HOI NGH! KHOA HQC C 6 N G N G H ^ SINK HQC TOAN QUOC 2013
TUY^N CHON CHONG NAM M 6 C SINH T 6 N G HP'P ENZYM TANNASE BANG PHL/aNG PHAP LEN MEN RAN
B§ng Hdng Anh*. NguySn Tliu VSn, Giang Th^ Viet; Phgm Hoai Thu, Ph?m Th] Thu —
Vi$n Cdng nghl$p thirc phimT6M TAT
Enzym tamuise (tannin acyl hydrolyase) U en^m xuc tac phan (Jng thi^y phln liSn kit esle cila phan tfr tannin giai ph6iig ghicoae vi a^t gallic, Siy li ei^ym c6 ohieu img dyng trong cdng nghiSp tb^c p h ^ va duoc phim. D|c bi$t trong c6ag a g h j ^ tfa^ic phim, Ang dung quan tr9ng nhat cua tannase la su dvmg cho v i ^ 1 ^ trong cic san phim do uong giiu tannin (tii hda tan, nu6c qua, nnni vang, do uong ci phe, bia...) d^ ngin vifc t ^ cjb dye v i gi&p nang cao chit luQng cim quan cua sin phim.
Enz/m tannase dugc sinh tong hgp chu yeu nhir cic chung nim mdc phirong phip len men iin vl phuong ph£p niy chu yeu t|0 enzym Qgo^i bio. Trong nghiSn cini niy, mgt sd cUlliig nam mdc phin 1^_ tiJr n g u ^ CO chit g ^ tannin trong tg nhiei^^
smi t$p giong vi nh^p ngo^i d i du^c n ^ £ n cuu kM ning tao enzym tannase tr^n mdi trufmg Tin. Ga tuy^ chgn dagc 02 chung nim mdc trong d6 c6 01 chung k^ hi^u MKv dugrc phin l|p tir tu nhiSn vi 01 ch&ng nhip ngoai k^ h i ^ TQ42 c6 khi ning tao enzym tannase cao nhit trong sd £ic chiing khio sit Khi Ifn men tren mdi tmong rin, ho^t l^rc enzym lin lugt U 10,2 v i 7,5 UI/ g ccr chit.
Dya tren m$t sd d|c diem hlnh thii v i sinh hpc phln tu £ xic djnh dugc c i hai chiing niy li Aspergfllus oryzae. Day la nhfing chiing nam mdc c6 n h i ^ t i ^ nang ling dyng Irong sinh tdng hqp enzym tannase nhjr pbuong p b ^ ISn men tin.
Tir khda: l&i men rin, tannase, Aspergillus
MdcAu
Enzym tannase (tannin acyl hydrolyase, .EC 3.1.1.20} xiic tdc phan i>ng thiiy phSn liSn k i t este trong phfln ti> tannin thdy phdn, gISi phdng glucose vd axit gallic. Sdn pham ci:ia phdn i>ng thOy phdn Id axrt gallic cd nhieu u'ng dgng quan trgng trong cfing nghiip th^c phSm vd duvc phSm (Batra A. efa/., 2005;Sabu A. ef aA, 200SJ. Enzym ndy c6n diF^c si>
dgng trong ru'p'u vang vl trong ru^u vang cdc chSt mau chu ySu Id tannin tuy nhl#n khi tilp xiic vdi khdng khf tht tannin s i bj oxy hda tgo dgc c|n cho san phim. Ngodi ra tannase c6n diro'c si> dgng b'ong sdn xuat cdc logi dh u6ng gidi khdt .cd chi>a cd phi, diing d l Idm trong bra vd nu^^ qua, I n djnh hdm lug'ng polyphenol cua malt dgi mgch v^ cdn dUM^c
diing nhu' m$t diu id d l xdc dinh clu tnic cOa cdc loal este axit gaclllc cd trong tg' nhien, tuy nhlSn i>ng dgng thi/ong mgi Idn nhlt cOa tannase Id trqng ngdnh sdn xult trd hda tan. Gan ddy, tannase c6n dug'c si> dgng cho vi^c phdn cdt cdc hgp ch^t polyphenol trong mdng t l bdo thuc v^t, dllu ndy r l t can thilt cho qud trlnh tiln xO 1;^ thO'c dn dio ddng v$t (Belmares R. ef al, 2004).
Enzym ndy cd thS dugc tgo td cdc nguin dgng v$t, thi,pc v$t, vi sinh v§t, tuy nhlSn tannase tCr vl slnh v|t thu&ng I n djnh hon to cdc nguin dOng v§t, thi/c v$t. Tannase sinh tong hg'p tCp nim mlc thud'ng hogt dgng trong khodng pH vd nhi^t
dd rOng. Da cd mOt s l nghign cuu cho thiy tannase sdn xuSt bdng phucrng phdp Idn men rln thi thudng uu vi|t horncdc phuong phdp len men chlm vd ISn men be m|l Idng do l l n men nhdr phuang phdp nay thi chii y l u tgo enzym ngogi' bdo vd hogt luc tgo enzym bing phuong phdp ISn men ran cQng cao hon, dieu ndy gliip thu^n Igi hon cho qud trlnh thu hii sdn phim (Purohit J.S. eta/., 2006). Trong nghiSn cthi ndy chiing-tdi da tuyln chgn m$t s l chiing nim rnlctucdc nguin khdo nhau (phdn Igp tir t u nhiSn, suu t|p gilng vi sinh vdt cdng nghigp, nhSp ngogi) vdl myc dich chgn dugrc chOng nam mlc c6 t i m ndng tgo enzym tannase hogt li/c cao de i>ng dgng trong ISn men tgo tannase theo phuong phdp lfin men rln. --^ . - . - . . • • •-.-.._ =^^^- NGUVeN U$U VA PHU'aNG P H A P
NguySn li^u
Nguln ph3n Igp nim mlc cd kha ndng sinh tong hg'p enzym' ba qud mac kham (m^t logi qud cd hdm lug'ng tannin cao, thudng cd d vCing nili phia bic), ba nho, IS, tao, sung, mit, chull, trd mgn, trd den, cdm gao, cOi cg...-
MQt s l chOng nam m l c cd trong b$ suu tgp gilng vi^inh v$t cdng nghigp cua \^$n Cdng nghigp Thi,rc phim, ky hi$u TV, TQ, A. No.1, A. No.2, A. No. 3. VA. _ _ MOI s i ChOng nim mlc nh|p ngogi k^ hifu TQ38, TQ 41, TQ 42 (Id cdc chCing trao dli'vdi phia d l i tdc Taing QuIc trong khudn khi de tai hgp tdc nghj i^nh thu).
Mdi tnrdng phdn l$p n i m mic PDA (nude chilt khoai tay 200g/1, glucose 20g/l, agar 20g/l).
Mfll tOFdng T1 (K2HP04 0,5g/l, KHzP04 0,5g/l, MgSO* 2g/l, NH4CI 3g/l, CaClz Ig/I, axit tannic 2 g/l, agar 20g/l).
Mdi tardng T2 (K2HPO4 0,05%. KH2PO4 0.05%, •MgS04 0,2%, NH4Ct 0,2%, CaClz 0,1%, axit tannic 0,2% b l sung vdo ggo hip chin d l c6 mdi trudng ran).
Mdi trudng TA (axit tannic 2g/I, agar 20g/l).
Mdi trudng ISn man r l n ( M t tannic 2%, glucose 0.1%. NH4CI 0,3%, KH2P04 0,2%, MgSO* 0,2%, FeSO^ 0,3%, CuSO*
0,2%, CaCb 0,1%, Tween 80 0,2% b i sung vdo ggo hap chfn).
Phuong phdp nghien cii'u
Phuvng phip phin l^p chungytim m6cHOI NGHI KHOA HQC C C N G NGH? SINH HQC T O A N Q U 6 C 2013
Cdc n g u i n nguySn l i ^ d u ^ thdi nhd ho#c bam ndt vd nudi trong d i l u kign t u nhiSn d l ISn m i c . Cdc churig m i c dug'c c l y t r s l i m e . trudng PDA d nhi$l dO 30=0 trong 72h vd t i l n hdnh phdn lap de thu d u ^ c cdc chung t h u i n khiet X i c d / n h ho9t dd onzym tannase (Mondal K C. et al.. 2001)
NguySn t i c : Phucmg phdp dua tren c o s d IhiJy phdn axft tannic bdi enzym tannase c6 trong m l u ^ n PhSn Uch s a n phliTT tgo thdnh iA d u d n g glucose vd axit gallic. XSc djnh cudng dO mdu tgo thdnh cua axit tannic cdn tgi v d i FeCIa Id c h l l h i l n mdu.
Mdt d o n vt hogt O0.ciia tannase Id k/^ng enzym c6 t h i xiic tdc thiiy phSn 1 mmol axit tannic sau 1 phut cr d i l u ki$n tiSu c h u i n .
Cho vdo 2 i n g nghiem m i i i n g nghi$m 0,3 ml axit tannic 0 , 1 % . Ong nghiem 1 la m l u d l i d i u n g : t>i sung thSm vdo 0.5 ml nude c i t Ong nghiem 2 Id m l u thf nghl$m: b i sgng thSm vdo 0,5 ml djch c h i l t enzym tho.TiSn hdnh tdc d e u hai 6 n g nghi$m r i i dem u * 60"C b i n g mdy i n nhift trong 30 phiiL Di>ng phdn dng bang cdch them vdo hai 6ng n g h i f m m o i i n g 3 ml BSA. Do BSA phdn ung vdi axit tannic tgo thanh kel tOa. Sau khi phdn Cmg k i t thiic thi nnang hal Ong n g h i l m di ly tdm vdi l i c d<t 4000 vdng/phiit trong 5 phiit ttiu d u g c k i t tiia. Tdch l l y p h l n k i t tua d hai i n g n g h i f m , phSn k i t tua ndy d u ^ hod tan b i n g 3 ml SDS - Trtethanolamine. Sau dd b i sung vdo m i i i n g nghifm 1 ml FeCb 0,01 M vi glQ- trong 15 phCit d l i n djnh mdu. Oo h i p Ihu quang cOa djch trong hai i n g nghifm d b u d c sdng A 530 nm.
X i c ainii khi ndng thdy phdn tannic bing each Oa vdng dtOyphSn tannic
NguySn t i c : Di/a IrSn khd nSng thuy phSn axft tannic cua tannase vdi FeCb Id c h i t h i l n thj mdu, dung djch F e C l j p h d n Cmg vdl nhdm F^enol cua axit tannk: Igo ra ntdu ndu.
Phuong phdp c&y c h l m d i l m : Cdc chOng n i m m i c dug'c c l y c h l m d i l m trSn mdi t n r d n g thi> n g h i f m T l , nudi cay d 30°C Irong 72h. O i dung djch FeCb 0,01M ISn b l m§t dfa thgch. Do vdng phdn gidi t h i h i f n hogt tfnh tannase d i a n ^ m m i c . D u d n g kinh vdng thiiy phdn dugrc xdc djnh b i n g gid tn D-d, trong dd D: d u d n g kinh vdng thOy phSn, d: d u d n g kfnh n i m m i c
Phuong phdp d g c l i : ChOng n i m m i c d u g c nudi trSn mdi tnrdng T2, sau dd c h i l t enzym tannase b i n g dung djch N a C I 0,5%. Sau fchi Ihu djch c h i l t enzym IhA, nhd 0,02 ml djch enzym vdo cdc dfa petri cd chda mdi t r u d n g TA da dtfcrc d g c lfi Inrdc vd dug'c nud) d 30°C trong 72h. D i dung djch FeCIs 0,01M ISn b l mgt dTa thgch. D o vdng ph3n gidi I h l h i f n hogl llnh tannase ciia n i m m i c . D u d n g kinh vdng thiiy phdn ducrc xdc djnh l i i n g gid tq D-d, trong dd D: d u d n g kfnh vdng thOy phdn, d: d u d n g kfnh (6 dye
Phuvng phip lin men rin
S u dgng mdi trudng ISn men r l n d l thuc h i f n qud trlnh ISn men. D i l u k i f n thuc h i f n qud trinh ISn men n h u sau: m d i tnrdng d u g r d l trong cSc khay g i kfch t h u d c 50 x 50 cm, nhift d$ ISn men 30°C, d$ I m mdi t r u d n g len men khoang 41,5%, dd ddy k h i i u t i t n h l t Id 1-4 cm k i t hgrp thSm thdng khf b i n g cdch ddo trOn d i o khodng 8 g i d / l i n , duy tri i m t r i n b l m f t cOa k h i i nudi b i n g cdch d f y vdi I m d l trdnh khfl b l m $ t giu- d f I m khdng khf frSn 9 0 % . TTidi gian nudi 4 0 gid, sau dd s d dgng dung djch d f m axetat pH 5 d l c h i l t thu d[ch c h i l t enzyme diing cho phdn Kch.
Dlnhtdit vl slnh vft bing glil trinh pr
Phdn dogn rADN ciia vi slnh v f t dugrc khuylch dgi bang m i i I T S I , NL4 frSn t h i l t b[ GeneAmp* PCRSystem 9700 ( P E Applied BkMystem. My) vdi chuong trinh n h i f t d u g c t h i l t I f p v d i pha b i l n tinh d 94''C trong 2 phiit k l t i l p Id 35 chu k^
nhift (94°C trang 30 gidy, 52 "C bnog 30 gidy vd 72 -C trang 1 phiit). Qud trinh k h u y l c h dgi d u g c hodn t i t * 72 X trong 7 phiit vd sau dd sdn p h i m PCR d u g c bdo qudn d 10 °C.
Sdn p h i m PCR sau khi khuylch dgi dug^; tinh c h l bdng QIAEX II (Qiagen. Due) theo k h u y i n cao cOa hdng Trlnh t i / fiDN a\K?c dpc bdng phuong phdp "dideoxy chain temnination' vdi v i f c s u dung kit AmpliTaq (Amersham) theo hud'ng dSn cua hdng vd cdc m i i I T S I , ITS4. Mdy dgc trlnh t u , model 377 o i a hang Applied Biosystem d u ? c st> dgng C a c chufli ADN d u g c so sdnh vdi GeneBank thdng qua giao d i f n tlm k i l m BLAST nucleotide-nucleotide dat tai National Center for Biotechnology Infwmatkm. Bethesda, My (http://www.ncbi.nlm.nih.gov). Cac c h u i i lidn quan d u o c c h u y i n tai v l sau dd x u ty blrjg p h l n m i m BbEdit vd so sdnh bang ClustaiX.
Danh sich c i c m ^ air dgng trong p h i n lo^i
TCCGTAGGTGAACCTGCGG GGTCCGTGTTTCAAGACGG TCCTCCGCTTATTGATATGC
K ^ QUA V A T H A O LUAN
P h l n I 9 P v i t u y l n c h g n c h i i n g nam m i c t^r nhiSn c 6 kha nSng s i n h t o n g h o p enzym tannase PMn Ifp eic ChOng nim mSc tynhldn ft> ede ngudn nguydn lieu khae nhau
Nguin nguySn l i f u thu t h f p d i u Id nhfmg vd vd thjt qud cd \^ d i d t cd hdm lugrng tannin cao d l vdi mong m u i n thu d u g ' c h f vt sinh v f t c6 khd ndng tgo enzym thuy phdn n g u i n c o c h i t dd.
HOl NGHI KHOA HQC CONG NGHI? SINH HgCTOAN QU6C 2013
- C ^ c j g u o n ngtjyen l i f u d u p e thdi-nhd h o f c bdm ndt vd nudi trong d i l u kifin t u nhien de m i c phdt t r i l n . Cdc chiing m i c d u g c c l y trSn mfli t r u d n g P D A d nhift d$ SCC t r o r i g ^ h ^ " p l T d r r i 5 p r S a u n h i l u l i n Cay"chuyln"trSn~cdc dTa P e t n 7 ^ 4 "
chung moc thuan da thu nhgn d u g c
HInli 1. Hlnh dnh k h u l n Igc m$t so chiing n i m m i c da phdn I f p
Cdc ChCing ndy t i l p tgc dug'c c l y c h l m d i l m trSn dTa petri chira mdi tordng TA d SO^C trong 72h. Cdc chOng n i m m i c dug'c kl h i f u theo t6n_cua c bdo t o . K i t qui cdc chiing cd khd nang sinh t i n g h g p tannase d u g c trlnh bdy trong bdng n g u i n nguySn l i f u vd mdu s
sau ddy:
Bdng 1. K i t q u i danh gid scr b f cdc chiing n i m m i c t u n h l i n co kha nang sinh t i n g h^p tannase K;^ til^u chung n i m m6c Khd nSng smh ting hipp tannase
T r i mgn M ^ k h a m Mac kham Mdckbam Nho Cdm ggo TfSden
CClicQ LS T^o mSo Chuii xanh
(-): Khdng od kha nSng sinh enzym tannase
(+): Cd khd ndng sinh enzym tannase ^ Theo bdng trSn Ihi 5 chiing m i c : S, CC, L, CX, SS khflng cd kha nang sinh tong hpp tannase, 9 chiing n i m m i c : M, TM, MK, MKx, MKv, N, CG, T D , T d i u cd khS ndng sinh t i n g hg'p tannase tren mdi trudng TA.
Lya chgn chdng cd kha nSng slnh tang h g p tannase c a o td" ngu6n tip nhien, suv tfp gidng vi nhfp ngo$l Hogt tfnh tannase cOa 9 chiing M, T M , MK, MKx, MKv. N. CG, TD, T, cdc chCIng cd trang s u u t f p g i i n g TV, T Q , A.No.1, A . N D . 2 , A.No.3, VA cd trong s u u t f p g i i n g vd n h f p ngogi TQ38, T Q 4 1 , TQ42 da d u g c so sdnh vS hogt tfnh tgo enzym
• tannase bang cdch cay chdm d i l m trSn mdi t m d n g thgch T l vd nudi d n h i f t do SO^C trong 72 g i d . B l dung dich FeCb 0,01 M lfin b l mgt dia thgch. Do d u d n g kfnh vdng phdn gidi t h i h i f n hogt tinh tannase cua cdc chCing n i m m i c , k i t qud d u v c trlnh bdy d Bang 2:
Bdng 2. K h i n i n g t^o enzym phan giai axit tannic ciia c i c chung n ^ m i c
K]? h i ^ chiing {lirdng kinh vdng thiiy phdn
(mm) K i t qua frfin mSI t n r d n g T l cho t h i y trong t i n g s i 9 Chung n i m m i c phdn Igp tg^ nhifin ^ 1 chung- MKv cd kha ndng tao enzym phan gidi axft tannic cd d u d n g kinh vdng phdn gidi Idn n h l t . Ngodi chiing MKv thi 2 chiing MK, MKx cung cd k h i ndng sinh t i n g hpp tannase cao hon dang k l so vdi cdc chiing cdn Igi vd cd d i l m Chung la deu d u g c phdn I f p tCr n g u i n nguySn l i f u qua mdc kham (hay cdn ggi Id me rCrng, m f t togi qud rat giau tannin cd n h i | u tgi vOng niii Cao B i n g ) , d i l u nay cho t h i y ddy Id nguySn l i f u gidu tannin rdt thfch hg'p Cho s u phdt trien cCra n i m mdc tgo enzym phdn giai tannin. Trong s i cdc chOng phdn t f p ditgc tCr n g u i n t y nhien chCing tfii da Ig'a chgn chCing MKv d u g c l y a chgn cho c^c nghiSn c d u t i l p ttieo.
Chiing tdi da tien hdnh ifin men tgo enzym tannase trSn mdi trudng ran T2 Id c o m h i p cd b i sung c h i t dinh d u d n g . Sau khi k i t thiic qud trinh Ifin men thi t i l n hdnh , c h i l t djch enzym tir mdi t r u d n g vd xdc djnh hogt tinh enzym tannase dinh tinh bang cdch O djch c h i l l enzym trong dta thgch TA trong 36 g i d . K i t qua vdng thiiy phdn thu d u g c Id 11 m m . B i l u ndy cho I h l y MKv Id chOng n i m m i c cd n h i l u hda h^n i j n g dgng trong nghiSn ci>u sinh t i n g hgp enzym tannase vi cd khd ndng tgo enzym tannase hogt l u c cap.
A.No.1 A No.2 A No.3 VA TQ38 TQ41 TQ42
HQI NGHI KHOA HQC C O N G N G H $ SINH HQC T O A N Q U 6 C 2 0 1 3
D i l v d i cdc chCing n&n m i c c d tmng- s u u t f p g i i n g thI cho khd
ChCing phdn i f p t u nhifin vd n h f p n g o ^ , d i l u nay cd ttil do ddy khflng phdi cdc chCing n i m m i c thfch hg'p cho sy phdt t r i l n trSn mdi trudng gidu tanrtin vd sinh t i n g hpp enzyme phdn gidi tannin. Offi vdi 3 chiing n h f p ngogi thi khd ndng tgo enzym tannase Id tutrng d i i l i t d f c b i f t Id chiing TQ42 dio d u d n g Wnh vdng thQy phdn dgt 9,5 mm. chf-dung sau c^ung phdn I f p t u nhiSn MKv.
Do v f y hai chiing MKv vd TQ42 d u ^ si> dyng cho cdc nghiSn ciru tiep theo v l dgc d i l m hlnh thdi \ ^ dinh tfin d l Idm c o s d cho vifc irng dgng treing nghifin cOu slnh long hgp enzym tannase Iheo phirong phdp ISn men r l n .
Xdc d j n h m f t s i d f c d i l m htnh thdi v d d j n h tSn 02 chung n i m m o c c d khd nang s i n h t i n g h g ^ e n z y m t a n n a s e cao
Dfe diim hinh thil vi d}nh tin chdng 7042 Kit qui blnh lir gen ARNr vOng ITS cua chung TQ42
TTGCCTCAGTAACGGCGAGTGAAGCGGCAAGAGCTCAAATTTGAAAGCTGGCTCCTTCGGGGTCCGCATTGTAATT TGCAGAGGATGCTTCGGGTGCGGCCCCTGTCTAAGTGCCCTGGAACGGGCCGTCAGAGAGGGTGAGAATCGCGTC TGQGATGGGGTGTCCGCGCCCGTGTGAAGCTCCTTCGACGAGTCGAGTTGTTTGGGAATGCAGCTCTAAATGGGT GGTAAATTTCATCTAAAGCTAAATACTGGCCG6AGACCGATAGCGCACAAGTAGAGTGATCGAAAGATGAAAAGCAC TTTGAAAAGAGAGTTAAAAAGCACGTGAAATTGTTGAAAGGGAAGCGCTTGCGACCAGACTCGCCTCCAGGGTTCA GCCGGCATTCGTGCCGGTGTACTTCCCTGGGGGCGGGCCAGCGTCGGTTTGGGCGGCCGGTCAAAGGCTCCCGG AATGTAGTGCCCTCCGGGGCACCTTATAGCCGGGAGTGCAATGCGGCCAGCCTGGACCGAGGAACGCGCTTGGGC ACGGACGCTGGCATAAT (557 bp)
K i r qud 80 sdnh vdl trinh t y tuong d i n g trang GenBank: T u o n g d i n g 100% vdi AB470911 cOa Aspergillus oryzae XJ29; T u o n g d i n g 100% EF409801 cOa AspergHlus llavus ucb035, d i l u ndy dua ra k i t l u f n chiing MKv t u o n g d i n g 100% vdi Aspe/gAfus oryzae/ Asperg^s Bams ( A o o ^ a a d u g c coi Id dgng t h u i n h d a ' cOa A. llavus).
Die dl4m hlnh thii eda c/iung 7 0 4 2
Klch thudc k h u l n Igc sau 7 ngdy nudi d y trfin mdi Inr^ng Czapeck: 55- 70 mm;
Mdu s i c k h u l n Igc: Trfin mfli tnrdng Czapeck, k h u l n Igc cd mdu vdng xdm d i n mdu ndu oliu, s p i n i m mdu t r l n g , b f n chft, x u l l h i ^ hgch'nim mdu tic. m f t trdi khdng mdu, sau ngd sang mdu vdng xdm n h g t k h u l n Igc x i p , bdo tCr m d u oliu tdi ndu d i u , k h u l n Igc cd dgng bflng x i p , c6 sgi khf sinh, cd x u l t h i f n m f t sd hgch n i m nhd. mdu trang.
Cdc d f e d i l m d u tnic h i l n vi: C u i n g sinh bdo t d ddi, p h l n g i n bgng t h i n g , chO y l u rdp, ftlnh thodng cOng c d c u o n g nhan, kfch thudc 500-2500 x 10-25pm. Bgng n h i l u hlnh dgng: g i n d u h o f c kSo ddi hlnh qua IS, kfch t h u d c (10)-20- 50
|im. Cd d 2 dgng t h i binh, khi cdn non dgng 1 t i n g Id chd yfiu, khi gla dang 2 t i n g chQ y l u . Kfch t h u d c ttii blnh s o d p Id 7-12 X 4-6 pm chiing bao phii khodng V4 d i n V* bgng, t h i blnh t h d d p hlnh chai nguoc, kfch t h u d c 7-12 3-5 pm Bdo ti> ttiudng cd rdp, c6 gai, cd n h i l u dgng: hinh dgng d u , g i n d u , hlnh tnrng. eiip kfch t h u d c 3- 7,5 (10) p m . Dya vdo phfin tfch hinh thai vd Wnh l y gen rARN wing ITS k i t tuf n chOng TQ42 ttiufc v l lodi Aspergillus oryzae.
D#c diim hlnh thil va djnh (Sn chdng MKv K4I qui tiinh ftr gen ARNr vdng ITS cOa chiing MKv
TGCGGAAGGATCATTACCGAGTGTAGGGTTCCTAGCGAGCCCAACCTCCCACCCGTGTTTACTGTACCTTAGTTGCT
^^^^^^I^^^^J^^^^^^^GACGGGCCCCAAAGGCAGCGGCGGCACCGCGTCCGATCCTCGAGCGTAT
GGGGCTTTGTCACCCGCTCTGTAGGCCCGGCCGGCGCTTGCCGAACGCAAATCAATCTTTTTCCAGGTGACCT y ^ 3 ( ? " T . ™ « ^ * ' ^ n J S l " ^ t : ^ ^ " 3 ^ " ^ ^ GenBank: T u o n g d i n g 100% v d i AB470911 o i a Aspergillus oryzae C^l' ^ ? ^ ^ ^ EF409801 cua Aspergillus flavus ucb035, d i l u ndy c h o k i t l u f n chOng IMKv l u o n g d i n g 1 0 0 % vdl Asper^lus oryzael Aspergillus llavus {A. oryzae dirge boi Id dgng T h u i n h d a ' d a A. flayus)
D$c aim hlnh thii cda chdng UKv
Kich thudc k h u l n Igc sau 7 ngdy nudi d y IrSn mdi turdng Czapeclc 55- 70 m m ;
HOI NGHj KHOA HOC C O N G N G H ^ SINH HQC T O A N Q U O C 2013
~ M d a ' s l r k h t i l n - i g c r . T r f i n moitordng-Czapeck, k h u l n - l g c c d m a u - v d n g - x a n r d S m T i d a - n a u T ) l i u r s g t T i l m T n d u - t r l n g r b f n - chgt, x u l t h i f n hgch n i m mau toi, mat trdi khdng mdu, sau nga sang mau v^ng xdm nhgL k h u i n Igc x i p , bdo tCr mdu oliu t d i ndu oliu. k h u l n Igc cd dgng bdng xdp, sgi khi sinh It, khdng thay h a d i nam xuat h i f n .
Cdc d f e d i l m d u tnic h i l n vi: C u i n g sinh bdo t i r ddi. p h l n g i n bgng ttiing, chii yeu rap, thinh ttiodng cOng co cudng nhan, Jtfgh thjrdc 500-2500 x 1j0-25pm. Bgng nhieu hlnh dgng: g i n . d u hogc keo dai hinh qud.JS, kfch t h u d c (10)^20- 50 pm. C d d 2 dgng t h i blnh. khi con non dang 1 t i n g id chu y l u , khi gid dgng 2 tang chO yeu. Kich t h u d c t h i binh s o d p la 7-12 X 4-6 p m chiing bao phii khodng Vi den V» bgng. t h i blnh ttiO- d p hlnh chai n g u y c , klch t h u d c 7-12 3-5 p m . BSo tCr t h u d n g c6 rdp. cd gai, cd nhieu dang: hinh dgng dia, gan d u , hinh trdng, elip kich t h u d c 3- 7.5 (10) p m . D y a vdo phSn Itch hlnh thai vd trinh t y gen rARN vCing ITS k i t l u f n chung MKv t h u f c v l lodi Aspergillus oryzae.
Hlnh 4. Hinh dnh cdc co- quan sinh a i n cua chung TQ42 a. I h l Hlnh 5. Hlnh Snh cac c o quan slnh sdn ciia chiing MKv a binh 2 tang. b. i h l blnh 1 ting, c lido ti> (a.b,c sau 7 ngdy nudi t h i binh 1 ting, b. t h i binh 1 vd 2 ting. c. bdo tir. (a.b.c sau 7 d y , bar 20 Mm), d. khuln Igc trSn moi tnrdng Czapeck sau 4 ngdy ngdy nuSi cay, bar 20 jim). d. khuln Igc IrSn mdi tnrdng n u d i d y . ' Czapeck sau 4 ngdy nuOl d y .
' K h d n d n g sfnh t i n g hg'p enzym c i i a cdc chOng t r e n md) trtpong ten m e n ran
Chiing n i m m i c TQ42 dd diroc si> dyng de nghiSn c d u thdnh p h l n mdi t o r d n g vd cdc d l l u k i f n t i i u u cho qud trlnh ten men r l n v d cho k i t aud Id dgt d u g c hogt l y e enzym tannase trSn mdi t n r d n g len men r l n Id 7,5Ul/g c o c h i t . Khi Sp dyng d l l u k i f n ndy d i i vdl chfing MKv thi dgt d u g c hogt luc enzym Id 10,2 U!/g c o c h i L Ddy Id k i t qud khd t u o n g d i n g so v d l d c nghifin c d u da t h y c h i f n tren I h l gidi (theo cdc nghiSn cCcu da cdng b i dgt khodng 4-13 U!/g co c h i t ) (Belmares R. et al., 2004; Sabu A. ef al., 2005).
Ngodi ra d l k i l m tra mirc d f an todn t h y c p h i m cCra d c chting g i i n g , sau khi k i t thiic qud trlnh ISn men r l n bdi 2 chiing n i m m i c , chCing tfli da giri m i u mdi t n r d n g ISn men di k i l m tra dgc td vi nam aflatoxin tgi Trung tdm phdn tfch v d gidm djnh thyc p h i m Q u i c gia (thufc V i f n Cflng n g h l f p T h y c p h i m ) . Ket qua cho t h i y khfing phdt h i f n d f e t i vi n i m trang m l u mdi trudng lfin men d i i vdi c d haixhOng n i m m i c , d i l u nay dam bao an todn t h y c p h i m khi si> dyng d c ' chiing n i m m i c ndy trong O'ng dyng ISn men sinh t i n g hgp e n ^ m tannase n h d phirong phdp Ifin men r l n . K ^ L U A N
T d cdc n g u i n phdn I f p t y nhien, s u u tap g i i n g vi sinh v f t vd n h f p ngogi da t u y l n chgn dug'c 02 chiing n i m m i c trang di5 CO 01 chOng ky h i f u MKv d u y c phdn ldp ti^ t y nhifin v d 01 chiing nhgp ngogi ky h i f u TQ42 cd khd nSng tgo enzym tannase cao n h l t trong s i cdc chung da khdo skt vd khi ISn men trfin mfli t a r d n g ran da thu d u y c hogt l y e enzym lan
!ugt Id-10,2 vd 7.5 Ul,' g co' c h i t . D y a trSn m f t s i Cfo d i l m hlnh Uidi vd p h u o n g phdp sinh hgc phSn tit di xdc d i n h "
d u y c c d hal chiing ndy Id Aspergillus oryzae, ddy Id nhO'ng chOng n i m m i c cd nhilu tiem ndng Ci-ng dyng trong sinh t i n g hgp enzym tannase n h d phuong phdp ISn men r l n .
T A I L I E U THAIVl K H A O
Belmares R COntreras-Esquivel J C, Rodriguez-Herrera R, Coronel A R, Aguilar 0 N (2004). Microbial prciduction of tannase. an enzyme wilh potential use in food industry. Lenesm-Wiss u-Technol. 37:857 - 864 ,;^.
Chdvez- Gonzdiez M, Rodriguez- Duran L V. Balagumsamy N, Prado^anagan A, Rodriguez R, Contreras J C, Aguilar C N (2011).
Biotechnological advances and challenges of tannase: An ovenriew. Food Biopnxess Technol DOI 10.1007/sl 1947-011-0608-5.
IbuchiS. Minoda Y, YamadaK(1972). Hydrolyzing pathway, substrate spedfidty and inhnbidon of tannin acyl hydrolase of Aspe^tf/us oryzae Ho 7. Agnculturai and Biolo^ai chemistry X {ay. ^5S3-^S6Z
Mondal K 0 . Baneijee D, Jana M, Pati B R (2001). Colonmetric assay method for determinalion of the tannin acyl hydrolase. Analytical Biochemistry 295: tea -171
Lekha P K, Lonsane B K (1994). ComparaBve litres, locaBon and pnsperties of tannin acyl hydrolase produced by Aspergillus niger PKL 104 in solid-state, liquid surface an submerged fermentaSons. Proeoss Biochemistry 29 (6): 497 - 503.
Paranthaman R. Vldyalakshmi R, Murugesh S, SIngaravadivel K (2009) Optimization of various culture media for tannasse production in submerged feffnentanon by AspergSlus flavus. Advances m Biologic^ Research 3 (1-2); 34 - 39.
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Rakesn K, Jrtender S. Randir S (2007) ProdocSon of tannase from AsperwBus njber under solid-state famentaSon using jami (Syzygium cumini) leaves. Microbiolo^c^ Research 162:384 - 390.
Jana A: Maity C. Hakier S K Mondal K C. Pati B R, Mohapatra P K (2012). Tannase producfen by Penic^iurn^uwgenum PAF6 in Krfid slate femenlaeon of tannin-rich plait rssklues following OVAT and RSM AppI Biochem aotec/iro/167:1254 -1269.
Pufohft J S, Dulta J R, Nanda R K. Baneije© R (2006). Straki improvement to tantiase production from OMairture of A5p«gfl/os ^ and Rtilzopua cyzae. aorasource Technology ST. 793 - 801.
Sabu A, Wran G S. Pandey A (2005). Purification and Characterization of tannin acyl hydrolase from Aspergillus niger'ATCC 16620.
Food Tec/Mo/. 43(2): 133 - 1 3 8
THE SELECTION OF MOLD STRAINS TO BIOSYNTHESIZE TANNASE BY SOLID STATE FERMENTATION
Dang Hong Anh*, Nguyen Thu Van, Giang The Viet, Pham Hoal T h u , Pham Thi T h u Food Industries Research InstSute
SUMMARY
Ttie tannase enzyme (tarmin acyl bydrolyase) catalyzes the hydrolysis of ester and depside tMJbds in hydrotysable tannins, releasing glucoie and gallic acid, this enzyme has nmiy applicaiioiu in tfie food industry and die phamiaceutical industry. Especially, in Uie food industry, the biggest commercial i^UcaCioDS of tannase are given in clarificaticHi of rich tannin iKverages 0 » s 1 w ' t^a, fiuit juice, wine, coffee-flavored soft drinks, beer etc...) to prevent sedimeaC and to improve quality of the products.
The t u n a s e is bioaynthesized mainly by molds. Tannase production by solid state fermentatiori is more advantageous than submerged fermeiitatioD due to the iKodnct of this process b ^ n g extra cellular e n ^ m e . In this research, some mold strains i ^ c h were iiolated &om rich tannin source in nature or taken fiom industrial microoiganism collectioD and inqxnted w e i e studied tannase productivity in solid Hate medium. Two mold strains w o e selected (one isolated fiom nature,-named MKv a n d o n e from imported lource, named TQ42) due to the highest tannase activity production among die studied strains, conelativcly 10.2UI/g aad 7.5 IH/g.
Base4 on some moridiological charmcteiistics and the molecular biology method, two strains were isolated as Asper^lhiS oryzae, diese mold strains have high potential application in tannase biosynthesis by sohd state fennentation.
Keywords: solid state fermentation, tannase, Asper^ba
•Author for correspondence: Tel: t84-4-38587B45; Email: [email protected]