Proteins from Liza suboiridis, a marine fish, were hydrolyzed using Alcalase 2.5L. The parameters studied were pH, temperature, ratio of enzyme to substrate concentration, and substrate concentration.

It was observed that the optimum conditions for the hydrolysis were pH 9 at 40°C, using an enzyme to substrate ratio of 16 Au/kg and a substrate concentration of 5%

R., Ahmad

Yu, S. Y.

Universiti Putra Malaysia Institutional Repository

251Asian Fisheries Science 10(1998):251-257Asian Fisheries Society, Manila, PhilippinesHydrolysis of Proteins from Liza subviridisS.Y. YU AND R. AHMADFaculty of Food Science and BiotechnologyUnioersiti Putra Malaysia43400 UPM Serdang, Selangor.AbstractProteins from Liza suboiridis, a marine fish, were hydrolyzed using Alcalase 2.5L.The parameters studied were pH, temperature, ratio of enzyme to substrate concentration,and substrate concentration.It was observed that the optimum conditions for the hydrolysis were pH 9 at 40°C,using an enzyme to substrate ratio of 16 Au/kg and a substrate concentration of 5%.IntroductionProteolytic modification of food proteins is a means for improving the useof this valuable food source. For decades, autolysates of fish have providedadded flavor and nutrients to the cuisines of Southeast Asia (McIver et al.1982). Among the uses of modified protein by chemical and enzymic hydrolysisare : to improve nutritional characteristics, retard deterioration, impart texture,increase or decrease solubility, add foaming or coagulation properties, addemulsifying capacity, prevent undesired interactions, remove off-flavors orodors, and remove toxic or inhibitory ingredients (Lahl and Grindstaff 1989;Feeney 1986; Yu and Tan 1990).The functional potential of fish hydrolysates has been studied by Millerand Groninger (1976) and Hoyle and Merrit (1994). Hydrolysis has been em-ployed as a possible means of upgrading the use of underutilized fish by Ghoshet al. (1991), Lewis (1981), Gildberg et al. (1989), Skorupa and Sikorski (1993)and Yu and Tan (1990), and as a method of recovering protein from processingby-products (Tarky et al. 1973; Jacobsen and Rasmussen 1984). Its potential foraccelerating the fermentation process of fish sauce has been ascertained fromthe work of Beddows et al. (1976), Beddows and Ardeshir (1979) in 'budu' fishsauce, and Raksakulthai et al. (1986) in fish sauce from capelin. Recent tech-nologies in fish processing such as mechanical deboning has made it possibleto use bony and small fish, where before such traits would have been a hin-drance to their utilization.In Malaysia, where such fish products as 'budu' and 'keropok' (fish crack-ers) are widely accepted, the potential exists for the use of fish hydrolysate inthe production of novel products or its incorporation in food products to upgradefunctional properties without the inherent fishy odor.252L. sub viridis is found among trash fish which are almost always con-verted to fishmeal. The use of L. subviridis as a substrate for hydrolysis willimprove the use of this low-value fish.This paper describes the hydrolysis of the proteins of L. subviridis usingthe enzyme Alcalase 2.5LMaterials and MethodsRaw MaterialsAlcalase 2.5L, a commercial protease isolated from the bacteria Bacilluslicheniformis, was a gift from Novo Industries AlS, Denmark. The declaredactivity of the enzyme was 2.5Aulg. One Au is equal to 430 InternationalUnits at 30°C (Adler-Nissen 1986).Fresh L. subviridis were obtained from a local supplier and transported tothe laboratory in ice. The fishes were degutted, deboned and washed in chilledwater before they were frozen for storage at -20°C until further use.The chemicals used were of analytical grade. The hydrochloric acid, petro-leum ether and copper sulphate were from BDH (UK), the sulphuric acid, so-dium hydroxide and selenium dioxide from Merck (Germany), the boric acidfrom R & M Chemicals (UK) and the sodium sulphate was from May andBaker (UK).Proximate AnalysesProtein contents were determined using the Kjeldhal distillation technique(pearson 1973). The digest was distilled using lOml of 40% NaOH and titratedwith 0.05N H2S04. The amount of sample used was 0.lg-0.15g. The conver-sion factor used was 6.25.Crude fat content was determined by extracting fat from a previously-dried sample using the Soxhlet method, with extraction time of 8 hours on anelectrothermal unit (Gerhardt, Germany). Petroleum ether was used as thesolvent. The fat content was calculated on a dry sample basis (pearson 1973).Ash content was determined by placing dried samples into a furnace set at450-500°C until a whitish residue was obtained. The ash content was taken asa percentage of the dried sample (pearson 1973).Moisture content of the fish protein was determined by placing the samplein an oven at 106°C until a constant weight was obtained. The moisture con-tent was calculated as a percentage of the dried sample (pearson 1973).The hydrolysis processHydrolysis was carried out using the pH-Stat method of Adler-Nissen(1986). The fish mince was homogenized (Braun no. 4249, Germany) with anappropriate amount of water at a low speed for 1 min. The enzyme in solutionwas added to the equilibrated fish slurry and hydrolysis carried out with con-tinuous stirring. Equilibration of pH was done by adding IN HCI or 3NNaOH and the temperature of the mixture was maintained by the use of awaterbath (Polytherm Type PY2, US). pH values were monitored using an253Orion 420 A2 Bench Top pH-meter (Ll.S)with an automated temperature-com-pensated electrode.The hydrolysis parameters used were based on literature (Adler-Nissen1986), where for Alcalase, the optimum conditions using soya isolate is S=8%,E/S=12 Au/kg, pH 9.0 and a temperature of 50°C. From these settings, theparameters were varied in turn to find the optimum conditions for L.sub viridis.Degree of hydrolysisThe total number of peptide bonds of fish protein was taken as 8.6 meq/g as reported by Lalasidis et al. (1978).The approximate value of pk of the amino acids of polypeptides is 7.5 at25°C (Steinhardt and Beychock 1964). Using the Gibbs-Helmholtz equation,the pk value at 50°C can be calculated as approximately 7. This means thatthe -NH2 groups are fully titrated and the equivalents of peptide bonds cleavedare equal to the equivalents of base consumed. Using the calculated values oflIocwhere ocis the average degree of dissociation of the -NH2 groups (basedon temperature)(Adler-Nissen 1986), the DH (degree of hydrolysis) is calculateddirectly from the base consumption in a pH stat hydrolysis technique usingthe following equation :DH = B x Nb x 11x lIMP x 1/h tot. x 100%where,B = base consumption in mLNb = normality of base (NaOH)oc= average degree of dissociation of the -NH2 groups(based on temperature) Adler-Nissen, 1986MP = mass of protein (N X 6.25) in gh.tot. = total number of peptide bonds in the protein substrateHydrolysis parametersFour process conditions were varied to ascertain optimum parameters forhydrolysis. These conditions were pH, temperature, enzyme to substrate ratio,and substrate concentration.The pH values used were 7, 8, 9 and 10. The three temperatures testedwere 30, 40 and 50°C while the enzyme to substrate ratios used were 12, 16and 20 Au/kg. The three substrate concentrations used were 5, 6 and 7%. Thedata were statistically analyzed using Duncan's multiple range test using SASsoftware (SAS Institute Inc., North Carolina, U.S).Results and DiscussionL. subviridis has 4.7% of crude fat, a protein content of 19.3%, an ashcontent of 1.9% and a moisture content of 77%.Temperature effects on the hydrolysis of L. sub viridis was carried out at30, 40 and 50°C (Figure 1). At 30°C, the hydrolysis was significantly lower (at5% confidence level) from that of 40°C and 50°C. At 40 and 50°C, the254hydrolyses curves were not significantly different at the 5% level of confidence,which meant that a working temperature of 40°C would be a more economicalchoice.The enzyme Alcalase is basically an alkaline enzyme, although it hasbeen known to work as effectively at a low pH of 6.3 (Jacobsen and Lykke-Rasmussen 1984). The effect of pH on the hydrolysis was studied at pH 7, 8,2015105oo 100 300 400200time (minutes)-- 30·C .... 40·C .......50·CHydrolysis conditions: ph 9, substrate concentration: 5%, enzyme to to substrate ratio:12 Au/kgFig. 1. The effect of temperature on the hydrolysis of L. sub viridis.time (minutes).....pH 7 +pH 8 *pH 9 BpH10Hydrolysis conditions: temperature: 40"C, substrate concentration: 5%, enzyme tosubstrate ratio: 16 Au/kgFig. 2. The effect of pH on the hydrolysis of L. suboiridis.9 and 10 (Figure 2). pH 9.0 gave a hydrolysis that was significantly higher atthe 5% level than that of the other pHs, including the higher pH of 10. Thisobservation could be typical of many enzyme-catalyzed reactions where pHcould cause changes in the reactants, among them being the possible denatur-ation of the protein structure of the enzyme or the disturbance of the ioniccharacter of the substrates which, in turn, could affect the ability of the sub-strate to bind to the enzyme (Boikess et al. 1986).time (minutes).....12 AU/kg +16 AuJkg '*20 AuJkgHydrolysis conditions: ph 9, temperature: 40"C, substrate concentration: 5%Fig. 3. The effect of enzyme to substrate ratio on the hydrolysis of L. suboiridis.20~ 15f/)'(i)>-"0-a>- 10s:'0G)G)6-5G)"0time (minutes)....5% +6% *7%Hyrolysis conditions: temperature: 40·. ph 9, enzyme to substrate ratio: 16 Au/kgFig. 4. The effect of substrate concentration on the hydrolysis of L. subvil'idis.255256The hydrolysis profile of L. sub viridis, as shown in Figure 3, was similarfor enzyme to substrate ratios of 16 and 20 Au/kg but were both significantlydifferent at the 5% level from that of 12 Au/kg. Hence, the lower ratio of 16Au/kg was chosen for further hydrolysis work.The substrate concentration of the hydrolysis mixture was constrained bythe thickness of the fish slurry which became too thick at more than 5% levelfor effective stirring. This is likely to be due to the good gelling capability offish protein. Stirring was difficult only at the initial stage. However, once theenzyme started to hydrolyze the protein, the slurry became thinner and easierto stir. Figure 4 shows the effect of the three substrate concentrations on thehydrolysis profile of L. subviridis. The profiles showed that the hydrolysis atthe three substrate concentrations were not significantly different at 5% levelof confidence.ConclusionPreliminary studies on the hydrolysis of L. sub viridis using the enzymeAlcalase 2.5L show that the optimum parameters for further hydrolysis workare pH 9, a temperature of 40°C and an enzyme to substrate ratio of 16 Au/kgat a substrate level of 5%.Acknow ledgementThis project was funded by the Ministry of Science, Technology and theEnvironment, Malaysia (project no. 03-02-04-0037),and through an STD 3 re-search project (project no. ERBT CT93-0207), sponsored by the EuropeanUnion.ReferencesAdler-Nissen, J. 1986. Enzymic hydrolysis of food proteins. Elsevier Applied Science Pub-lishers, London and New York.Beddows, C.G., M. Ismail and K.H. Steinkraus. 1976. The use of bromelain in the hydroly-sis of mackerel and the investigation of fermented fish aroma. Journal of Food Tech-nology. 1:379-388.Beddows, C.G. and A.G. Ardeshir. 1979. The production of soluble fish protein solution foruse in fish sauce manufacture. 1. The use of added enzymes. Journal of Food Tech-nology 14:603-612.Boikess, R., K. Breslauer and E. Edelson. (eds.) 1986. Elements of Chemistry - Generalorganic and biological. Prentice-Hall, New Jersey.Feeney, R.E. 1986. Tailoring proteins for food and medical uses: state of the art and inter-relationships. In Protein tailoring for food and medical uses. (Eds. R.E. Feeney andJ.R. Whitaker). 1-40. Marcel Dekker Inc., New York.Ghosh, S.W., M.D. Alur and D.P. Nerkar. 1991. Hydrolysis of fish protein by Bacillusmegateriuni cells immobilized in radiation-induced polymerized wood. Journal of FoodScience and Technology 19(2):89-90.Gildberg, A., I. Batista and E. Strom. 1989. Preparation and characterization of pep tonesobtained by a two-step enzymatic hydrolysis of whole fish. Biotechnology and AppliedBiochemistry. 11:413-423.Hoyle, N.T. and J.H. Merrit. 1994. Quality of fish protein hydrolysates from herring(Clupea harengus). Journal of Food Science, 56(1):76-79.Jacobsen, F. and O. Lykke-Rasmussen. 1984. Energy-savings through enzymatic treatmentof stickwater in the fish meal industry. Process Biochemistry 19:165-169.257Lahl, W.J. and D.A. Grindstaff. 1989. Species and seasonings: Hydrolyzed proteins. Pro-ceedings of the 6th SIFST Symposium on Food Ingredients - Applications, Status andSafety, 1989. Singapore: Singapore Institute of Food Science and Technology, 51-65.Lalasidis, G., S. Bostrom and L.B. Sjoberg. 1978. Low molecular weight enzymatic fish pro-tein hydrolysates: chemical composition and nutritive value. Journal of AgriculturalFood Chemistry 26:751-756.Lewis, N.F. 1981. The isolation of proteins from low priced fish. Fleischwirtsch. 61:9.McIver, R.C., R.I. Brooks and G.A. Reinceccius. 1982. Flavour of fermented fish sauce.Journal of Agricultural Food Chemistry. 30:1017-1020.Miller, R. and H. Groninger. 1976. Functional properties of enzyme-modified acylated fishprotein derivatives. Journal of Food Science. 4:268-272.Pearson, D. 1973. Laboratory techniques in food analysis. Butterworth and Co. Ltd. London,31-37.Raksakulthai, N., Y.Z. Lee and N.F. Haard. 1986. Effect of enzyme supplementants on theproduction of fish sauce from male capelin (Mallotus villosus) Canadian Institute ofFood Science and Technology Journal 19(1):28-33.Skorupa-K. and Z.E. Sikorski. 1993. The potential for utilization of low-grade fish materialfor the production of microbiological peptones. Przemysl-Spozywezy. 47(6):159-161.Steinhardt, J. and S. Beychok. 1964. Interaction of proteins with hydrogen ions and othersmall ions and molecules. In : The proteins. (ed. H. Neurath) pp. 139-304. AcademicPress, New York.Tarky, W., O.P. Agarwala and G.M. Pigott. 1973. Protein hydrolysate from fish waste. Jour-nal of Food Science. 38:917-918.Yu, S.Y. and L.K. Tan. 1990. Acceptability of crackers (keropok) with fish protein hydroly-sate. International Journal of Food Science and Technology. 25:204-208.Manuscript "eceived 28 November 1996; accepted 13 October 1997.

Hydrolysis of proteins from Liza subviridis

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Hydrolysis of proteins from Liza subviridis

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