KHOA HQC CdNG NGHE

KHOA HQC CdNG NGHE

IXIGHIEIXI CLUJ K H A IXlAlXIG T H U Y PHAIXI PROTEIIXI TACH CHIET TLT PHIJ P H A M CA TRA {Pangasius hypaphthatmus]

BAIXIG EIXIZYM iXIEUTRASE io

^{o f I .}

Nguyin Thi T h V , Nguyin Cdng H4*

T6MTAT

Dl nang gia tri sit dung ngudn phu phdm trong quy trinh san xuat ca Tra phi le cong nghi&p. nghien cihi kha nang thuy phan protein dugc tach chiet tir phu p h ^ ca Tra frong do cd su dung enzym neutrase duoc thuc hien. Ket qua cho thay hai tham sd dong hoc V,n„ va K„ cua enzym neutrase tr&n co chat protein tir phy pham ca Tra duoc xac dinh lan luot la 0,190 junol tyrosin/phiit va K,„ = 0,254 g protein/10 ml tiiong ung. Vdi ty 16 khdi lupng enzym va co chiit \k 0,5 mg enzym: 0,694 g protein thi tdc do phan ung cua enzym U ldn nhat Khi thdi gian thuy phto keo dai ttr 30 din 240 phiit hieu qua thuy phan tSng nhanh vao giai doan dau va sau dd tang cham 1^ cho din khi flat miic cao nhat tai 240 phut (6,359% theo ty 1^ Tyr^Tyrj va 3,244% theo phuang phap O P ^ , vi vay thoi gian thuy phan hieu qua dupc chon la 240 phut. Ngoai ra, kit qua pban tich protein duoc thuy phan the hien trfin gel SDS-PAGE con cho thay hau hit protein tir phy pham ca Tra da dupc tbuy phan thanh dang peptit phSn tu lugng th^lp cung nhu hOn hop cac axit amm v^

mat do cao trong khoang thoi gian 240 phiit Kit qua nghien ciiu cho thdy co thl thuy phan protein dugc tach chilt tir phu pham ca Tra bang enzym tnmg tinh sir dung lam nguon d^im dl hap thu bd sung cho khau phdn tbirc an frong chan nuoi gia siic.

Tfrkhda: C^Tra, hiiu sudt thuy phin, neutrase, protein thiiy phan, phu pham ca Tra, SDS-PAGE.

1 . BAT VAN BE

Hi^n nay cd rat it nghidn cmi vl chl biln protein thuy phan d l lam thiic Sn gia sue, dac bi?t la ngudn protein tir ngudn phu ph£m ea Tra. d gdc dp dinh dudng, nlu protein diroc thuy phan d dang mach ngan nhu peptit hay axit amm thi kha nang dupc ngudi va vat nudi hap thy vdi hieu su^t cao la cd thl.

Dac bilt, ddi vdi heo eon, trong giai doan mdi sinh khi hd enzym tilu hda chua du manh thi viec bd sung ngudn dam nay se giiip kha nang hip tiiii ciia heo se tdt hon. Theo Abdida^ez va ddng tae gia (2013) thi d l thuy phan protein cd i h l su dung phuong phap hda hpc thdng thudng la dung dung mdi hoac hda chat mgnh, viec sir dung nhu vay Iam san phdm khdng phii hpp trong san xuat ihuc pham, lam giam gia in dinh duong, nghdo eae tinh chat chuc nang va khdng dupc sii dung trong ihuong mai.

Theo Ovissipour va ddng tac gia (2010), nlu su dung enzym d l thiiy phan protein cd till cai thien dac tinh hda ly, tinh chai chiic nang, cam quan va gia iri dinh dirdng. Tren tiiuc i l , cd rilt nhilu loai proteaza va c h l pham thuong mai khac nhau duoc sii dung d l thuy

Dai hpc Can Tho 80

phan protein nhu bromelain, neutrase, alcalase, proiamex hay papain.

Enzym neutrase ed ma sd EC la 3.4.24.28.

Neutrase, mdt san pham ngoai bao ciia vi khuan Bacillus subtihs dupc phan lap 1 ^ ddu tidn vao nhung nam 1950. Neutrase la mdt protease kim lo^ii.

Trin mdi don vi neutrase Udn k i t vdi mdt ion kem v^

bdn ion canxi. Ch^i boat hda la Ca^*, Mg^*, Mn^, Fe^*, Al^. Khi thuc hien qua trinh thuy phdn lien kA peptit, neutrase bat dau ihuy phan tu ddu amin (N- termmal). Do dd, day la enzym thich hpp d l thiiy phan cac protem bien tinh cd ngudn gdc ddng v^t Vdi nhung ly- do ndu tren, muc dich nghidn ciiu cua chling idi la nghien ciiu kha nang thuy phan protein tii phu pham ea Tra bang enzym neutrase nhdm tao ra ngudn dam d l hap tiiii va cd gm iri hon irong vi&

san xudt ihiic an ehan nudi gia siic trong giai do^in ddu sau khi sinh da dupc tiiuc hidn.

t. VAT l i u VA PHUONGPHAP NGi^ CUU 2.1. Chuin bi n g u y ^ v | t li|u

Phu pham ea Tra (dau, vay, xuong va ndi tang) dirpc thu mua tir Nha may Caseamex, iai khu Cdng nghidp Tra Ndc, Can Tho dupc van chuyin v l phdng ihi nghiem cua Bd mdn Cdng n g h | thuc pham, Khoa Ndng nghiep va Sinh hpc iing diing, Trudng Dai hoc NONG NGHIEP VA PHATTRIEN N 6 N G T H 6 N - KY 1 - THANG 11/2015

KHOA HOC CdNG NGHfi Can Thff d l tiln hanh thi nghiem. Ngudn phu pham

nay sau dd dugc loai bd ndi tang, riia sach d l loai bd mau va mo rdi dugrc hap so bp trong 15 phiit va loai bd xuong. Phdn protein thu hdi dugc xay nhuyin, chia thanh nhilu phdn nhd va dupc tru a -20°C d l thue hidn thi nghidm thiiy phan. Enzym neutrase cd ngudn gdc tu Merck. Cae hda chat chuan nhu tyrosin, ca^in, cystein chuan cd ngudn gdc tir Sigma AMch.

2.2. Xac dinh cac tham sd ddng hpc Vn„ va KQ, cua enzym papain irin co ch& protein lir phu phdm cdTra

D l xae dinh hai tham sd ddng hpc V^^ va K^

cua enzym neutrase trdn co chat protein tii phu pham ca Tra, ihi nghidm dupc tiln hanh d dieu kien pH va nhidt dp idi im cua enzym neutrase ldn lupt la 50"C va pH 6,5. Su dung 0,5 mL enzym neutrase tiong dung dich ddm phdt phdt vdi ndng dp la 1 mg/mL, ca ehdt la protein dugc tach chiet tir phu pham ca Tra sau khi ra dong dupc pha trong dung dich dem phdt phat (pH 6,5) vdi ndng dp tang ddn iu 0,1 g din 6 g/10 ml. TTidi gian phan iing duoc tiiuc hien trong 30 phut Kit qua dupc ghi nhan la ham

* lupng tyrosin sinh ra trong 30 phut phan iing.

2 ^ . xac dinh cap ty 1| enzym/ca chA va thdi gian tiitiy phan h i ^ qu4 nhdt

Tir dd thi phuorng irinh Michalis - Menten kit , hpp vdl kit qua thdng ke su khac biei vl tdc dp phan , ling cua enzym tr&n co chat la protem tu phu pham ( ca Tra cd t h l xac dinh dupc ty sd giua ndng dp

\ enzym va co chdt tdi tm tiii nghiem nay. Dl xac dinh ' dupc ndng dp ca chat cao nhat cd t h l dung eho qua trinh thuy phan protein, tien hanh tSng iy sd giua ndng dp enzym va ca chat theo cac he sd 1,2 ,1,4 va ' 1,6. Tir cap E/S tiiich hop, khao sat d cac mdc tiidi ' gian khac nhau de xac dinh tiidi diem ihuy phan dat hieu suat cao nhaL Cac tiii nghiem dugc khao sat vdi 3 Ian lap lai tren cung mpt ngudn nguyen heu.

2.4. Xdc dinh hdm hipng tyrosin sinh ra C i y ^ Ham lupng tyrosm smh ra trong qua trinh tiiuy phan tiieo phuong phap Anson cai tien (Anson, 1938). Phuong p h ^ nay dua ti^n su tiiuy phan protein cazeui bang enzym cd b'ong dich nghi&n eiiu, ' tilp dd lam vd hoai enzym va kit tiia protein chua bi

• thiiy phan bdng dung dich axit tricloraetic (TCi^.

' Dinh hipng tyrosine tao tiianh tiong phan iing tiiiiy phan bang phan iing man vdi thude thu Folm. Dua

vao dd ihi ehuan oia tyrosin d l tinh Iupng san pham do enzym xiic tac tao nln.

2.5. Xdc dinh hdm hiong tyrosin tdng (J^^

Xac dinh ham lupng tyrosin tdng bang phuong phap tiiuy phan bang axit HCl 6 N (ZHU Xian va cs., 2008). Theo dd, can chinh xac 1 g phu pham, them dung dich HCl 6 N tiieo ti Id 1:30 (wrv), tiln hanh ihuy phan d 110"C tiong 24 gid. Hdn hpp sau ihiiy phan dupc loc qua giay lpc, sau do pha bang 30 lan vdi nudc cdt Hiii 5 ml dung dieh da pha loang thdm vao 10 ml NaOH va 3 ml Folin da pha loang 5 lan, Idc manh va de yen 10 phiiL Hdn hgp dupc do d budc sdng 660 nm. Tir dudng ehuan tyrosm cd i h l xac dinh ham lupng tyrosin tdng cd trong mdu nguyen hdu.

2.6. Xdc dinh h i | u sudi thuy phan theo ^ 1|

tyrosin O V r ^ y r t (%))

Hieu suat thuy phan iheo lupng ^ o s i n sinh ra (Tyr,p/Tyr^ dupc tinh theo cdng thiic:

TyTt/Tyrt (90 = (Lupng tyrosine smh ra x 100)/

Luong tyrosine idng

2.7. Xdc dinh h i | u sudt thuy phdn dam amin tiieo phuong phdp OPA

Ham Iupng dam amin sinh ra dupc xac dinh iheo phuong phap OPA (Nielsen, 2001). Theo dd, cac nhdm amin eiia axit amui hoac d dau mui cac peptit se phan ling tgo phiic vdi Ortho- phtiialdialdehyt (OPA) va diihiotiireti)l (DTT) hoac mercaptoethanol ed mang nhdm -SH tu do irong phan tii, phiie iao ihanh cd kha nang hdp ihu d bude sdng 340 nm.

2.8. Phuong p h ^ xdc dinh dd dm, hdm lupng protein va chdt b l o tdng

Xac dinh am dp cua phu phdm cd Tra nguydn lieu bdng phuong phap sdy l g phu pham din khdi lupng khong ddi su dung may say hdng ngoai MA35- Sariorius 35, xac dinh ham Iupng dam tdng sd trong thit ca Tra nguydn lieu bang phuong phap Kgeldahl va xac dinh ham lupng chat beo trong thii ca nguydn heu bang phuong phap Soxlehi (AOAC, 2010).

2.9. Dilndi SDS-PAGE

Protein tir tiiit de va phu phdm dupc chuan bi nhu sau: phoi iron 20 pi protein cd ndng dp 2 mg/ml vdi 20 pi dung dich d^m pha mdu (glyxin mdu ddm) va cho vao dng eppendorf, sau dd lde dlu va dim each thuy d 95°C, 5 phut trude khi cho vao moi gilng NONG NGHIEP VA PHAT TRIEN NONG THON - KY 1 - THANG 11/2015 ⁸¹

KHOA HOC CONG NGHE

vdi i h l tich 20 pi gel polyacrylamit 10%. Dilu chinh ehl dp dien di la 25 mA/40 V trong thdi gian khoang 2 gid. (jel sau khi ehay dien di dupc nhudm vdi Bromphenol Blue R-250.

2.10. Phan tich sd h | u

Thi nghidm dugc khao sat vdi 3 ian lap lai tren cimg mpt nguydn lieu. Kit qua dupc xii I^ bdng phdn mim ling dung MS. Excel 2010. Tinh toan ihdng ke, phan tich phuong sai, kilm dinh LSD bang phdn mim Siagraphic 15.1.02. Xac dinh cdc ihdng sd ddng hpc V ^ , K^ bang phdn mem SAS 9.1.3 Portable.

8. K B QUA VA IHAO LUAN

3.1. Xdc djnh cdc tiidng sd ddng hpc ciia enzym neutrase tr6n co chdt phu phdm cd TVa

Dpng hpc enzym cd lien quan mat thiet den tdc dd phan iing iheo ihdi gian; mdi quan hd giua ndng dp enzym va ndng dd ca chat; idc dp phan iing cue dai Y„ax' hdng sd tdc dp phan iing K„ vd anh hudng cua cac yeu id hda ly din qua trinh xuc tac (Nguyin Cdng Ha va Le Nguyin Doan Duy, 2011). Do dd, cung mdi enzym se cho dpng hpc khac nhau ddi vdi mdi CO chdi khac nhau. Kit qua khao sdt ddng hpc enzym neutrase tr&n phu pham cd Tra the hien trdn hinh 1 cho ihay phan iing jciic tac qua trinh thuy phan protein tir phu pham ea Tra ciia neutiase tuan theo phuong trinh ddng hpc Miehaelis-Menten (V=

0,254 [S]/ 0,190 + [S]) vdi dp tin cay R^ = 0,993. Dieu nay phan anh mdi quan he giiia ham Iupng tyrosin sinh ra iren mdi don vi thdi gian iheo su gia tang ndng dp co chdt Hdm lupng tyrosin sinh ra iang nhanh nhat trong khoang nong dp co chat 0,02 - 0,35 g proiein/10 ml. Khi ndng dp co chdi irong khoang 0,40 - 0,60 g proiein/10 ml van idc phan ung ed xu hudng dn dinh, dilu nay dugc cho Id tdc dd phan iing enzym khong phu thupc vao ndng dp ca chat khi ndng CO chdt cao. Nhu vay khi ndng dp co chat iang idi mdt gia tri nao dd, tdc dp dn dinh eua phan ung dai idi mpt gia tri nhat dinh, idc do ay gpi la tdc dp cue dai Vjnajt cua phan ung. Tuy nhien su lien he nay chi gidi han den mdt ndng dp nhat dinh, nlu tiep iuc tang ndng dp co chat se lic e h l ngupc lai boat dpng xuc tac cua en^mi. Dilu nay gidi thich vi sao khi tdc dd phan iing dat cue dai thi enzym d irang thai bao hda CO chat va luc ndy phan iing khdng tang dupc nua.

Theo mpt nghien ciiu ciia Pagan va ddng tac gid (2013), khi thuy phdn protein tu xuang heo, hdng sd phan Iy hieu qua ciia neutrase tren loai co chdi nay

^^ NONG NGHIEP VA PHAT

dupc xdc dinh Id 0,036 g protein/10 mL Nhu v ^ hdng sd K„ ciia enzym neutrase trdn eo ehdi la ^ phdm cd Tra ldn hon nhieu so vdi iren ca chdi xuoi^

heo. Nhu vay tir k i t qua thi nghidm cho thay enzym neutrase cd di luc tren ca chdi protein tir phu p h ^ ca Tra thdp hon so vdi iren ca chat xuong heo trra^

nghien ciiu ciia Pagan vd ddng tac gia (2013). Cung iu dd thi cho thdy, enzym neutrase dat din ^ ^ phan ling cue dai d miic co chat tiong khoang 0,52- 0,69 g proiein/10 mL Do vdy, ty l l khdi lupng enzym : eo ehat dupc chpn cho tin nghiim tilp theo la 0,5 mg E : 0,694 g protein.

lichaelii-Ntitei.leiUDic ro tio

Hmh 1. Dd thj bilu diln sif phu thude ciia tdc dd phan ling vdo Ididi lupng c a d i ^ 3.2. Tl l l giiia lupng enzym vd ndng dO co ch^

(E/S) idi uu

Qua trinh ihuy phan ung dung trong quy mo san xuat edng nghiep chi tinh rieng cho enzym va ca chat phai dam bao duoc hilu suat tdi uu ma tilt Idem dupc hda chat cflng nhu lupng enzym su dung. Di^

nay dam bao dupc Ipi ich kinh i l eho qua tiinh san xuat eung nhu ha gid thanh san phdm. \^ ngudn phu phdm la tirong ddi ddi dao lai re tiln ndn vide tilt kilm lupng enzym su dung ma hidu sudi tiiiiy phan vdn dat hilu qua cao la muc tieu ciia nghidn ciiu. Tif ty Ie khdi lupng enzym; ca chdi (mg E : g Pro) idi uu cd dupc tu tiii nghiem 1, bdng each tang ddng thm khdi lupng enzym va co chat thdng qua viec nhan voi nhung he sd tang dan 1,2, 1,4 va 1,6 trong mdt th^

tieh dung dich ddm phdt phat pH 6,5 ed dinh (10 mO nham khao sdi su anb hudng cua ndng dp hdn hpp phan ling ldn hidu suat thiiy phan ciia enzym. K^t qua khao sdi dupc trinh bay d cdc bang 1 va 2. Hieu suat thuy phan theo ^ o s i n dupc tinh tiieo luong tyrosin sinh ra sau khi thiiy phan bdi enzyme so vdi lupng tyrosin idng sd trdn 100 g chdt khd troiffi nguyen lieu. Cdn Iupng tyrosin ^ n g sd dupc xitt TRIEN NONG THON - KY 1 - THANG 11/2015

KHOA HOC CdNG NGHl

dinh iheo phuong phap thuy phan bdng axit HCl 6 N cua Zhu et al. (2008), tiieo Ta Himg Cudng (2014) trin cimg mdt don vi khdi lupng nguydn h|u. Theo khao sai, luong tyrosin trfin 100 g chdi khd irong nguyen lilu la phu phdm ea tra cd gia tri Id 2,365 g.

Kit qua khao sdi nay eua de idi cung cho thdy lupng tyrosin tdng sd d phu phdm ca Tra cung gdn gidng vdi luong tyrosin tdng sd dupc nghidn ciiu trdn ndi t^ng loai cd tuylt (Adpenser persicu^ eiia Ovissipour va ddng tac gia (2009) (2,34 g/100 g ehat khd nguydn lidu).

Bang 1. Hi|u sudt Ihuy phan (tinh theo ham lupng tyroan) ciia neutrase 6 cdc hg sd nhan khde nhau

H6 so 1,0 1,2 1,4 1,6

Cgp enzym : ca ch^t (mg E : g Pro) 0,5 mg E: 0,694 g Pro 0,6 mg E: 0,833 g Pro 0,7 mg E: 0,972 g Pro 0,8 rag E: 1,111 g Pro

Hifeu su^t thuy phan {%, theo ham

luong tyrosm) 2,652+0,102"

2,273±0,110' 1,883±0,107=

1,510*0,158"

Vi ihl, ndng dd cua hdn hpp phan iing ihich hpp cho hidu sudi ihuy phan cao nhdi Id miic ban ddu (he sd 1,0), tiic Id cap khdi lupng E: Pro Id 0,5 mg E:

0,694 g Pro, trong 10 mL dung dich ddm phdt phdi pH 6,5. Do dd, khdi lupng enzym va co chat nay dupe chpn eho ihi nghiim tilp tiieo.

3.3. I h d i gian thiiy phdn tdi uu

Nhdm khao sat su dnh hudng eua ihdi gian phan ling lln hieu sudt thiiy phan eua en^mi trung iinh, tir khdi lupng enzym: ca chdt tdi uu dupc chpn tir ihi nghidm irudc (0,5 mg E : 0,694 g proiem/10 mL, tiln hdnh tang ddn thai gian gian thuy phan tir 30 din 240 phuL Kit qua thi nghiem dupc trinh bay trin bang 3 vd hinh 2.

Bang 3. Hilu sudt thuy phdn cua neutrase tiieo thdi gian phdn iing

(a, b, c, d: Cic chd cai khic nhau tiong cung mdt cot thi hiin sir khac biit cd f nghia dmuc dp tin

Vdi k i t qua d bang 1, dii d ty lg khdi Iupng enzym: co chat tdi uu, khi hdn hpp phan iing cd ndng dd cdng cao se cdng lam giam hidu sudt thuy phan cua neutiase. Dilu nay dupc t h l hidn thdng qua su giam m ^ h hidu suat thiiy phan theo ^ o s m e tir 2,652% d h i sd nhan la 1,0 (tiic 0,5 mg E va 0,694 g Pro ti-ong 10 mL dung dich dim phdt phai) xudng edn 1,510 d hg sd nhan la 1,6 (tiic 0,8 mg E va 1,111 g Pro tix)ng 10 mL dung dich dem phdt phat). Su khac bi|t nay Id cd y nghia thdng k l d miic dp tin cdy 95%.

Bdn canh dd, khi hidu sudt thuy phan xac dinh bdng phuong phdp OPA (pp OPA), nhu kit qua khao sat dupc trinh bay d bang 2, hidu suat tiiuy phan cflng giam , khi edsu tang ldn vl ndng dp cua hdn hpp phan ling.

Bdng 2. Hi|u sudt tiiuy phdn dia neuti:ase 6 cdc h | 5 nhdn khde nhau

sci 10 t?.

14 1,6

C}p khdi luong E : Pro (mg E : g Pro) 0,5 mgE: 0.694 g Pro 0,6 m g E : 0.833 g Pro 0.7 mc-E: 0.972 g Pro 0.8 mgE: 1,111 g Pro

Hi^u su^t thuy phan {%, theo ham luong

dam amin, theo phuong phap OPA)

0,553±0,031''' 0,577±0,040"

0,602±0,026- 0,498+0,046'

Thai gian phan ung (phiit)

0 30 60 90 120 180 240

Hiiu suat thuy phan (%, theo

tyrosin) V 2,702±0,074' 3,542+0,002' 4,821+0,075'' 5,228±0,126' 6,055+0,118' 6,359+0,021*

Biiu suat thiiy phan (%, theo phuong phap

OPA) 0,854+0,064'

»

1,173±0,020=

1,651+0,071'' 2,121+0,128°

2,800+0,108"

3,244±0,146"

(a,b: Cac chu cii khic nhau tiong ciing mot cdt tiii hiin su khic biit cd y nghia d miic dp tin cSy 95%)

NONG NGHIEP VA PHAT TRIEN NONG T H 6 N

(a, b, c, d, f, e.Cic chd cii khic nhau tiong ciing mdt cdt la su khac biet cd y nghia d miic dd tin cay 95%)

Kit qua khao sat cho thdy, higu suat thiiy phdn tinh theo phuong phap OPA cua enzym neutrase irIn CO chdt phu pham cd tra tang nhanh irong giai doan khoang 120 phut tiiiiy phan ban ddu (ttr 0,854% d 30 phiit Ign 2,121% d 120 phiit) sau dd tang chlim va d 240 phiit ihi hilu sudt ihuy phan 6^t cao nhdt (3,244%). Theo Hou and Zhao (2011), neutiase Id mdi enzym phan cdt protein iai cac lien k i t hinh tiianh bdi cac axit amin ky nudc, trong khi dd, so sdnh vdi trypsin, enzym chi phan edi protein iai cae liln k i t hinh thanh bdi nhdm cacboxyl ciia lyzin hoac acginin. Ndn khi so vdi trypsin, neuiralizen iao ra cdc peptit cd mach ngdn hon. Mat khac, khi thdi gian ihiiy phan cdng ddi thi nhung peptit mach ngdn va axit amin sinh ra cdng nhilu. Chinh nhung doan peptit ndy vd ede axit amin k i t hpp vdi ihudc ihu OPA, hap till] dnh sdng d budc sdng 340 nm cho k i t qud higu sudt thuy phdn tang ddn.

- K Y I -THANG 11/2015 83

KHOA HOC CONG NGHE

al thiiy (Am {'o. tbeo KTOsine) at !huv phan ("«. tbeo phinmg pbap OP.A)

Hinh 2. Higu suat thiiy phan ciia neutiase theo ihdi gian phan iing

Ket qua tir hinh 2 ciing cho Ihay hieu sudt thuy phan tinh theo tyrosin cung tang dan theo thoa gian phan ling. Tuy nhien, hieu suat thiiy phan tang nhanh tiong giai doan khoang 120 phiit ddu (tir 2,702% a 30 phut len 5,228% cr 120 phiit) va sau do tang cham den khi hieu suat thiiy phan dat cao nhat d 240 phut phan ung (6.359%). Thdi gian thuy phan cang dai, hieu sudt thiiy phan cang tang nhimg mdi llic mpt cham. Dieu nay cd the dugc giai thich la do qua trinh Ihiiy phan sinh ra cac peptit mach ngdn va cac axit amin hoa tan vao dung dich phan ung. Do su tang len ngay cang nhieu cua cac peptit mach ngdn nay va cac axit amin uc chl hoat tmh cua enzym nen thdi gian thuy phan cang dai thi hoat tinh enzym cang giam (Nguyin Xuan Trinh et al.. 2009).

Kit qua dien di d hinh 3 cho thay, neutiase thiiy phan protein tir phu pham ca Tra tiong thoi gian 30 phiit va 90 phiit chua triet dl nhu trudng hpp cung enz>'m nay nhung vdi co chat ia Ihit de ca Tra tiong thi nghiem ciia Ta Hiing Cudng (2014). Ban dau protein chua bi thuy phan la nhung mach peptit co tiong lupng phan tii ldn tir 30 din 250 kDa. Qua qua tnnh thuy phan bdi neutiase, mpt enzyme voi dac tinh phan cat protein tai cac lien ket hinh thanh bdi cac axit amin k\' nudc. da tao ra nhung doan peptit ngan (Hou and Zhao, 2011) cho nen voi khoang thdi gian thuy phan 30 phiit, san pham thuy phan chu ylu la cac peptit mach ngdn hon ban ddu voi tiong Iupng phan tu duoi 50 kDa. Va sau do. ven khoang thdi gian thiiy phan la 90 phut, cac peptit mach ngdn hon va cac axit amm vdi trong lupng phan tii dudi 3D kDa dupc san smh ra nhieu hon.

84

Theo nghien ciiu a i a Cao Xuan Thuy va dd tac gia (2011) khi su dung enz>-m alcalaza 2.4 L d^

thuy phan phu phdm tir ca Tra cung cho thdy rang thdi gian thiiy phan cang dai, san phdm cua qua trinh thiiy phan tiong dd co cac peptit mach ngan san smh ra cang nhilu. Tuy nhien, phan ung xay ra vdi t6c do tuong ddi Idn trong giai doan ddu. khoang tir 30 d(in 120 phiit thuy phan, the hien qua su tang len dang ke chi sd phdn tiam peptit hinh thanh (Peptide fonn yield) ttr 12,87 den 22.10%. Nhung sau dd, khi thoi gian thiiy phan keo dai den tien 150 phut thi tdc dp phan ung cham lai kem theo do la chi sd phdn tram peptide hinh thanh hdu nhu thay ddi khong dang ke, tir 22.10% a 150 phiit chi tang len den 22.20% d 210 phiit. Hien tuong nay cung dupc cac tac gia giai thich la do khi keo dai thdi gian thuy phan, Iupng co ch^t ngay cang giam va lupng san pham hinh thanh ngay mot nhilu. Chinh nhiing san phdm cua qua trinh thuy phan nay da lic che hoat ddng ciia enzym, lam cho tdc do phan ung bi giam. Cung tren co chat !a phu phdm ca Tra va enzym su dung la alcalaza 2,4 L Nguyen Phuoc Minh (2014) da khao sat khoang thiri gian thiiy phan la 210 phiit thong qua ham lupng ni to do axit amin sinh ra (g/L). Kit qua cung cho thay rang ham Iupng ni to axit amm sinli ra nhanh tiong giai doan dau (4,60 g/L a 30 phut len din 6,62 a 120 phiit) nhung khi thdi gian thuy phan keo dai hon 181^

phiit, qua trinh san sinh ra ni to do axit aniin nay cham lai (6,92 g/L d 180 phut va 6.90 g/L d 210 phiit). Do do, thdi gian thuy phan hieu qua nhdt ciia enzym neutiase duoc chon la 240 phiit.

S C M> 30 90 "i^ C 30 ^f\ c ^_^

Neutrase-De Neutrase-PP Papain- Bromelan-

Hinh 3. SDS-PAGE cua dich protein thtiy phan j bdi neutrase

Day S: Protein chudn; Day C: dung dich protein chua thuy phan; day 30, 90: dung dich protein sau NONG NGHIEP VA PHAT TRIEN NONG THON - KY 1 - THANG 11/2015

KHOA HOC CdNO HGHE 30, 90 phut thuy phan; Neutrase -Dl: Neutrase tiiuy

phan trin eo chdt thii d e ; Neuti^se-PP, papain-PP, brome!ain-PP: Neutralizen, papain va bromelain thiiy phan trin ca chat phu pham d l tdi dang khao sdt

4. K^ LUAN

Hdng sd phan ly hieu qua K„ va idc dp phan ling cue dai V , ^ cua enzym neutrase dugc xdc dinh la 0,254 g protein va 0,190 ^mol tyrosui/phuL Ty 11 e n ^ m : co chat d l idc do phan ung thiiy phan dia enzym dai cue dai la 0,5 mg enzym: 0,694 g protem phu pham cd Tra trong 10 mL dung dich dem phdt phat Thdi gian thiiy phan d l enzym cho higu sudt thuy phan cao nhdt Id 240 phtit. Nhu vay, neutrase kha thich hpp cho vile san xudi mdt hdn hpp peptit va axit amin d mu;c cao va d mdt ty Ig can ddi.

LOICAMQN

Nghiin cmi duqc tai tio tu Quy phat trien Khoa hoc va Cdng nghe Qudc gia (Nafosted) tiin ca sd cua di tii "Nghiin cuu phdi hop cac che pham pwtein tiiuy phan va betaglucan ttr phu pham cua cic nhi miy sin xuat ci Tra phi li va bia cdng nghiep de tdi uu hda khau phan thdc an tiong chSn nudi heo tai dong bing sdng Cuu Long" Ma sd lO&NN 052013.68 nam 2014.

TMlATHAMKHM

1. Abdulazeez S. S., Ramamoorthy B., Ponnusamy P., 2013. Proximate analysis and production protein hydrolysaie fi-om King fish of Arabian Gulf (3oast - Saudi Arabia, bit J. Pharm. Bio.

Sci. Vol 3 (1).138-144.

2. Anson, M. L , 1938. The estimation of pepsin, trypsin, papain and cathepsin with hemoglobin. J.

Gen. Physiol. 22,79-89.

3. AOAC (2010). Official methods of analysis (15*ed.). Washuigton, DC: Assoaation of Official Analytical Chemists. USA.

4. Cao Xuan Thuy, Tran Bich Lam and Ha Thanh Toan, 2011. Hydrolysis of Pangasius bjpophthaimushy-pToduct to peptide form. Pood Bioprocess TechnoL 8/2011.

5. Hou, Y. and X. H., Zhao, 2011. Luniied hydrolysis of two soybean protein products with trypsm or neutrase and the impacts on theu solubility, gelation and fet absorption capaciQr.

Biotech. 10(2).Pp. 190-196.

6. Nguyin Cdng Ha, Le Nguyin Doan Duy, 2011. Giao trinh: Ky thuat thuc pham 3. Nhd Xuat ban Dai hoc Can Tha.

7. Nguyin Xuan Trinh, Tran Hai Yin va Trdn Thi Anh Thu, 2009. Nghien cihi cdng nghg san xuat dich dam, bdi ea tir phu pham cd tia va ca basa. Luan van idi nghigp dai hpc. Dai hpc Cdng nghg Sdi Gdn.

TP. HCM,

8. Nguygn Van Mudi, Chung Thi Thanh Phupng, Thdi My Ngan, Trdn T h l Hiln, Tran Tdn Khanh, Lam Hda Hung, 2013. Anh hudng ciia ti Ie mo- va phu gia bd sung den dac tinh cau tnic a l a xuc xich dupc c h l biln bi tiiit de cd Tra. Tap chi Khoa hpc - Trudng Dai hpc Can Tha. 26. ti. 188-195.

9. Nguygn Phudc Minh, 2014. Ultilization of Pagansius hypophthalmus by-product io produce protein hydrolysaie using Alcalase enzyme. / HarmonizRes uiAppL Sd 2 (3). 250-256.

10. Nguyen Thi Thuy, Nguyen Tan Loc, Lindberg, J. E. and Ogle, B. (2007). Survey of ihe production, processing and nutritive values of catfish by-product meals in the Mekong delta of Vietnam.

Uvest Res. Rural Dev. 19 (9).

11. Nielsen, P. M., Petersen D. and Dambmann C , 2001. Improved method for determining food protein degree of hydrolysis. Journal of food Science.

Vol. 66. No. 5, pp. 642-646.

12. Ovissipour M., Benjakul S., Saferi R, Motamedzadegan A., 2010. Fish protein hydrolysates production from yellowfin tuna Thunnus albacares head using Alcalase and Protamex. Int Aquatic Res.

2:87-95

13. Pagan, J., A , Ibarz, V., Falguera and R, Benitez, 2013. Enzymatic hydrolysis kinetics and nitrogen recovery in ihe protein hydrolysaie production fi-om pig bones. / Food Engineering.

119(3): 655659

14. Ta Hung Cudng, 2014. Nghign ciiu che biln bdt protein ihuy phan tu thit de ca Tra. Luan van cao hpc chuygn nganh Cdng nghg thuc pham. Khoa Ndng nghigp vd Sinh hoc ling dung. Trudng Dai hpc Can Tho.

15. Tdng cue Thuy san, 2013. Hdi nghi idng kit sdn xuat, tigu thu cd Tra nam 2012 vd trien khai nhigm vu nam 2013 (25/01/2013).

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Amino Acids Production from Fish Proiems Hydrolysis m Subcritical Water. Chinese Journal of Chemical Engineering, 16(3) 456460.

NONG NGHIEP VA PHAT TRIEN NONG THON - KY 1 - THANG 11/2015 ⁸⁵

KHOA HOC C 6 N G NGHig

THE HYDROLYSIS CAPABUnY OF PROTEIN EXTRACTED FROM TiiA FISH BY-PRODUCT USING ENZYME NEUTRASE

Nguyen Thi Thuy^, Nguyen Cong Ha^

^Ck>llege of Agriculture and Applied Biology, Can Tho University Summary

To improve the capacity of using by-products from Tra fish fillet industry, study on the hydrolysis of protein extracted from Tra fish by-products usmg commercial neutiase was done. The results showed that when using optimal condihon of pH and temperature for reaction of neutrase, Y^„ and K^, of enzyme reacted on the protein from Tra fish by-products was consequently 0.190 junol fyrosine/minute and 0.254 g protein/10 ml. The result from the hydrolysis showed that with the ratio of enzyme and substrate as 0.5 mg E : 0.694 g protem. Beside, when prolonged the time for hydrolysis from 30 to 240 mmutes, the degree of hydrolysis increased correspondingly and achieved 6.359% as Tyr,p/Tyri as well as 3.244% following OPA method. So, the time for hydrolysis using neutrase was chosen at 240 mmutes. Moreover, flie bydrolyzed protein results which showed on SDS-PAGE proved that most of flie protein bydrolyzate from Tra fish by-products were bydrolyzed well and formed in high molecular weight peptides intensity but low mtensity in amino acid mixtures. The results indicated that neutralization can be used well for the hydrolysis of protein from fish by-product to form a mixture of ammo acid as well as low molecular weight peptides. And, the product can be used for animal feed as protem source.

Keywords: Degree of hydrolysis, hydrofyzate, neutrase, tra Ssh, Tra Ssh by - products Ngudi phdn bign: PGS.TS. Nguyin Duy LSm

Ngdy nhdn bdi: 22/4/2015 Ngdy tiidng qua phdn bign: 22/5/2015 Ngdy duygt dang: 29/5/2015

NONG NGHIEP VA PHAT TRIEN NONG THON - KY 1 - THANG 11/2015