THE EXTRACTION AND ACTIVITY OF CRUDE ENZYMES FROM COWTAIL RAY ( Trygon sephen ) VISCERA . )

The viscera are usually wasted in the processing of cowtail ray, although they may contain many valuable substances such as proteolytic enzymes. This study investigates the possibility of using these enzymes to produce fish protein hydrolysates. The enzymes were extracted by ensilation using a urixture of 50%, propionic and 50% forrnic acids at a 3Yo (vlw) inclusion rate at 40';C over a 5 day periods. Cnrde enzyrnes were produced by precipitation with ammonium sulphate. The activity of the extracted enzymes was investigated by fish meat hydrolysis conducted at 450C and 600 C, and pH 2.0, 5.0 and 8.0 for 4 hours. The ratios of crude enzyme to fish nreat were 5rlo and l0%. h was shown that the optimum hydrolysis was at 600C for 3 hour at a ratio of clrrde enzyme to fish ureat of 5% at all pHs.


INTRODUCTION
The viscera of frsh are an important digestive organ, functioning as a grinder and liquefrer of ingested foods.It contains a number of enzyrnes that aid in the breakdown and subsequent absorption of foods.The high levels of enzyrnes in the viscera make it very susceptible to autolysis, which is the frrst process that takes place when a frsh dies.However, visceral enzymes are partly responsible for flavour development during curing and fermentation of aquatic food products.They also play significant roles in viscera silage process, which is facilitated by an addition of acids to reduce the pH of the viscera to that close to pH optimum for digestive enzymes activity.If the ternperature is in the range of 25-40'C, the protein of the viscera liquefies within one week and is ready for further utilisation.
Hall el ol. (1985)and Raghunath & McCurdy (1990) indicated that both exopeptidases and endopeptidases were active in the autolysis of viscera silage.In the acidic conditions such as in silage, it is likely that endopeptidases such as pepsin-like enzyrnes, which have acidic optirna, dominate the reaction.However, Raghunath & McCurdy (1990) also observed trypsin-like activity.Although it has an alkaline pH optirnum, which is possibly not active in acid silage, this enzyrne is reported to be quite stable in acid conditions (Hjehneland & Raa, 1982), Reece ( 1988)   (1990) were aminopeptidase, cathepsin B and cathepsin C. Thus, silage is also able to extract enzymes from viscera, although some changes in the activity rnight occur.
The uses of frsh proteolytic enzymes in food processing have been investigated by a number of groups of workers (Simpson & Haard 1984; Stefansson & Steingrimsdottir 1990; Yoshinaka   et al., 1983).Acidic fish proteases from cold water species have been investigated as a rennet substitute in cheese processing (Brewer et al.,  1984; Shamsuzzaman & Haard, 1985; Haard,  1992).Fish pepsins have been industrially produced and used for the production of salmon and orange roughy caviar in some countries (Almas, 1990;Xu et a|.,1996).
The viscera ofcowtail ray are up to 10% oftotal body weight and are wasted in the processing of dried salted products in Indonesia, The present paper deals with the use of crude visceral enzymes from cowtail ray (Trygon Bephen) bo hydrolyse fish meat and the possibilities of producing fish protein hydrolysate using these fish enzymes.

MATERIALS AND METHODS
Cowtail ray viscera were obtained from producers of salted and dried ray at Labuhan Maringgai, Lampung, South East Sumatera, Rays landed at Labuhan Maringgai are usually caught A. Poernonto in Lampung waters by bottom set long lines druing 4 day fisNng trips.The frsh are kept on-board in wooden fish holds with minimal icing (Ariyani ef   al., 1993).The experiment was conducted twice and all chemicals were of analytical grade.
Separation and washing of viscera were done at the processing sites.The viscera were transported (by road and sea) in ice at a 1:1 (w/w) ratio to the laboratory of the Slipi Research Station for Marine Fisheries, Jakarta.The journey tirne was approximately eight hours.On arrival at the laboratories.the viscera was once more washed in fresh water, frozerr and stored at -450C until used.
Enzymes were extracted from the ray viscera as follows.The viscera were chopped and placed into glass containers (75091.To the chopped viscera was then added a mixture of propionic and formic acids (1:1, v/v) at concentration of 3o/o (v/w).
Previous studies had shown that at this level, the acids were able to preserve and liquefu the viscera (Poernomo & Buckle, 1993).The mixtures were stored at 400C, and stirred daily.After 5 days the mixttue was centrifuged (2000:rg, 10 min, 4oC).The silage liquid was mixed with ammonium sulphate, and the protein that precipitated between2S-5O% saturation was redissolved into 10 mM phosphate buffer pH 7.0 and dialysed overnight against the same bulfer solution.After further centrifugation, the supernatant was collected and used as a crude enzyrne preparation.
To assess the ability of the extracted enzymes to hydrolyse fish rneat, a test was conducted using minced flesh frorn Indian mackerel (Rastrelliger hanagurtal with a proxirnate composition of: 75.Lo/o moisture, 23.3% protein and 1.5%, fat (wet weight basis).
The method to hydrolyse fish meat was adopted frorn Liu & Pigott (1981).To prepare the substrate for hydrolysis, Indian mackerel fillets (159) were mixed with one volume of distilled water (15 mL) and hornogenised in a Waring blender.The crude enzyme was added at levels of 5 and 10olo (w/w) with respect to protein content of the crude enzyme extract and minced fish.The mixture was adjusted to pH 2.0, 5.0 and 8.0 using 2N HCI or 2N NaOH and incubated at 45uC and 600C for 4 h with continuous stirring.During this time the non-soluble nitrogen level was monitored.

ANALYTICAL METHODS
Moisture content was measured using the rnethodof Chng (1992a).Sarnplesof about 5gwere weighed in a tared dish, and dried in a forced air oven at 105"C for 24h.The weight loss after drying 40 was considered as moisture content.Dried material ftom the analysis of moisture content was ashed in a muffle furnace at 55trC overnight or until white (Chng, 1992b).The white material after ashing was weighed and considered as the ash content of the material.
The Kieldahl method was used to measure the protein nitrogen content of samples of about 2g (Kirk & Sawyer,f99f).The catalyst used for digestion was a mixture of 9 parts (w/w) of potassium sulphate.anhydrous and 1 part (w/w) ofcopper sulphate anhydrous.Tbe indicator used in the titration of the distillate was a mixture of 1 part (v/v) of O.Lo/o methyl red in 95% ethanol and 2 parts (v/v) of 0.27o bromocresol green i\ 95o/o ethanol.The protein content was obtained by multiplying the protein nitrogen content by a factor of.6.25.
Soluble protein in the crude enzyme preparation was measured by the method of Lowry el o/. (1951)using the modification of Bailey (1967) in which sodium citrate was used instead of sodium tartrate.Bovine serurn albumin (BSA)  was used as the standard protein.The estimation of protein content from spectrophotometer readings was done according to the method of Fat was extracted by the Soxhlet apparatus for 16b using petroleum ether (Boiling Point 40- 60oC) as solveut, and the weight of material in the flask after evaporation of solvent was calculated as the fat content.
pH was measured using a pH meter (Digital Ionalyser Model 6014, Orion Research Inc. ) calibrated to pH 4 and 7 with bulfer solutions.
The pH of the solid material was measured from the liquid dispersion of the sample (1:1, w:v), while that of silage was done by direct measwement using a calibrated pH meter stick (Gallenkamp).
Soluble nitrogen of silage liquid was measured by adding 3 portions of 20o/o trichloroacetic acid (TCA), mixed for 5 min and filtered (Whatman filter paper No. 3).The nitrogen content was measured in the filtrate by the micro-kjeldahl method (Kirk & Sawyer, 1991).In fish hydrolysates, the method of Liu & Pigott (1981)   was adopted.A volume of aliquot was taken from the minced fish and crude enzyme rnixture, and then mixed with 2 volumes of 2oo/o trichloroacetic acid for 10-15 minutes, and filtered through Whatrnan No. 3 frlter paper.The soluble nitrogen was measured as above.The results were corrected for the initial soluble nitrogen level.The degree of hydrolysis (DH) was ealculated according to Hoyle & Merritt (1994)  A viscometer (LVT Model, Brookfield Eng.tab. Inc. ) was used to measure the viscosity of the silage at ambient ternperature.Liquefaction of silage was defined as the prolrcrtion (w/w) of liquid to the total sample.Liquid silage was separated by centrifrrgation at 2000 g for 10 min using a Beckman J2-2L centrifuge (Beckrnan Instrurnents Inc. ).
Enzyme activity was measured in the liquid silage stored at 40"C at day 5 using hemoglobin as substrate and the methods of Rick (1963a,b) for pepsin-like activity and for trypsin-like activity.One Pepsin Unit (PU) or one Trypsin Unit (TU) were defrned as the amount of enzymewhich hydrolysed hemoglobin at such an initial rate under the standard conditions (total volume 6 mL, containing 0. 1g hernoglobin, temperature gb.S0C), that the arnount of tricholoroacetic acid-soluble hydrolysis products formed in one minute, gave the same optical density with the phenol reagent as for 1 mmole tyrosine (Rick,l963a,b), Experimental data were statistically analysed in factorial design using a computer statistical RESULTS AND DISCUSSION

Enzyme Extraction
The proximate composition of cowtail ray viscera is shown in Table 1.
Upon the addition of acids, the pH of the chopped viscera sharply decreased from 8.5 to 5.1, while the colow changed from reddish white to greyish white.It is likely that this colour change was due to oxidation of some pigments.Other ehanges are shown in Table 2.
The enzyme activities in the liquid silage were 0.05 TU and 0.08 PU for trypsin-like and pepsinlike enzymes respectively, and increased 8 and 13 fold, respectively after amrnonium sulphate precipitation and dialysis.This shows that the enzymes in the silage were still active at least after 5 days storage at 40oC.Reece (1988) found that activity of acidic proteases in silage made from cod, mackerel and salmon viscera were ahnost constant during 5 days storage.Prolonged storage at up to 200C, however, led to gradual loss of protease activity frorn mackerel and salmon, but this was not observed in cod viscera.Raa & Gildberg ( 1976)  and Gildber g & Raa (197 7 ) observed that protea ses in cod viscera silage were stable for at least 9 days aL 27oC.
Separate study showed that two pepsin-Iike and one trypsin-like enzymes were identified from the liquid cowtail ray viscera silage (Poernomo,  1997).These enzymes were reported to have molecular weight of about 30,000 dalton for both the pepsin-like enzymes and about 45,000 dalton for the trypsin-lihe enzyrne, respectively.

Fish Protein Hydroly sis
The results of frsh protein hydrolysis by crude enzymes are shown in Figure 1.At 450C, the hydrolysis of fish protein at all pHs and enzyrne to frsh meat ratios was very slow resulting in a low level of soluble nitrogen ( 10-309'0 of total nitrogen).The levels of soluble nitrogen at 600C was relatively constant during the first 2h, sharply increased in the next hotu, therr leveled off AII slurries at 600C, except that at pH 2.0 with 5% ratio of crude enzyme, had soluble nitrogen of about 60% (of total nitrogen) after 4h hydrolysis.The slurry at pH 2.0, at 60"C and 5'% r atio of crude enzyme had frnal soluble nitrogen of about 50% (of total nitrogen).Statistical analysis showed that all factors applied in this study and their interaction signifrcantly affected the level of soltrble nitrogen of the hydrolysate (p=0.0),except for interaction between temparature and enzyrn ratio (P = 0.59); temperattue, time and enzyme ratio ( P = 0. 10) and ternperatrrre, tirne, pH and enzyme ratio (P = 0.38).
The present study shows that at 600C the crude enzymes applied were able to hydrolyse frsh protein producing about 60% soluble nitrogen at pH 5.0 and 8.0, and altnost 5O9', at pH 2.0.The similar resrilts for different pHs were rnost probably due to the high buffering capacity of fish meat which tended to adjust the pH of the shuries to close to the original fish meat pH.Additionally, as protein hydrolysis is accornpanied by a release or uptake of H* (Sorensen, 1908), the pH of the mixture changes, except in the region around pH 5-6 where the uptake and release ofprotons cancel each other (Adler-Nissen, 1986).Thus since the pH of the frsh meat arrd enzylne mixture in the present study was not maintained, pH 2.0 would eventually increased, and pH decreased to close to the original fish meat pH.
In a test of frsh meat hydrolysis by bacterial proteases, Rebeca et al. (1991) found that nitrogen solubilisation was faster during pH-controlled hydrolysis than without pH control.They also observed that a decrease in pH, from 9.5 to 8.0 for the slurries using alkaline protease and from 7.5 to 6.5 for the slurries using neutral proteases, occurred during the first hour of hydrolysis, which corresponded to a 15-30% reduction of protease activity.
Another possible reason why the level of soluble nitrogen in the first 2h was low is the relative resistance to proteolysis of native fish protein (Reeck, 1971).Protein hydrolysis by pepsin and trypsin was easier in slightly cooked fish than in raw or fully cooked fi sh (Saha, 1940), and according to Rupley (1967) small amount of denatured proteins were sufficient to influence the kinetics of proteolysis.This also explains why hydrolysis at 45"C was slower than that at 600C since it is likely that at 45oC the protein did not suffer as much heat denaturation as at 600C.Mackie (1982a) reported that in fish protein hydrolysate "the degree of hydrolysis as measured by the proportion of trichloroacetic acid soluble nitrogen will be in excess of 50% of the total nitrogen of the fistt''.The hydrolysates produced at 6SC in the present study can be said to have met the above definition.In the present study the hydrolysis at 600C seemed to cease after 3h.This was a typical hydrolysis pattern of fish muscle in which there is always abort 20o/o of the total nitrogen remaining insoluble even when a further amount of enzyme is added (Mackie, 1982b).Similar results were reported by Shahidi et al. (1995) in the hydrolysis of capelin protein.No increase in the release of soluble protein in capelin hydrolysis was observed when a fi.rrther proteolytic enzyme was added after the hydrolysis reached the stationary phase.They suggested that the hydrolysis was probably inhibited by the hydrolysis products or by cleavage of all susceptible peptide bonds by the enzyme.

CONCLUSIONS
Proteolytic enzymes were extracted from cowtail ray viscera using a mixture of 50o/o propionic and 50% forrnic acids added at the rate of 3% and allowed to hydrolyse for 5 days at 4OC.The liquid silage contained acidic and alkaline active enzvmes which J Su.45"Ct O l0%.4-5t'C: f sYu.60'rC: V l0%, 60oC DH = Dcgrce of hydrolvsis (sec tcxt for details) 15 0 tE showed activity of 0.4 Trypsin Units and 1.0 Pepsin Units, respectively.The ability of these enzymes to hydrolysis fish protein has been demonstrated and may be useful in the production of frsh protein hydrolysates.Optirnum hydrolysis was at 6OC for 3 hour at a ratio of crude enzyme to fish meat of 5% (w/w, with respect to protein content of both the enzyme and fish), program developed by Hadi et aI. (lgg6), IFR Journal Vol.IV No.I, I gg8 Figure l.Hl'drollsis of fish nteat bv silage crude enzyules at different proportion of crude enzymes to meal.pH arrd tenrpcralurc.