KHOA HOC CONG N G H l
AI\1H H U O I V G CLIA T Y L E G I U A EIMZYIVIE A L C A L A S E V A P A P A I N DEIXI P U A T R i l V H T H L I Y PHAIM SLUM CA M A P
{carchardinus tiussumieri\
Dinh Hiiu D6ng^, VQ Ngpc BOi^ Nguyfin Thi My Trang*
T6MTAT
Bai bao nay trinh bay ket qua nghiSn ciiu ve anh hirong oia ty le giua enzyme alcaiase va papain d^n ham luong protein hoa tan, ham lucmg peptid, Naa, N-^HS, chondroitin sulphate tao thanh trong qua trinh thuy phan sun ca map (Carcharhinus dussumien) bang hdn bop enzyme alcalase-papain. Ket qua nghien ciiu cho thay ty le enzyme alcalase/papain trong hon hop enzyme alcalase-papain thich hqp cho qua trinh thuy phan sun ca map la 60/40. Sau 10 gia tbuy phan sun ca map bang hdn hop enzyme alcalase-papain voi nong do enzyme 0,296, nhiet dp thiiy phan SCC, thuy phan opHtu nhien (6,8), khdi luong mhi 2 kg va ty 1? nude bd sung 2 lit, dich thuy phan cd ham luong protein hda tan, peptid, Naa, chondroitin sulphate va N[jj|3 cao gap 6,54 lan, 3,26 lan, 7,91 lan, 78,17 lan va 1,25 lan so vdi ban ddu.
TLT khda: HSn hop alcaiase - papain, protein, peptide, Naa, Ni^i^
phan.
chodroitjn sulphate, sun ci map, thtiy
I.IWrDAU
Sun ca map cb chua ham luong chondroitin sulphat cao hon cac loai dong vat khac [5, 8. 9].
Chondroitin sulphat la thanh phdn co ban cau tao nen sun khop va cau tao nen cac to chiic soi chun giiip cho su v^n dgng linh boat va tinh dan h6i trong boat dbng khop. Chondroitin sulphat con giiip lam tang san xuat chat nhay va kha nang boi tron cua dich khop. Vi vay, chondroitin sulphat duoc su dung d^ hd tro dieu tri cac benh ly v^ xuong khop, ban che qua trinh thoai hoa khop. M6 sun noi chung va sun ca map noi ri&ng thuong duoc cau tnic boi cac protein khong tan trong nuoc [8, 9]. Chondroitin sulphat thuong hen kgt voi protein bang lien ket o- glycosid tao thanh mpt proteoglycan nam trong cau tnic ctia mo sun nen con ngudi rat kho bap thu cac chat tir mo sun [3, 8, 9]. De thu nhan cac chat tir sun ca map dinh huong cho viec sii dung lam thuc pham h6 tro phong chong thoai boa sun khop, da hen hanh nghien cuu su dung enzyme protease trong thuy phan sun ca map tuoi. Trong bai bao trinh bay ket qua nghien cuu ve anh hudng cua ty le giua enzyme alcaiase va papain den qua trinh thuy phan sun ca map bang h6n hpp enzyme alcaiase - papain.
2 . NGUYBU VAT UEU VA PHUDNG PHAP NGHBV CUU 2.1. Nguyfen v|t U^u
' Truong Dai HQC Cong nghi?p Th\rc pham TP. HCM
• Truong Dai hoc Nha Trang
2.1.1. Sun camap
Ca map trdng (Carcharhinus dussumleri (Muller
& Henle, 1839)) dugc thu mua nguyen con tai cac t^u khai thac tai vimg bi^n Khanh Hoa. Sau thu mua, thu toan bp vay ca, sun ca va van chuydn v^ phong thi nghiem. Tai phong thi nghiem, ti^n hanh xu ly lo^
bo thit, mo lien k^t, lam sach, cap dong va bao quan dong b -2QPC d^ dimg trong suot qua trinh nghifin
Hinh 1. Hinh anh v^ vi sun ck m|.p (Carcharhmus dussumleri) tru6c vA sau khi xiJr 1^
2.1.2. Enzym alcaiase
Enzym alcaiase 2.4L la che pham protease thuong mai do Hang Novozyme - Dan Mach cung cap. Alcaiase thuoc nhom enzyme serine endopeptidase co cac dac tinh ky thuat nhu: pH tiiich hop trong khoang 6 • 8, nhiet dd thich bop 30 - 65"C.
hoat tinh 2,4AU/g dupc bao quan a 0 - 5"c 2.1.3. Enzym papain
KHOA HOC C d N G NGHE
Papain thuong mai co hoat tinh > 2,0 mAnsonU/mg (co chat hemoglobine, pH 6, nhiet dp 35,5°C) do Merck - Due san xuat Papain la mpt enzyme chiu duoc nhifet dp tuong doi cao. O dang nhua kho, papain khong bi bi^n tinh trong 3 gio b 100"C, con b dang dung dich, papain bi mat boat tinh sau 30 phut o 82,5°C. Papain co pH thich hpp 4,5 - 8.5, de bi bi^n tinh o pH<4,5 va o pH >12.
2.2. Phuang phdp nghifin cihi 2.2.1. Cic phuong phip phin ti'ch
* Dinh luong nito- axit amin (N^J: tiieo TCVN 3708-1990 Thuy san - Phucmg phap xac dinh ham lupng nito amin.
* Dinh luong nita ammoniac (NfjHa): theo TCVN 3706-1990 Thiiy san - Phuang phap xac dinh ham lupng nita ammoniac.
* Xac dinh hoat do protease theo phmmg phip Anson [1]
Nguyen tac ciia phuong phap; ditng protein casein lam co chat xac dinh boat d6 thuy phan protein cua enzyme protease trSn co so dinh lupng san pham dupc tao thanh trong phan ung bang phan ling man vdi thudc thu Fohn - Ciocalteau. Dua vao d.6 thi chuan tyrosine de dinh lupng tuong img vai lupng san p h ^ tao thanh duoi tac dung cua enzyme.
- Dinh lupng protein hoa tan theo phuung phap Lowry[l,2]:
Nguyen t^c cua phuong phap la cac axit amin co vdng thom, Tyr va Trp co mat trong protem se phan ung voi thudc thu FoUn-Ciocalteau tao thanh phuc chElt mau xanh den co d6 hap thu cue dai o buoc sdng 650nm. Dua vao duang chuan protein co the dinh lupng ham luong protein.
- Xac dinh bim lupng peptid [1, 7]: ham lupng peptid dupc dinh luang dua vao duang chuan tyrosine. I i y 1 g mlu tiiuy phan, cho them 9 ml nuoc cat sau do khuay deu trong khoang 5 -10 phiit roi ly tam lay dich trong de xac dinh ham lupng peptid nhu sau: lay 2 ong nghidm sach 1 ong tiii nghiem va mot 6ng doi chimg. Ong thi nghiem: hut chinh xac 2 ml dung djch lpc o tren cong voi 2 ml tnchloacetic acid CrCA) 2096 de 30 phiit roi loc qua giay lpc ttiu dich lpc. lAy mdt ong nghiem sach cho vao 1ml dich lpc + 5 ml dung dich Na^COg 0.4 M ldc d^u, r6i cho vao 1
ml Folin de 20 phut so mau 6 buoc song 660 nm. Ong doi chimg: lay 1ml dung dich TCA 10% + 5 ml NagCOa 0,4 M + 1 ml fohn de 20 phiit dem so mau.
Tinh ket qua: dua vao duimg c h u ^ de tinh luong tysosin tuong ung.
Ham lupng peptid duoc tinh theo cong thuc:
Pepnd (mg/ml) = " ' J ' " > ' ^ ^ " ' , ^^ p^a lohig ml man
2.2.2 Phuong phip dinh luong chondroitin sulfate (CS) bing phuong phap so miu theo Famdale vi cdng sir {"i, 6]
Nguyen ly: Dua tren su thay doi trong quang pho hap thu cua DMMB (1,9 Dimethyhnethylene) khi tac dung vol chondroitin sulfate (glycosaminoglycan sulfate) o buoc song 525nm.
Dua vao duong chuan ctia chondroitin sulfate A feoc sulfate gdn a vi tri C 4 (chondroitin-4-sulfate), CS4) voi DMMB de xac dinh ham lupng chondroitin sulfate. Phuang phap nay co do nhay cao, co the dinh tinh va dinh lupng ham lupng CS 6 miic ^g.
2.2.3. Phuongphip bd tri thi nghiem Bo tri thi nghidm d^ lua chon enzyme protease thuy phan sun ca map trdng dupc trinh bay 6 hinh 2.
Vi va sun ca map da xu; ly dupc ra dong, rua, xay nho bang may xay va dupc sir dung de lam nguyen heu ngbien ciiu thuy phan bdng hfln hop enzyme alcaiase •»• papain. Tien hanh 6 mau thi nghiem thiiy phan hdn bop sun ca map bang hdn hop enzyme alcalase+papain voi ty le alcalase/papain khac nhau:
mdu 1: thuy phan bdng hdn hpp enzyme alcaiase va papain theo ty le 80/20; mdu 2: 70/30; mau 3: 60/40;
mdu 4: 50/50; mdu 5: 40/60 vd mau 6: 30/70. Cac mdu thiiy phan d&u sir dung 2kg bdn hpp sun ca map, ty le hdn hop enzyme sir dung la 0.2% va lupng nuoc bo sung la 2 lit, pH thuy phan Id pH tu nhien cua hdn hpp sun ca map (pH 6,8) vd nhiet dp thuy phan 50°C. Sau cac khoang thai gian: 0 gid, 2 gia, 4 gio, 6 gio, 8 gio va 10 gicr thuy phan, tien hanh lay mdu dich thuy phan de danh gia ham lugng protein hoa tan, ham luong pepbd, ham lupng N^.„ NSH3 va hdm lupng chondroitin sulphate tao fhanh. Ket qua danh gia la co so de lua chon ty le giiia alcaiase va papain thich hop trong hon hop enzyme dimg cho qua trinh thiiy phan sun ca map.
NONG NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANG 6/2020 61
KHOA HOC C 6 N G KGWt
Sun ca map ti/ori
Rira
HZ
Thuy phan bang hon hcfp alcaiase + papain vo"! ty le alcalase/papain khac nhau
X
i I i i , i_
80/20 70/30 60/40 50/50 \ 40/60 | 1 30/70
Lira chpn ty le alcalase/papain phu hcfp
Hinh 2. Sa d6 Ihi nghidm lua chpn t^ 1^ alcalase/papain thich hpp cho thiiy p h ^ vi sun cA m$p 2.3.'nii^tbiv^h6ach^t
- Thiet hi: su dung cac thiet hi hien co tai Trung tam Thi nghiem Thuc hdnh ~ Truong Dai hpc Nha Trang va Truang Dai hpc Cdng nghiep Thuc phdm - TP. HCM: May so mau UV-VIS DR6000 - Hach (My);
hi on nhiet MEMMERT WNB14 - Dire; may ly tam Ignh tdc dp cao HERMLE Z36HK - Diic; be thdng phan tich ham lupng nito/protein theo phuong phdp Dumas ^ l i c ) ; bon nude dieu nhiet Memmert WNB22 (Birc); ndi tiiiiy phan dung tich 30 lit (Viet Nam),...
- Cac hoa chat sir dung trong thi nghiem d^u la hod chat tinh khiet do Hang Merck - Diic cung cap.
2.4. Phuang phdp xu 1^ sd li§u
Mdi tbi nghiem deu tien hanh lap lai 3 ldn dpc lap va so lieu la ket qua trung binh ciia cdc lan thi nghidm. Ki^m tra su khac biet giira cac so heu thdng ke bang phan m6m Statgraphics Centurion XVII dial.
3. KET QUA NGHDI CUU VA THAO LUAN
3.1. Anh hu6ng ciia thdi gian thiiy phan vd ty 1§
giira alcalase/papain toi hdm lugog protem hda tan Tien banh 6 mdu thi nghi&m thuy phan hdn hop sun ca map bang bdn hpp enzym alcaiase - papain vdi ty le khac nhau: 80/20, 70/30, 60/40, 50/50, 40/60 va 30/70. Sau cac khoang thdi gian: 0 gid, 2 gid, 4 gio, 6 gid-, 8 gid va 10 gid tiiiiy phan, ti^n banh lay dich tiiiiy
phan d^ danh gid ham lupng protein hoa tan K^t qua duoc the hien tren hinh 3.
Tir ket qua phan tich d' hinh 3 cho thay theo thdi gian ham lupng protein hda tan tao thanh trong t^t ca cac mdu thuy phan thiiy phan sun ca map bang h6n hop alcalase-papain deu tang nhung miic dp tang khac nhau tiiy thudc vao ty 1^ alcalase/papain su dung trong thi nghidm va thdi gian Idy mdu. Cu thi, d giai doan sau 2 gio thuy phan, ham lupng protein hda tan cua dich thuy phan sun ca map bdng hon hgp enzyme alcalase-papain vdi ty le alcalase/papain bang 60/40 cao gap 2,84 lan so vai ban dau va cao gap tuong ling 1,30 ldn, 1,16 lan, 1,12 lan, 1,24 lan va 1,33 lan so vdi ham lupng protein hda tan tao thdnh 6 mdu thuy phan bdng enzyme hdn hpp alcaiase - papam tiieo ty le 80/20, 70/30, 50/50, 40/60 va 30/70. O thdi diem sau 10 gid thuy phan, ham lupng protein boa tan cua dich thuy phan sun cd map bdng hdn hop enzyme alcaiase papain vdi ty 1^
alcalase/papain bdng 60/40 cao gap 6,54 lan so vdi ban dau va cao gap tirong ling 1,33 lan, 1.16 lan, 1,10 lan, 1,21 lan va 1,27 lan so vdi ham lugng protein boa tan tao tbanh d mdu thiiy phan bdng bon hgp enzyme alcaiase + papam vdi ty le alcalase/papain tuong ung la 80/20, 70/30, 50/50, 40/60 va 30/70. Ket qua nay cho thay hdn hop enzyme alcabi--.- - papain voi ty 1^
alcalase/papain bang 60/40 cd kh.i wn^ thiiy phan hdn hpp sun ca map manh me nhat -.o - ,n thiiy phan
KHOA HQC C O N G N G H £
sun ca map bdng hdn hpp enzyme alcaiase - papain vdi ty Id alcalase/papain khac 60/40.
Thm gian Ihiiy phan (gjo) Hinh 3. Anh huong cua thdi gian thuy phan vd t^ 1$
alcalase/papain d^n hdm lupng protem tao thdnh trong dich thiiy phdn vi s i ^ cd mdp Ket qua nghidn ciiu cua chung tdi cung cd nhirng net tuong ddng vdi mdt sd nghidn ciiu da cdng bd tiirdc day. Cu the, nam 2003, Vu Ngpc Bdi khi nghidn cuu vd qud frinh thuy phan protein ca mdi bdng enzyme protease cho rdng su dung mdt hdn hpp enzyme protease de thuy phan ca thit cd mdi se tot hon mdt enzyme protease va qua trinh thuy phan phu thudc vao ty Id phdi trdn gitra cac enzyme thanh phan [1].
Tir nhiing phan tich b tren cho thay khi xet theo khia canh ham lupng protein hda tan cao thi su dung hdn hpp enzyme alcaiase papain vdi ty le alcalase/papain bdng 60/40 de thuy phdn sun ca map se tao ra dich thuy phan cd chiia ham lupng protein hda tan cao hon khi su dung hdn hop alcaiase - papain vdi ty 1§ alcalase/papain khac vdi ^ le 60/40.
3.2. Anh huong cua thdi gian thuy phan vd ty 1^
giiia alcalase/papain tdi hdm Iirgng peptid hda tan Tien hanh 6 mlu thi nghiem thuy phan hdn hop sun ca map bdng hdn hop enzym alcaiase - papam vdi ty Id khac nhau: 80/20, 70/30, 60/40, 50/50, 40/60 va 30/70. Sau cac khoang thdi gian: 0 gid, 2 gid, 4 gid, 6 gid, 8 gid va 10 gid thuy phan, tien hanh lay mdu dich tiniy phan de danh gia ham luong peptid hda tan Ket qua dupc the hien tren hinh 4.
Tir kft qua phan tich d hinh 4 cho thdy cung gidng nhu su thay ddi cua ham luong protein trong qua trinh thiiy phan, theo thai gian thuy phdn ham lupng peptid tao tiianh trong tat ca cac mdu thiiy
phan deu tang va mtic dp tang ciing khac nhau tiiy thupc ty le giiia enzyme alcalase/papain. Trong dd, hdm lupng peptid cua mdu thuy phan hdn hpp sun ca map bdng hdn hop enzyme alcaiase - papain vdi ty le enzyme alcalase/papain la 60/40 ludn tang nhanh han theo thdi gian thuy phdn vd tang cao ban so vdi cac mdu tiiuy phan khac. Cu the, sau 2 gid thuy phan, ham luong peptid cua mdu thuy phdn sir dung hdn hop enzyme alcalase-papain theo ty le 60/40 cao gap 2,55 lan so vdi ban ddu va cao gap tuong img 1,35 lan. 1,19 lan, 1,12 ldn, 1,23 lan va 1,28 lan so vdi ham lupng peptid tao tbanh d mdu thuy phan hdn hop sun ca map bdng hdn hop enzyme alcalase-papain vdi ty le tuong img Id 80/20, 50/50, 40/60 va 30/70. Tuang tu nhu vay, sau 10 gid thuy phan, hdm lupng peptid cua mdu thuy phan sir dung hdn hgp enzyme alcalase-papain theo ty le 60/40 cao gap 3,26 lan so vdi ban dau va cao gap tuong ling 1,34 Ian, 1,19 ldn, 1,11 ldn, 1,23 ldn vd 1,28 ldn so vdi hdm lupng peptid tao thanh d mdu thuy phan hdn hpp sun cd map bdng hdn hop enzyme alcalase-papain vdi ty le tuong ung Id 80/20. 50/50. 40/60 va 30/70.
iBOOOO
8-""»
^ 2 0 0 0 0
ELI JO 00 5.
- 1 0 0 . 0 0
2
"" so 00
/•
t
J_.
•- so:o
• - + - •
—^f"^
• - -0 30
l-
• • s a j o
• JCTO
-•
i
Ilioi gian t b ^ pban (gc) Hinh 4. Aitii hirong cua thdi gian thuy phdn vd ty 1$
alcalase/papain d^n hdm Itrpng peptid tao thdnh trong dich thiiy phdn vi syn cd mdp Tu cac phan tich b tren cho thdy khi xet tiieo khia canh hdm lupng peptid tao thdnh cao trong djch thuy phan thi su dung hdn hap enzyme alcaiase - papain vdi ty le alcaiase so vdi papain la 60/40 la phu hop.
3.3. Anh hudng ciia thdi gian thuy phdn vd ty 1$
giira alcalase/papain Mn hdm Iirgng Naa
Tien hdnh 6 mdu tiii nghidm thiiy phan hdn hop sun ca map bang hdn hpp enzym alcaiase - papain vdi ty Id khac nhau: 80/20. 70/30. 60/40, 50/50,40/60 va
NONG NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANG 6/2020 63
KHOA HOC CONG NGHE
30/70. Sau cac khoang thdi gian: 0 gid. 2 gid. 4 gid, 6 gid, 8 gid va 10 gid thuy phdn, tien hdnh lay mlu dich thuy phan dd danh gia ham luang NNH3- Ket qud duoc thd hidn trdn hinh 5.
dung hdn hpp enzyme alcaiase - papain dd tbuy phan sun ca map vdi ty le alcalase/papain la 60/40 thi hdm luong nito acid amin tao tiianh frong dich thuy phan sun cd map cao nhat vd cao ban cdc mdu thi nghiem khac.
3.4. Anh hudng ciia thdi gian thiiy phan vd t^ 1$
giiia alcalase/p^)ain tdi hdm lugng chodroitin sulphate t^o thdnh
Tien bdnh 6 mdu tbi nghidm thiiy phdn hdn hpp sun ca map bdng hdn hop enzym alcaiase - papain vdi ty le khac nhau. Sau cac khodng thdi gian: 0 gid, 2 gid, 4 gid, 6 gid, 8 gid va 10 gid thuy phan. tien hanh lay mdu dich thuy phan dd danh gid ham lupng chodroitin sulphate. Ket qua duoc thd hien fren hinh 6.
IlniEillllki^pliaiilgici)
Hinh 5. Anh hirdng cua thdi gian thuy phdn va ty' 1§
alcalase/papain d^n hdm lugng Naa tao tiidnh frong
> djch thuy phdn vi sun cd mdp Tir kdt qua phdn tich d hinh 5 cho thay gan gidng nhu su thay doi cua hdm luang protem vd peptid frong qua frinh thuy phan. theo thdi gian thuy phdn hdm luong Naa tao thdnh frong tdt cd cdc mdu thuy phan deu tang va tiiy thudc vao ty le gitra enzyme alcalase/papain. Trong dd, hdm lupng Naa ciia mdu thuy phan hdn hop sun cd map bdng hdn hpp enzyme alcaiase - papain vdi ty le enzyme alcalase/papain Id 60/40 ludn tang nhanh hon theo thdi gian thuy phan va tang cao hon so vdi cdc mdu thuy phan khac. Cu thd, sau 2 gid thiiy phan, ham lupng Naa cua mdu thuy phan sir dung hdn hgp enzyme alcalase+papain theo ty le 60/40 cao gap 4,30 ldn so vdi ban ddu va cao gap tuong ling 1,35 lan, 1.15 ldn, 1,09 lan, 1,12 lan vd 1,23 ldn so vdi ham lupng Naa tao thdnh d mau thuy phan hdn hpp sun cd map bang hdn hpp enzyme alcalase-papain vdi ty le tuong img Id 80/20, 50/50, 40/60 va 30/70. Tuong tu nhu vg.y, sau 10 gid thiiy phan, hdm luong Naa cua mau thuy phdn su dung hdn hgp enzyme alcalase-papain theo ty le 60/40 cao gap 7,91 lan so vdi ban dau va cao gdp tuong ling 1,34 ldn, 1,16 ldn, 1,09 ldn, 1,12 ldn vd 1,22 lan so vdi bdm lupng Naa tao thanh d mdu thiiy phan hdn hop sun cd map bang hon hop enzyme alcalase-papain vdi ty 1^ tirong ling la 80/20, 50/50. 40/60 vd 30/70.
Tir nhimg phan tich d fren khi xet tiieo khia c ^ h ham lupng nita acid amin tgo thdnh cao thi sir
Hmh 6. Anh hudng cua thdi gian thuy phdn vd ty 1§
alcalase/papain d^n hdm lupng chondroitin sulphate tao thdnh frong dich thuy phdn vi sun cd map
Tir ket qua phan tich d hinh 6 cho thay theo tiidi gian thuy phan ham luong chondroitin sulphate tao thanh frong tat ca cdc mdu thuy phan deu tang va tiiy thudc vdo ty le giira enzyme alcalase/papain. Trong dd, ham luang chondroitin sulphate cua mdu thuy phan hdn hop sun ca map bang hdn hpp enzyme alcaiase - papain vdi ty le enzyme alcalase/papain la 60/40 cung ludn tang nhanh hon theo thdi gian thiiy phan va tang cao ban so vdi cdc mdu thuy phdn khde.
Cu thd, sau 2 gid thuy phan, bam lugng chondroitin sulphate cua mdu thuy phan sii dung hdn hpp enzyme alcalase-papain theo ty le 60/40 cao gdp 23,94 ldn so vdi ban ddu va cao gap tuong ling 1,08 lan. 1,04 lan, 1,05 ldn, 1,09 lan va 1,15 lan so voi ham lupng chondroirin sulphate tao thanh d mdu tbuy phan bdn bop sun ca map bang hdn hgp enzyme alcalase-papain vdi ty le tuong img la >fi/;>o. 50/50,
KHOA HOC CONG NGHE
40/60 va 30/70. Tuong tu nhu vay, sau 10 gid thiiy phan, hdm lupng chondroitin sulphate cua mdu thuy phan su dung hdn hpp enzyme alcalase-papain theo ly le 60/40 cao gap 78.17 lan so vdi ban ddu va cao gap tuong ling 1,07 lan, 1,04 lan, 1,05 ldn, 1,08 lan va 1,10 lan so vdi ham lugng chondroitin sulphate tao thdnh d mdu thiiy phan hdn hpp sun ca map bang hdn hop enzyme alcalase-papain vdi ty le tuong img Id 80/20, 50/50, 40/60 va 30/70.
Tir nhiing phan tich d tren khi xet theo khia canh ham lugng chondroitin sulphate tao thdnh cao thi su dung hdn hop enzyme alcaiase - papain de thiiy phan sun cd map vdi ty le alcalase/papain Id 60/40 thi ham lupng chondroitin sulphate tao thdnh trong dich thiiy phan sun cd map cao nhat vd cao ban cac mdu thi nghiem khac.
3.5. Anh hirdng cua thdi gian thuy phdn vd ty 1^
giira alcalase/papain tdi hdm lugng N^^HS Tien hdnh 6 mdu thi nghiem thuy phan h6n hpp sun ca map bdng hdn hop enzym alcaiase - papam vdi ty le khde nhau. Sau cac khoang thdi gian: 0 gid, 2 gid, 4 gid, 6 gid, 8 gid va 10 gid thuy phan, tien hdnh lay mlu dich thiiy phan de ddnh gia ham Itrpng N^HS Ket qua dugc the hien tren hinh 7.
Hinh 7. Anh hudng ciia thdi gian thuy phdn vd ty Id alcalase/papain d^n hdm lugng NNHB tao thanh frong
dich thiiy phSn vi sun cd map Tir ket qua phan tich b hinh 7 cho thay ham lugng NNH3 tao thdnh frong cdc mlu thuy phdn sun cd map bang hdn hpp enzyme alcalase-papam deu tang theo thai gian thuy phan nhung miic dp tang chdm va khde nhau khdng nhieu. Cu the, sau 10 gid thuy phan, cac mlu thdy phan sun ca map bdng hdn hop enzym alcaiase - papain vdi ty le khac nhau;
80/20, 70/30, 60/40, 50/50, 40/60 va 30/70 deu cd ham lupng N^HS tang trong khoang tir 1,25-1,26 lan so vdi ban ddu vd su chenh lech ve ham lupng N^HS giua cac mdu thi nghiem khdng cd y nghia fhdng ke.
4. KETLUAN
Tir cac ket qua nghien ciiu d fren cho phep nit ra ket luan: ty le alcalase/papain frong hdn hop enzyme alcalase-papain thich hpp cho qud trinh thuy phan sun ca map la 60/40. Sau 10 gid thuy phan sun ca map bdng hdn hpp enzyme alcalase-papain vdi ndng dp enzyme 0,2%, nhiet dp thuy phan SO^C, thuy phan d pH tu nhien (6,8), khdi luong mlu 2 kg vd ty le nude bd sung 2 lit, dich thuy phan cd ham lupng protem hda tan, peptid, Naa, chondroitin sulphate va NNH3 cao gap 6,54 ldn, 3,26 ldn, 7,91 ldn, 78,17 Ian vd 1,25 lan so vdi ban dau.
TAI UEU THAM KHAO
1. Vu Nggc Bdi (2003). Nghien cuu san xui't protease tir Bacillus subtihs vi su dung dd sin xui't dich dam tbuy phan tu ca tap. Bao cdo de tai nghien cim khoa hpc cap Bd Gido due va Ddo tao. Ma sd:
B2000 - 33 -33, Trudng Dai hgc Thiiy sdn Nha Trang.
2. Vu Ngpc Bdi (2004). Nghien cuu gui trinh thuy phan protein ca bing enzyme protease tu B.
subtihs, Luan dn tien si Smh hpc chuyen ngdnh Hda sinh, Trudng Dai hgc Khoa hpc Tu nhien, Dai hgc Qudc gia TP. Hd Chi Mmh.
3. Tran Canh Dinh va cdng su (2010). Nghien cuu ling dung sin xui't thu nghiem chondroitin vi glucosamin tu nguyen lieu thuy san. Bao cao tdng kdt de tdi nghien ciiu khoa hpc thudc chuang trinh CNSH thuy san, Vien Nghien cuu Hai san, Hai Phdng.
4. Dang Van Hgp, Dd Minh Phung, Vu Nggc Bdi, Nguydn Thuan Anh (2010). Phan tich kiem nghiem thuc pham thiiy sin. Nxb Khoa hgc va Ky tiiuat. Ha Ndi.
5. Bruyere 0 . & Reginster J. Y. (2007).
Glucosamine and chondroitin sulfate as therapeutic agents for knee and hip osteoarthntis. Dmgs Aging, 24(7); p. 573-580.
6. Famdale W. R., Buttle D. J. & Barrett A J.
(1986). hnproved quantitation and discrimination of sulphated glycosaminoglycans by use of
NONG NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANG 6/2020 65
KHOA HOC C Q N G N G H £
dimethylmeti^ylene blue. Biochim. Biophys. Acta., on a wide range of biological systems.
883: p. 173-177. Complementary Therapies in Medlcuie. 17: p. 56-62.
7. J. Jayaraman (1998). Laboratory manual in 9. Shil S. C , You S. J.. Anb K. & Kang C. W.
biochemistiy, Wiley Eastern Limited. (2006). Study on extraction of mucopolysaccharide- 8. Robert M. Lauder (2009). Chondroitin Protein containing chondroitin sulfate from chicken sulphate: A complex molecule witii potential impacts ^^^^ cartilage. Asian-austialasian joumal of annual
sciences, 19(4): p. 601604.
EFFECTS OFTHE RATIO BETWEEN ALCALASE AND PAPAIN ENZYME TO THE PROCESS OF SHARK CARIUAGE (Carcharhmus dussumleri) HYDROLYSIS
Duih Huu Dong, Vu Ngoc Boi, Nguyen Thi My Trang Summary
In this paper, we present tbe results of the study on the effect of tbe ratio between enzyme alcaiase and papain on the content of soluble protein, peptide, Naa, N^^, and chondroitin sulphate formed in hydrolysing shark cartilage (Carcharhmus dussumien) by alcalase-papam mixture. The results showed that tbe suitable ratio of alcalase/papain in the mixture of alcalase-papam enzyme for shark cartilage hydrolysis is 60/40, After 10 hours of shark cartilage hydrolysis by alcalase-papain mixture at enzyme concentration of 0.296, temperature of 50"C, natural pH (6.8), sample weight of 2 kg and the addition of 2 liter water ratio, the hydrolyzate has the content of protein, peptide, Naa, chondroitin sulphate and NNHS higher than 6.54 times, 3.26 times, 7.91 times, 78.17 times and 1.25 times higher than the original.
Keywords: Mixture of alcalase-papain enzyme, protein, peptide, Naa, N^^^, chodroitin sulphate, shark je, hydrolysis.
Ngudi phan bi#n: TS. D6 Van Nam Ngdy nhlai bdi: 20/3/2020 Ngdy thdng qua phdn bi#n: 20/4/2020 Ngdy duy^t dang: 27/4/2020
