Tp Khoa hc - Cụng ngh Thy sn S 2/2016 THONG BAO KHOA HOẽC NGHIấN CU NG DNG ENZYME PROTAMEX THY PHN C TRCH (SARDINELLA GIBBOSA) THU DCH M ENZYMATIC HYDROLYSIS OF HERRING FISH (SARDINELLA GIBBOSA) USING PROTAMEX ENZYME TO PREPARE SOLUBLE PROTEIN SOLUTION Trn Th Bớch Thy1, Th Thanh Thy2 Ngy nhn bi: 09/4/2015; Ngy phn bin thụng qua: 19/01/2016; Ngy duyt ng: 15/6/2016 TểM TT Dch m thy phõn ó c nghiờn cu t protein cỏ trớch bng enzyme Protamex pH t nhiờn vi t l nc/nguyờn liu l 1/1 Kt qu nghiờn cu cho thy iu kin thy phõn thớch hp l: nhit 50oC, t l enzyme - c cht 0,5% (w/v), thi gian gi thy phõn, hiu sut thu hi nit, hm lng nit ammonia v hm lng nit acid amine dch thy phõn thu c ln lt t l 70,73%, 63,04%, 1,57 g/l v 12,88 g/l T khúa: Cỏ trớch, Protamex, dch m thy phõn ABSTRACT The hydrolysis of herring to prepare protein hydrolysate solution was studied The hydrolysis process was carried out using Protamex enzyme at natural pH with a rate of water/material is 1:1 Research results show that the best hydrolysis temperature is 50oC, the most suitable rate of enzyme - substrate is 0,5% (w/v) and the most appropriate hydrolysis time is hours The hydrolysis degree, nitrogen recovery, the content of ammonia nitrogen and the content of amino acid nitrogen obtained in the hydrolysis protein solution are 70,73%, 63,04% 1,57 g/l, and 12,88 respectively Keywords: Enzymatic hydrolysis, protein hydrolysis, herring sh, Protamex I T VN Kh nng khai thỏc hi sn bin Vit Nam khong 2.147.444 tn, ú cỏ ni nh chim 51,13 % tng tr lng cú th khai thỏc Trong cỏc loi cỏ ni nh thỡ t l cỏ trớch chim 16,46% tng tr lng cỏ ni nh (Nguyn Vit Ngha, 2005) Sn lng cỏ trớch chim mt s lng ln nhng kh nng ch bin v tiờu th sn phm ch bin t cỏ trớch cha tng xng vi tim nng Vi thnh phn dinh dng v sn lng cao, cỏc sn phm ch bin t cỏ trớch l cỏ ti ụng lnh, cỏ khụ, úng hp v lm nc mm, em li hiu qu kinh t cha cao Vn nghiờn cu to sn phm giỏ tr gia tng t ngun cỏ trớch nhiu nhng cha c ch bin hp lý l rt cn thit nõng cao hiu qu s dng ti nguyờn, mang li thu nhp nhiu hn cho ngi dõn S dng enzyme thng mi thy phõn cỏ trớch nhm nõng cao hiu qu kinh t v a dng húa cỏc sn phm t loi cỏ ny c coi l mt nhng phng phỏp hiu qu nht Cỏc enzyme c s dng ph bin cỏc nghiờn cu v thy phõn bng enzyme thng l Alcalase, Neutrase, Protamex v Kojizyme (Nguyen v cng s, 2011) Protamex c bit n l loi enzyme cho : Khoa Cụng ngh thc phm - Trng i hc Nha Trang 1, TRNG I HC NHA TRANG 93 Tp Khoa hc - Cụng ngh Thy sn sn phm thy phõn ớt ng nht v ú ó c la chn cho nghiờn cu ny Trong cỏc thụng s sinh húa ca quỏ trỡnh thy phõn thỡ mc thy phõn (DH) l mt nhng thụng s quan trng nht vỡ nú trc tip nh hng n chiu di peptide, giỏ tr dinh dng, v cỏc tớnh cht cm quan ca sn phm thy phõn Hn na, DH t l thun vi tan ca thy phõn v ú tỏc ng n kh nng tiờu hoỏ ca cỏc protein (Nguyen v cng s, 2011) Ngoi ra, thụng s v hiu sut thu hi protein, hm lng NH3 cng khỏ quan trng vỡ chỳng cung cp thụng tin hu ớch v sn phm thy phõn Nghiờn cu tỡm ch thy phõn thớch hp thu dch m hũa tan giu acid amine t protein cỏ trớch l c s quan trng tip tc phỏt trin sn xut nhiu dũng sn phm giỏ tr gia tng nh: cỏc loi nc chm cao cp, b sung dinh dng cho nhiu loi thc phm, ng dng nụng hc, y dc II VT LIU V PHNG PHP NGHIấN CU Vt liu nghiờn cu 1.1 Cỏ trớch i tng cỏ trớch c thu mua ti cng cỏ Hũn R - Thnh ph Nha Trang Cỏ cũn S 2/2016 ti nguyờn, sỏng búng, mựi c trng, khụng b dp nỏt tn thng, kớch c 19 ữ 20 con/kg, cỏ c ra, loi b cht, bo qun v chuyn v phũng thớ nghim bng thựng xp cỏch nhit ữ 4oC Ti phũng thớ nghim, m bo tớnh ng nht, cỏ c ra, rỏo, xay nh (bi mỏy nghin tht TA57/ DSN 947000 vi mc ng kớnh l sng l 4,5mm), trn u, phõn chia, bao gúi bng bng bao PA hỳt chõn khụng, lnh ụng v bo qun nhit -20 2oC 1.2 Enzyme Protamex Protamex c mua ti cụng ty Novozyme (an Mch), Thnh ph H Chớ Minh Protamex thuc nhúm endopeptidase, cú ngun gc t vi khun Bacillus, c t chc FAO/WHO cho phộp s dng Protamex cú hot ghi trờn nhón l 1,5 AU (Anson Units)/g, hot ng thớch hp khong pH = 5,5 ữ 7,5 v to = 45 ữ 65C Protamex cú th b mt hot tớnh 30 phỳt ti 55oC (122oF) pH bng v 10 phỳt ti 85oC (185oF) pH bng Nhit bo qun tt nht ca Protamex l ữ 5oC Phng phỏp nghiờn cu 2.1 S quy trỡnh v b trớ thớ nghim Hỡnh S quy trỡnh v b trớ thớ nghim 94 TRNG I HC NHA TRANG Tp Khoa hc - Cụng ngh Thy sn S 2/2016 - Nguyờn liu cỏ trớch: l mu c thu v TCVN 3703:2009; Hm lng nit tng s: x lý nh mc 1.1, ró ụng ữ C, 15 gi theo TCVN 3705-90; Hm lng NH3: theo o - Cỏc thụng s thớch hp c xỏc nh bng thc nghim c in: xỏc nh thụng s th nht bng thớ nghim c nh cỏc thụng s khỏc (da trờn s k tha), cho thụng s cn tỡm bin i tỡm giỏ tr thớch hp Sau tỡm c thụng s th nht thỡ c nh thụng s ny v lm tng t tỡm thụng s th hai Tip tc nh vy n tỡm c tt c thụng s cn tỡm Trong quỏ trỡnh lm thớ nghim, hot enzyme c kim tra thng xuyờn, kt qu cho thy hot enzyme hu nh khụng thay i theo thi gian bo qun (thi gian tin hnh nghiờn cu tng i ngn), nờn thụng s t l emzyme/c cht c tớnh theo %.- Xỏc nh nhit thy phõn thớch hp: b trớ ttp = 40 ữ 60oC, bc nhy = 5oC; t l enzyme - c cht (E/S) = 0,2%, thi gian gi, W/NL = 1/1, pH t nhiờn ca cỏ - Xỏc nh t l enzyme c cht: b trớ TCVN 9215:2012; Hm lng nit acid amine: theo phng phỏp formon; Thnh phn axớt amin c phõn tớch theo Kechaou v cng s (2009).; thy phõn DH c xỏc nh bng phng phỏp DNFB theo Nguyen v cng s (2011); HSTH = Lng nit tng s sn phm thy phõn (g) ì 100/Lng nit tng s nguyờn liu em thy phõn (g) 2.3 Phng phỏp x lý s liu Mi thớ nghim c thc hin song song ba ln, mi ln ba mu S liu c x lý bng phn mm thng kờ SPSS 16.0, tớnh toỏn trờn phn mm Microsoft Ofce Excel 2007 (giỏ tr ca p < 0,05 c xem l cú ý ngha v mt thng kờ) III KT QU NGHIấN CU V THO LUN Kt qu xỏc nh thnh phn húa hc c bn ca cỏ trớch E/S = 0,1 ữ 0,7%, bc nhy = 0,1%, nhit Bng Thnh phn húa hc c bn tỡm c thớ nghim trc, thi gian ca cỏ trớch gi, W/NL = 1/1, pH t nhiờn ca cỏ Xỏc nh thi gian thy phõn thớch hp: b Thnh phn T l (%) so vi lng t Nc 73,87 0,01 E/S tỡm c thớ nghim trc, W/NL = 1/1, Protein 19,25 0,04 pH t nhiờn ca cỏ Lipid 2,53 0,03 Tro 0,96 0,01 trớ T = ữ gi, bc nhy = gi; nhit , - Bt hot enzyme 95 C vũng o 15 phỳt - Cỏc ch tiờu ỏnh giỏ: thy phõn (DH), hiu sut thu hi nit (HSTH), m ammoniac (NH3) - Thụng s c chn l thớch hp thớ nghim phi tha tt nht iu kin Bng cho thy cỏ trớch cú hm lng protein tng s cao (19,25%), giỏ tr ny tng ng vi hm lng protein cỏ trớch mt s nghiờn cu khỏc (Bruce, 1924; Shigeo, 1958) cao hn so vi mc (17ữ21%), ng thi: DH cao nht, HSTH cao nht, NH3 cao hn hn so vi mt s loi thy sn khỏc 2.2 Phng phỏp phõn tớch hm lng lipid thp (2,53%), xp vo loi cỏ mc hp lý m: theo TCVN 3700-90; Tro: phng phỏp nung 600oC; Hm lng lipid: theo nh sũ (8ữ9%), moi (13ữ16%) v c (11ữ12%); gy rt thớch hp cho vic sn xut dch m thy phõn TRNG I HC NHA TRANG 95 Tp Khoa hc - Cụng ngh Thy sn S 2/2016 Kt qu xỏc nh cỏc thụng s thớch hp cho quỏ trỡnh thy phõn protein ca cỏ trớch bng enzyme Protamex 2.1 Kt qu nh hng ca nhit n hiu qu thy phõn bng enzyme Protamex Hỡnh nh hng ca nhit thy phõn n DH Hỡnh nh hng ca nhit thy phõn n HSTH Kt qu th hin trờn hỡnh 2, 3, cho thy nhit thy phõn cú nh hng ln n DH ( thy phõn) Khi tng nhit thy phõn t 40oC n 50oC thỡ mc DH tng ỏng k t 46,5% n 57,7% Giỏ tr DH ny gim ỏng k nhit tng lờn 55oC v 60oC Xu hng ny cng xy tng t vi HSTH c th hin trờn hỡnh Theo ú, tng nhit t 40oC n 50oC thỡ HSTH tng ỏng k t 40,69% n 50% Sau ú, giỏ tr ny gim nh tng nhit lờn 55oC (46,54%) v gim ỏng k tng nhit lờn 60oC (38,53%) Kt qu ny cng tng ng vi kt qu thy phõn t cỏ hi Atlantic (Liaset v cng s, 2001) Khỏc vi trng hp DH v HSTH, kt qu nghiờn cu cho thy hm lng nit amoniac gim ỏng k tng nhit t 40oC n 50oC (1,53 g/l - 1,19 g/l), tip tc gim tng nhit lờn 55oC v 60oC (hỡnh 4) iu ny cú th gii thớch tng nhit thy phõn thỡ tc phn ng cng tng lờn Hỡnh nh hng ca t l E/S n DH 96 TRNG I HC NHA TRANG Hỡnh nh hng ca nhit n hm lng NH3 cỏc phõn t enzyme cú ng nng ln hn, tng cng kh nng tip xỳc gia enzyme Protamex v c cht, ú quỏ trỡnh thy phõn s c tng cng v t cc i ti nhit ti thớch ca enzyme Protamex l 50oC Tuy nhiờn tng nhit thy phõn vt quỏ 50oC, hot tớnh ca enzyme Protamex s b gim v ng thi enzyme cng b bin tớnh ti nhit ny Kt qu giỏ tr DH v hiu sut thu hi nit gim xung Khi nhit tng t 40oC n 60oC thỡ hm lng nit amoniac gim iu ny l khong nhit ny, s tng nhit lm c ch hot ng ca vi sinh vt nờn hm lng nit amoniac gim T kt qu phõn tớch trờn cho thy nhit thy phõn thớch hp cho quỏ trỡnh thy phõn cỏ trớch l 50oC 2.2 Kt qu nh hng ca t l enzyme c cht (E/S) n hiu qu thy phõn bng enzyme Protamex Hỡnh nh hng ca t l E/S n HSTH Hỡnh nh hng ca t l E/S n hm lng NH3 Tp Khoa hc - Cụng ngh Thy sn Hỡnh 5, 6, cho thy tng t l enzyme t 0,1% - 0,5% thỡ giỏ tr DH tng mt cỏch ỏng k t 53,3% - 65,6% Tuy nhiờn giỏ tr DH tng khụng ỏng k E/S tng t 0,5% n 0,7% Kt qu ny cng c ghi nhn bi hiu sut thu hi nit (hỡnh 5) Khi tng E/S t 0,1% n 0,5% thỡ HSTH tng mt cỏch ỏng k t 45,41% n 56,57% Khụng cú s khỏc cú ý ngha thng kờ v s thu hi nit cỏc mu t l enzyme 0,5%, 0,6% v 0,7% Bờn cnh DH v HSTH thỡ hm lng nit amoniac dch thy phõn cng l mt thụng s cn quan tõm Hỡnh ch nh hng ca E/S n hm lng nit amoniac dch thy phõn Nhỡn chung, tng E/S thỡ hm lng nit amoniac cú xu hng tng Tuy nhiờn, t l enzyme t 0,1% n 0,5% thỡ hm lng nit amoniac tng khụng ỏng k iu ny cng cho thy hm lng nit amoniac tt c cỏc mu mc thp Hỡnh nh hng ca thi gian n DH S 2/2016 chim t 7,4% n 9,48% Cỏc cụng trỡnh nghiờn cu trc õy cng ó cho thy cú s hũa tan nit di tỏc dng ca enzyme quỏ trỡnh thy phõn v t l thu hi nit sn phm thy phõn, cng nh s ct t cỏc liờn kt peptide tng theo nng enzyme (Wachirattanapongmetee v cng s, 2009; Motamedzadegan v cng s, 2010) DH tng tng E/S cú th c gii thớch l tng t l enzyme thỡ tỏc dng ct mch s tng dn n DH tng Khi mc DH tng dn n HSTH tng (Liaset v cng s, 2002; Haslaniza v cng s, 2010) T kt qu phõn tớch trờn cho thy t l enzyme so vi c cht l 0,5% thỡ thy phõn v hiu sut thu hi nit cao v hm lng nit amoniac mc cho phộp Vỡ vy, chn t l enzyme thớch hp l 0,5% 2.3 Kt qu nh hng ca thi gian n hiu qu thy phõn bng enzyme Protamex Hỡnh nh hng ca thi gian n HSTH Hỡnh 8, 9, 10 cho thy tng thi gian thy phõn t gi lờn gi thỡ giỏ tr DH tng ỏng k theo thi gian thy phõn C th, gi u thy phõn tng t 62,67 % (3 gi) lờn n 70,87% (6 gi) Sau ú, DH tng chm mc dự thi gian thy phõn kộo di thờm gi na Khụng cú s khỏc cú ý ngha v thy phõn gia cỏc mu vi thi gian gi v gi Cỏc cụng trỡnh nghiờn cu trc õy cng ó ch rng DH tng theo thi gian thy phõn (Souissi v cng s, 2007; Chun v cng s, 2006; Amiza v cng s, 2012; Hỡnh 10 nh hng ca thi gian hm lng NH3 Wachirattanapongmetee v cng s, 2009; Ovissipour v cng s, 2010; Shamloo v cng s, 2012) iu ny c gii thớch nh sau: Thi gian thy phõn phi m bo enzyme cú th phõn ct cỏc liờn kt c cht, to c sn phm cui cựng mong mun theo mc tiờu ca ti Thi gian tỏc ng kộo di thỡ enzyme cú iu kin thy phõn protein cỏ trớch trit Nhng nu kộo di thi gian thy phõn quỏ mc s to iu kin cho vi sinh vt gõy thi hot ng lm sn sinh nhiu sn phm cp thp nh: NH3, H2S, TRNG I HC NHA TRANG 97 Tp Khoa hc - Cụng ngh Thy sn indol, scaptol nh hng n cht lng sn phm i vi nh hng ca thi gian thy phõn n HSTH th hin hỡnh 8, kt qu nghiờn cu cho thy, thi gian thy phõn tng t gi n gi thỡ HSTH tng ỏng k t 48,35% lờn n 60,66% iu ny phự hp vi cỏc cụng trỡnh nghiờn cu trc õy cng ó cho thy s hũa tan nit (hay protein) di tỏc dng ca enzyme quỏ trỡnh thy phõn tng theo thi gian thy phõn (Motamedzadegan v cng s, 2010; Haslaniza v cng s, 2010) Khi tip tc tng thi gian hn gi thỡ khụng lm tng HSTH Hm lng nit amoniac tng theo thi gian thy phõn C th t gi n gi thy phõn thỡ hm lng nit amoniac tng nh khụng ỏng k (hỡnh 9) Thi gian thy phõn tng dn n cỏc liờn kt peptide b ct mch cng nhiu, S 2/2016 to nhiu peptide v acid amine Vỡ vy, tng thi gian thy phõn thỡ DH v HSTH tng Sau ú, cng tng thi gian thy phõn thỡ DH tng chm Hm lng nit amoniac cú xu hng tng theo thi gian thy phõn l thi gian thy phõn cng di thỡ vi sinh vt gõy thi cng cú iu kin hot ng hn nờn hm lng nit amoniac to cng nhiu NNH3/NTS l 9,36% n 10,21% thp hn nhiu so vi TCVN 5107: 2003 ca sn phm nc mm c bit (NNH3/NTS < 20%) T kt qu phõn tớch trờn cho thy thi gian thy phõn thớch hp cho quỏ trỡnh thy phõn cỏ trớch bng enzyme Protamex l gi Xỏc nh hiu qu ca ch thy phõn tỡm c p dng th nghim ch thy phõn thớch hp, kt qu c th hin bng v Bng ỏnh giỏ cht lng dch m thy phõn protein cỏ trớch STT Ch tiờu Cm quan Húa hc Kt qu Mu sc Cú mu vng nht Mựi Mựi c trng ca dch m thy phõn, d chu V Hi cú v ng Trng thỏi Dch lng Naa 12,88 g/l NTS 15,41 g/l NNH3 1,57 g/l Naa/NTS 83,58% Bng cho thy, dch m thy phõn thu thp hn nhiu so vi TCVN nc mm cao c cú mu vng nht, trong, cú mựi c m (NNH3/NTS 20%) Sn phm dch m trng ca dch m thy phõn t cỏ Hm thy phõn l bỏn thnh phm, sn xut lng nit tng s v nit acid amine ca dch thnh phm hon ton cú th kim soỏt c thy phõn l cao, t l nit acid amine trờn nit lng NNH3/NTS mc cho phộp nờn dch m tng s chim ti 83,58% NNH3/NTS =10,19% c ng dng lnh vc thc phm 98 TRNG I HC NHA TRANG Tp Khoa hc - Cụng ngh Thy sn S 2/2016 Bng Thnh phn acid amine ca dch m thy phõn protein cỏ trớch Tờn acid amine Hm lng (g/l) Tờn acid amine Hm lng (g/l) Alanine 0,79 Aspartic acid 0,39 Glycine 0,67 Methionine* 0,57 Valine* 0,98 4-Hydroxyproline Leucine* 2,01 Glutamic acid 0,78 Isolecine* 0,88 Phenylalanine* 0,98 Threonine* 0,68 Lysine* 1,34 Serine 0,69 Histidine* 0,88 Proline 0,35 Hydroxylysine Tyrosine 0,67 Tryptophan 0,22 TAA 12,88 TEAA 8,32 TEAA/TAA 64,6% (*): Acid amine khụng thay th, TAA (Total amino acids): Tng acid amine, TEAA (Total essential amino acids): Tng acid amine khụng thay th Kt qu phõn tớch thnh phn acid amine bng cho thy, dch m thy phõn protein cỏ trớch cú giỏ tr dinh dng cao, giu acid amine khụng thay th (64,6%) Cỏc acid amine chim hm lng cao dch m thy phõn protein cỏ trớch l: Leucine (2,01 g/l), Lysine (1,34 g/l), kt qu ny trựng vi nghiờn cu thy phõn cỏ nc s bi Protamex ca Chun v cng s (2006) Mt s nghiờn cu khỏc v thy phõn u cỏ ng (Nguyn Th M Hng, 2012) v thy phõn u cỏ hi (Sathivel v cng s, 2005) cng cho thy dch m thy phõn thu c t nghiờn cu ca cỏc tỏc gi ny cú hm lng acid amine khụng thay th cao IV KT LUN Ch thy phõn thớch hp thu dch m giu acid amine t protein ca cỏ trớch bng enzyme Protamex l: nhit 50oC, t l enzyme - c cht 0,5% v thi gian gi Sau gi thy phõn, thy phõn t 70,87%, hiu sut thu hi t 60,66% Sn phm thy phõn cú giỏ tr dinh dng cao, giu acid amine khụng thay th (chim 64,6% tng lng acid amine) Sn phm thu c cú th c s dng sn xut nc chm, bt dinh dng hoc b sung vo thc n chn nuụi TI LIU THAM KHO Ting Vit Nguyn Th M Hng, 2012 Sn xut sn phm thy phõn protein t u cỏ ng võy vng bng protease thng mi Tp Khoa hc - Cụng ngh Thy sn, s 2/2012, tr 25-30 Nguyn Viờt Ngha, 2005 Nghiờn cu tr lng v kh nng khai thỏc cỏ ni nh (ch yu l cỏ nc, cỏ trớch, cỏ bc mỏ, ) bin Vit Nam Bỏo cỏo khoa hc, Vin Nghiờn cu Hi sn TRNG I HC NHA TRANG 99 Tp Khoa hc - Cụng ngh Thy sn S 2/2016 Ting Anh Amiza, M.A., Kong, Y.L., Faazaz, A.L., 2012 Effects of degree of hydrolysis on physicochemical properties of Cobia (Rachycentron canadum) frame hydrolysate International Food Research Journal, 19(1): 199-206 Bruce, J R., 1924 Changes in the Chemical Composition of the Tissues of the Herring in Relation to Age and Maturity Biochem J 1924; 18(3-4): 469485 Chun, C., Mouming, Z., Xiaofang, Z., Jiaoyan, R., 2006 Protein degradation of extensive enzymatic hydrolysis of decapterus maruadsi Transactions of the CSAE, Vol.22 No.1 Haslaniza, H., *Maskat, M Y., Wan Aida, W M and Mamot, S., 2010 The effects of enzyme concentration, temperature and incubation time on nitrogen content and degree of hydrolysis of protein precipitate from cockle (Anadara granosa) meat wash water International Food Research Journal 17: 147-152 Kechaou, E.S., Dumay, J., Donnay-Moreno, C., Jaouen, P., Gouggou, J.P., Bergộ, J.P., Amar, R.B., 2009 Enzymatic hydrolysis of cuttlesh (Sepia of cialis) and sardine (Sardina pichardus) viscera using commercial proteases: Effects on lipid distribution and amino acid composition Journal of Bioscience and Bioengineering.107(2): 158-164 Liaset, B., Nortvedt, R., Lied, E., Espe, M., 2002 Studies on the nitrogen recovery in enzymatic hydrolysis of Atlantic salmon (Salmo salar, L.) frames by ProtamexTM protease Process Biochemistry, 37: 1263-1269 Motamedzadegan, A., Davarniam, B., Asadi, G., Abedian, A., 2010 Optimization of enzymatic hydrolysis of yellown tuna Thunnus albacares viscera using Neutrase Int Aquat Res, 2: 173-181 10 Nguyen, H.T.M., Sylla, K.S.B., Randriamahatody, Z., Donnay-Moreno, C., Moreau, J., Tran, L.T., Bergộ, J.P 2011 Enzymatic hydrolysis of yellown tuna (Thunnus albacares) by-products using Protamex protease Food Technology and Biotechnology 49 (1): 48 - 55 11 Ovissipour, M., Benjakul, S., Safari, R., Motamedzadegan, A., 2010 Fish protein hydrolysates production from yellown tuna Thunnus albacares head using Alcalase and Protamex Int Aquat Res, 2: 87-95 12 Shamloo, M., Bakar, J., Mat Hashim, D and Khatib, A., 2012 Biochemical properties of red tilapia (Oreochromis niloticus) protein Hydrolysates International Food Research Journal, 19(1): 183-188 13 Santhivel, S., Smiley, S., Prinyawiwatkul, W., Bechtel, P.J., 2005 Functional and Nutritional Properties of Red Salmon (Oncorhynchus nerka) Enzymatic Hydrolysates Journal of Food Science, 70(6): 401-406 14 Shigeo, S., 1958 Chemical studies on herring meat Hokkaido University Collection of Scholarly and Academic Papers, 8(4)_P319-345 15 Souissi, N., Bougatef, A., Triki-Ellouz, Y., Nasri, M., 2007 Biochemical and Functional Properties of Sardinella (Sardinella aurita) By-Product Hydrolysates Food Technol Biotechnol 45 (2) 187194 16 Wachirattanapongmetee, K., Wachirattanapongmetee, K., Thawornchinsombut, S., Pitirit, T., Yongsawatdigul, J., Park, J.W., 2009 Functional Properties of Protein Hydrolysates Prepared from Alkali-Aided Protein Extraction of Hybrid Catsh Frame Trends Research in Science and Technology, (1), 71-81 100 TRNG I HC NHA TRANG