Tgp chi Khoa hgc - Cong nghe Thuy sdn Sol/^ij
KET QUA NGHIEN COU DAO TAO SAU DAI HOC
ANH Hl/ONG CUA LOAI ENZYME VA DIEU KIEN THUY PHAN DEN HOAT TINH KHU GOC T l f o O DPPH CUA PROTEIN ARTEMIA THUY PHAN
THE EFFECT OF ENZYMES AND HYDROL YSIS CONDITIONS ON DPPH RADICAL SCA VENGING A CTIVITY OF AR TEMI A PROTEIN HYDROL YSA TES
Nguyen Hong Ngdn', Nguyen Anh Tuan', Huynh Nguyen Duy Bag'
Nydy nhan bai 12/9/2012, Ngay phjn bien ihong qua: 12/10/2012, Ngay duyetdang, 15/3/2013TO.M I \ T
Nghien ciiu nay nhdm khdm pha hoat tinh khu goc tu do 2.2-diphenyl-l-piciylhydrazyl (DPPH) cua Protein Artemia Ihuy phan (APH) Prolein cua Artemia dtroc thuy phdn bdng cac enzyme Protamex, Neutrase, Flavourzyme vd Alcalase tmng 5 giir a SCC, v&i ty le enzyme/ca chdt (E/S) Id 0.3 75 A U/g nguyen lieu Tdt cd cdc enzyme deu lao ra APH co hoa!
tinh khit gdc tit do DPPH Protein Arlemia thuy phdn ben enzyme Protamex co hoat tinh khir goc tu do DPPH cao nhdt nin dd chon enzyme Protamex de nghien ciru dnh hucmg cua dieu hen thiiy phdn Kel qud cho thay ty li E/S. nhiel do, pHva thai gian ihuy phan protein Artemia bdng enzyme Protamex co dnh hu&ng den khd nang khu goc tif do DPPH ciia APH lao thanh
Tli khoa arienua, i'ii:r}me. cliiing oxy hoa, protein, thuy phdn ABSTRACT
The present study wai aimed lo investigate the 2.2-diphenyl-I-picrylhydrazyl (DPPH) radical scavenging aclmli' of Artemia Protein Hydrolysale? (APH) The Anemia protein wai hydrolyzed with Protamex. Neutrase, Flavourzyme and Alcalase for 5h at 5lfC. al an enzyme/substrate (E/S) ratio of 0375 AU/g matenal. All the enzymes yielded APH that possessed DPPH radical scavenging activity Protamex derived hydrolysates having ihe highe.st DPPH radical scavenging activity and was therefore used lo investigate ike effect of other hydrolysis conditions Time, temperature. pH and E/S ratio were shown to Influence DPPH radical scavenging activity of Protamex-derived .4PH
Keywords artemia. enzymes, antioxidant, protein, hydrolysates
I. DAT VAN D 6
Artemia la mpt loai giap xac nho co the sfing 6' nhCrng vitng nuac man co nflng do mufii dao dong Idn tir vai phan nghin den 250%o Vdi chu trinh sinh fryang ngdn, sau 10 - 15 ngay Artemia co the dat giai doan trudng thanh va tham gia sinh san Artemia CO the smh san lan ddu sau 8 ngay phat tnen Ivl6j ldn de khoang 300 trii'ng hoac con, vdi chu ky de 4 ngay/lan vi vay luong stnh I<h6i Artemia thu dugc rat Idn |1,2]. Artemia la loai nguyen lieu thuy san co ham luang protein cao, chiim 67,8% trpng luang I<h6. Hien lai, cac nghien cifu che bien Artemia con
rat han che dac biet la che bien Artemia thanh thifC pham cho ngudi chi/a di/gc quan lam Artemia cliu yeu dugc su' dung dudi dang sinh khoi ttiai dJng lam thirc an nuoi du triing tflm cang xanh, cua, tom bien va ca canh [5], Phan smh khoi dy thira tiifn chua dugc sii' dung mot each hieu qua, do vay viec nghien ciru che blfln Artemia thanh cac san pham thuc pham la can thiet nham nang cao hieu qua su dung loai nguyen Neu thuy san giau protein nay
Hien nay, co nhieu nghten cuu tach chiel cac chdt cfl hoat tinh sinh hoc, dac biet la d c chaloi hoat tinh chong oxy hoa tii' protein cua dong v^l
' Nguyen Hong Ngan Lop Cao hoc Cong nghe Sau thu ho^ch 2009 - Tru'ang Bai hoc Nha Trang
^TS Nguyen Anh Tuan, ^TS Huynh Nguyin Duy Bao Khoa C6ng nghe Thye pham - Tru'ong Oai hoc Nha Trang
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Tgp chi Khoa hoc - Cong nghe Thuy sdn Sol/2013
thuy san [7, 9, 15]. Protein thuy phan c6 ehi>a c^epeptide, acid amin e6 kha nang ch6ng oxy hoa dirq/c sir dyng^rOng rai trong cac linh vyc nhu Ihyc phim, thye pham chu-c nSng, my phim [11,14].
Vi v|y, nghien eCru kh^ nang eh6ng oxy hoa eiia protein Artemia thOy ph^n d l sir dyng Irong ITnh vyc thye phim cho eon ngu&i Ik mot hudng di mai gop phan sir dung hi$u q u i han nguon sinh khoi Artemia. IVIyc tieu cOa nghign ciru nay la danh gia inh hudng cua logi enzyme v i dieu ki#n thCiy phan din boat tinh khir goe ty do DPPH eua APH.
' II. OOl TU'g'NG, VAT LIEU VA PHU'aNG P H A P N G H I £ N CO'U
1. Nguyen vat lieu va h6a chat
Goc l y do 2,2-diphenyl-1-plcrylhydra2yl (DPPH) ' mua tir c6ng ty hba chat Sigma-Aldrich, Hoa Ky.
Cic hfia chat khae diing trong nghien ciru nay la I09I dgt tiSu chuin diing cho phan tieh.
' Dya v i o vi^e sd" dyng enzyme trong cae nghiSn i ci>u ve hogt tinh ching oxy hoa eua prtein thuy - phan tir sinh vat biln [6, 7,10], nghien ciru nay su dyng eae c h l pham protease thuang mgi bao g i m : Neutrase, Alcalase, Flavourzyme va Protamex cua hang Novo - Ban Mgch. Neutrase l i logi metallo endoprotease du^c thu nhgn tir vi khuin Bacillus amylollquefaciens; Alcalase l i loai endoprotease du(?c thu nhan ttr vl khuin Bacillus licheniformis;
PnDtamex ket hQ'p ea hat logi edoprotease v i exopeptidase dugc thu nh|n tir vl khuin Bacillus licheniformis va Bacillus amylollquefaciens;
Flavourzyme ket hgp ca hai logi edoprotease v i exopeptidase thu nhin tir nam Aspergillus oryzae.
2. Chuan bj protein Artemia thuy p h i n Artemia duyc thuy phin bang eac enzyme thu'ang mgi bao 96m Protamex, Neutrase, Alcalase v i Flavourzyme. Q u i trinh thuy phan duge giif 6 SO'C trong be on nhi^t trong thdi gian 5 gid. Hoat dO ciJa enzyme so vdi ea chat l i 0,375 AU/g. Hon hgp sau khi thiiy phan dua di b i t hogt enzyme d nhiet dp 85°C trong 10 phut, r l i Ipe tach bo phan b i thu dich protein Artemia thiJy phan (APH).
3. Phiramg phap phan tich
Kha ning khi> goc ty do DPPH cua APH xie djnh theo phuang phap cua Fu v i cOng sy (2002) [8,12].
Djeh thuy phin duge pha loang 5 tan bing nirOc elt. D6i vdi m l u do cho vao Ing nghigm 1,8ml nude elt, 0,2ml m^u tuang irng, 1ml methanol, 1ml DPPH Do djeh Uiuy phan cd m i u nen ta ein chuan
bj m i u blank. Mau blank cung ehuin bj tirang ty nhu'ng thay 1ml DPPH bing 1 mt methanol. 6ng ehuan gom: 2ml nude + 1ml methanol + 1ml DPPH.
Lie 6ku roi dem ti trong tmng toi d nhiet dp phong.
Sau 30 phut dem dl do dp h i p phy d hw6c s6ng 517 nm tren thlel bj UV-Vis mint 1240.
4. Phuvng phap xv ly so lieu
So lieu trinh bay trong bai bap nay l i g i i tn trung binh ciia 3 Iln thi nghigm ± SD (dO lech chuan). Tinh g i i tri trung binh v i ve do thj si> dung phan mem Microsoft Excel 2003. Sy khac biet co y nghTa ve mat th6ng k6 (p < 0,05) ciia c i c g i i trj tnjng binh dirge phan tieh tren phan mem thong ke R phien ban 2.15.1. Cac k i t y a, b, c, d, e, f khae nhau tr§n d6 thi the hi#n s6 lieu khae nhau co y nghTa d mdc p<0,05.
III. KtT QUA NGHIEN CLfU VA T H A O L U ^ N 1. Anh hu'dng ciia lo^i enzyme thiJy phan den hogt tinh khip goc tir do DPPH cOa APH
Anh hudng cQa logi enzyme thuy phan den hogt tinh khu goc l y do DPPH eua APH dugc trinh biy tren hinh 1.
N a u t i a H Flavounyme Enzyme thuy phin Hinh 1. A n i l htrcrng cua loai enzyme thuy pbSn d i n hoat
tinh k h v goc t i j do DPPH cua APH Tir ket qua nghiin eiru d d l thj Hinh 1 cho Ihly APH thuy phan bing cac loai enzyme khae nhau co hoat linh khir gic ty do DPPH khae nhau (p < 0,05).
APH thuy phan bang enzyme Protamex co boat tinh khir goc l y do DPPH cao nhit, tilp d i n l i APH thuy phin bing enzyme Alcalase va Flavourzyme APH thiiy phan bing enzyme Neutrase co hoat tinh khir goc tu do DPPH thap nhit. Enzyme Protamex thupe nhom endopeptidase ed kha nang phan c l l mgch protein tao thinh c i c peptide cd hogt tinh khu g l e t y do DPPH nen APH thiiy phan bing enzyme Protamex co hoat linh khir goc l y do DPPH cao han APH thuy phin bing enzyme Alcalase,
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Tgp chi Khoa hgc - Cong nghe Thiiy sdn S6l/m
Flavourzyme v i Neutrase. Vi hogt linh khd goc ty do DPPH eiia APH thiiy phan bang enzyme Protamex eao nhat nen da chpn enzyme Protamex de nghien edu anh hifdng eiia dieu kiin thuy phan.
2. Anh huOTig cua ty le enzyme Protamex/ca chat den hoat tinh khir goc t y do DPPH cua APH
Anh hudng eua ty le enzyme Prolamex/ca chit d i n hogt tinh khu goc tu do DPPH cCia APH dugc trinh baytrSndl thj hinh 2.
uim
0,075 0,150 0,225 0,300 0,375 0,450 Ty 1$ enzynte/cff ch&t ( A l / g ) Hinh 2. Anh hvfrng ciia ty le enz>me/co' chSl den hoat tinhkhir goc t v do DPPH ciia APH
Tu ket qua nghiSn cdu d do thj Hinh 2 cho thay khi tang ty le enzyme Protamex/ca chit tif 0,075 - 0,300 AU/g thi hogt linh khu- goe t y do DPPH eiia APH lang l&n (p < 0,05). Ro ring rang trang cOng dieu kien nhiet dg va thdi gian thCiy phin, khi lang ty t§ E/S thi toe dO thiiy phan ting len (3, 4], lugng peptide tgo ra trong APH nhieu nen hogt tinh khir goe ty do DPPH tang. Tuy nhiin.
khi tiep tyc tang ty 10 enzyme Protamex/ea chat tip 0,300 - 0,450 AU/g thi hogt tinh khd goc ty do DPPH cua APH lai giam di (p < 0,05). Hien tugng niy cd t h i giii thich nhu sau: Hoat linh khir goc ty do DPPH cCia APH chii yeu l i do eie peptide tham gia [6], khi lang ty If enzyme/ca ehat qua eao se lam thiiy phin cae peptide d i n den hogt tinh khir goc ty do DPPH eua APH giim. Hogt tinh khCr goc l y do DPPH ciia APH dgt eao nhat l i 84,54 ± 1,45% d t^
l# enzyme Protamex/ea chat l i 0,300 AU/g, n&n d i chon ty le nay de nghien euu anh hudng eCia nhigt d$, pH va thdl gian thiiy phan den hogt tinh khd goc ty do DPPH eOa APH.
3. Anh huwng ciia nhiet d? thuy p h i n den hogt tinh khip goc t y do DPPH cua APH
Anh hudng eua nhiet dO thuy phan den hoat tinh khif goe ty do DPPH cua APH dugc trinh biy tren fll thj hinh 3.
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i 1 I
Mitft d 9 thuy p h i n (%) Hinh 3. Anh hirdng ciia nhiet d(i thiiy phSn den hoat linb
khir goc t ^ do DPPH ciia APH
Tir k i t q u i nghi§n cuu d do thj hinh 3 cho thjy khi tang nhi0t dp thiiy phan td 30 - 5 0 ^ . hogt tinh ! khii' gic l y do DPPH cua APH thu duge tang I6n j (p < 0,05). Nhung khi tilp tyc tang nhigt do thiiy phin Id 50 - 60"C, hoat tinh khd goc ty do DPPH ciia APH thu dugc giim d i n . Trong trudng hgp rdy, thuy phan d 5Q''C thu dugc APH co hogt tinh ktii goe ty do DPPH eao nhit (88,46 ± 0,67%) nen iJa ehpn nhiel dp niy d l nghien edu inh hudng ciia pH v i thai gian thuy phin d i n hogt tinh khu gic ti/ do DPPH ciia APH bang enzyme Protamex.
4. Anh hu'dng ciia pH d i n hoat tinh khOgdcfy do DPPH cua APH
Anh hudng eiia pH din hoat tinh khu gic tir do DPPH cua APH du^c trinh biy tren hinh 4.
Hinh 4. Anh hiromg ciia pH d i n boat tinh khfr g^c t\rd(i D P P H cna A P H
Tir k i t i n h hudng (p < 0,05) flln hogt tinti khd goc ty do DPPH cua APH. Dilu niy la do ^ lam thay doi bin chit cua phin dng enzyme, anh hudng den c l u tnic protein eiia enzyme va ttiay d l i trgng t h i i ion hoa eua ca chat lim inh hi/ong den hoat dp eua enzyme, toe dp phin trng v i ttianh phan san phim thiiy phin [9,13]. Chinh vl v|y, hoet tinh khd goc t y do DPPH cua APH thay dli khi thay flli pH eua mdi trudng thiiy phin. ThOy phin tronB moi tnfdng eo pH = 7,0 thu duge APH cd hogt linh khd goe t y do DPPH cao nhit (83,96 ± 1,30%) ^
Tgp chi Khoa hgc - Cong nghe Thuy sdn So 1/2013
d i chgn pH n i y d l nghiin cdu anh hudng cOa thdigian thuy phan d i n hogt tinh khd g l e ty do DPPH cua APH bing enzyme Protamex.
5. Anh hvdng ciJa thoi gian thiiy phan den hoat tinh khiJ goc ty do DPPH cOa APH
Anh hudng cua thdi gian thuy phin flln boat linh khd goc t y do DPPH eiia APH dugc trinh bay tren hinh 5.
Thfrl gian thuy phin (gid') Hinh 5. Anh hirong ciia IhM gian thiiy phSn din ho^l tinh
khir g^c tv do DPPH ciia APH Tu k i t q u i nghien edu d flo thj hinh 5 cho Ihly khi kio d i l thdl gian thuy phan t d 0 - 4 gid, hogt linh khd goc ty do DPPH eCia APH lin ding ke (p < 0,05). Tuy nhien, hoat linh khd goe ty do DPPH cOa APH lai giam di khi tiep tyc keo dii thai gian
thiiy phin han 4 gid. Hien tugng niy c6 the giii thieh nhu sau: Trong giai dogn dau eua qua trinh thiiy phan bing enzyme, prolein bj thuy phan tgo thanh cac peptide cd hogt tinh khu" gic ty do DPPH.
Khi k i o dai thdi gian thiiy phan, eae peptide tilp tyc bj thuy phin nen hogt tfnh khu g l e l y do DPPH cOa APH giam di [9].
III. K £ T LUAN VA KI^N NGHj 1. Ket luin
Protein Artemia thuy phin bdi enzyme Protamex cd hogt tinh khu g l e ty do DPPH eao ban so vdi cie enzyme Neutrase, Flavourzyme v i Alcalase trong ciing flilu ki§n thiiy phan. Khi su dung enzyme Protamex, dieu ki$n thiiy phin thich hgp de thu duge APH co hoat tinh khu goc lu do DPPH cao l i : Ty Ig E/S = 0,300 AU/g, pH = 7, thCiy phan 4 gid d 50°C.
2. Kien nghi
Nghien cdu them ve moi quan he giira muc d0 thity phan va hogt tinh ching oxy hoa ciia APH.
Gk nang cao g i i In sd dung cua Artemia, cin tiep tyc nghien cuu su dung djeh flgm thdy phin protein Artemia fll lim Ihyc phim chuc ning.
TAI L i f u THAM K H A O Tieng Vift
1 Nguyin Thj Nggc Anh va egng tac viSn, 1997. Danh gia tiem nang thu sinh khoi Artemia tren mpng muoi VTnh Chau Bao cio khoa hgc hgi nghj cong ngli? sinh hgc toan quoc. NXB Khoa hoc Ky thuat. Hi Ngi, tr. 410 - 417.
2 Nguyin Van Hoa, 2005. Nang cao hieu qua ciia viec nuoi sinh khoi Artemia tren mgng muoi, Bao cao de lai nghien ciiu khoa hgc cip h^, Tnrdng D^i hgc Can Tho.
3 Nguyen Dire Lirgng, Cao Cuong. Nguyen Anh Tuy^l, 1^ Thj Thuy Tien, Ta Thu Hang, Huynh Nggc Oanh, Nguyin Thiiy Hirong, Phan HiSn, 2004. Cong nghe enzyme NXB Dai hgc Quoc gia TP Ho Chi Minh
4 Trin Thj Luyfin, 2006 Cac phin ling co ban va biln &hi ciia thye phSm trong qua trinh cong nghe. NXB Nong nghidp, TP.H6 Chi Minh, ir.9-22.
5. Ha Thanh Toin, 2004 Kha nang su dung sinh kh6i Artemia de san xuit thiie an cho thuy san. Bao cao de lai nghiSn cihi khoa hgc cip l)g, Vien Nghien ciiu va phat tnen CNSH, Trucmg Dai hgc Can Tho.
TlengAnh
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