wormhoudt et al.,1989; Van Wormhoudt and Dirksen, 1990) that stimulate the secretion of digestive enzymes and the production of hormones controlling satiety.
Several other classes of molecules can be generated by enzymic digestion of fish proteins: peptides with opioid-like activity ( Piot et al. 1992; Zhao et al. 1995, 1996) are found in several types of fish hydrolysates. Cellular growth factors present in hydrolysates of cod muscle stimulate significantly division and proliferation in cell cultures of fibroblasts. Fish protein hydrolysates are also known as possible substitute of usual protein hydrolysates-based substrates for microbial cultures (Gildberg et al., 1989; De la Broise et al., 1998
Two projects were designed for addressing these questions. The first one ( FAR UP 3.654) was mainly directed on the enzyme activities already present in these wastes and on their potential use in various processes of economical significance. The project was particularly aimed to the studying of the specific properties, which render then proper to be included in new industrial processes, such as the production of soluble peptides, production of peptones, development of flavours, etc. With respect to these objectives, several enzymes from crustacean and fish (tuna, mackrel, sardine, blue shark) wastes for which sizable stocks of viscera are actually available have been considered.
The second project ( FAIR CT 97 3097) aims to explore the possibility of obtaining biologically active peptides from extracts or hydrolysates of marine food processing waste with the ability to be used for enhancing growth and disease resistance of animals, as attractants or flavourants in feed and food and possibly as pharmacological tools. The current work programme of this project aims to control the conditions for the obtention of peptides retaining biological activities, the identification of bioactive sequences in hydrolysed products by searching for immuno reactivity based on vertebrate peptide recognition, biological testing in cell cultures :bacteria, vertebrate and invertebrate dissociated cells (crustacean, molluscs), growth hormone and immune response enhancement, electrophysiological tests on isolated intestine organs (opioid molecules,AC enzyme inhibition).

MATERIAL AND METHODS

Proteolysis: A major element of the waste from the fishing industry is the gastrointestinal tract of fish which contains a variety of proteases together with lipases and carbohydrases required for digestion of food. The enzymes have been used as endogenous sources of catalysts for the production of silage from fish waste (Atlantic cod, sardine, tuna), especially under acidic conditions to control bacterial growth. Adjusting hydrolysis conditions to allow the action of, for example, trypsins, chymotrypsins, cathepsins, collagenases, elastases, etc. result in different hydrolysis patterns and the production of different groups of peptides (for details, see : Final report contract UP-3.654).
Purification and identification of hydrolysates. The peptides are characterised by their molecular weight distribution and amino acid composition. Hydrolysates giving optimum levels of activity are fractionated by HPLC and active sub-fractions are analysed by Electro Spray Mass Spectrometry and other relevant techniques for information on amino acid composition and sequence.
Immunostimulating activity. The immunostimulating activity is measured as the ability of macrophage to produce oxygen radicals by the reduction of nitroblue tetrazolium after triggering with phorbol myristate acetate (PMA).
Opioid peptides. The opioid activity is tested on the contractions of the longitudinal muscle of a segment of isolated guinea-pig ileum. In this isolated organ preparation, opioid activities are evaluated in terms of an inhibition of electrically induced contractions, in so far as these proves antagonizable by the opiate antagonist naloxone (0.2 mM).
Gastrin-CCK.. Gastrin radioimmunoassays (RIA) are performed using a rabbit anti-serum, synthetic 125I-gastrin as a tracer and synthetic gastrins as standards.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

Museum National d'Histoire Naturelle
Address
Place De La Croix
29182 Concarneau
France

Participants (5)

Instituto de Investigacao das Pescas e do Mar
Portugal
Address
Avendida De Brasilia
1449-006 Lisboa
NORWEGIAN INSTITUTE OF FISHERIES AND AQUACULTURE LTD
Norway
Address
University Campus Breivika
Tromsö
Universite de Bretagne Occidentale
France
Address
Pole Universitaire Gorgeu, Creac'h Gwen
29000 Quimper
University of Plymouth
United Kingdom
Address
Drake Circus
PL4 8AA Plymouth
Université de la Rochelle
France
Address
23,Avenue Marillac
17042 La Rochelle