You are on page 1of 6

O TH TUYT Xc nh nng cc thnh phn x da ..

XC NH NNG CC THNH PHN X DA, CM GO, BT U TNG V UR CC CHNG NM MC Aspergillus awamori VTCC-F296, A.niger VTCC-F128 V A. oryzae MN SINH TNG HP NHIU MANNANASE
o Th Tuyt1*, V Th Thu Hng1, L Vn Knh2 v Quyn nh Thi1
1

Vin Cng ngh sinh hc; 2 Vin Khoa hc v K thut Nng nghip min Nam *Tc gi lin h: Quyn nh Thi - Vin Cng ngh sinh hc - H Ni Tel. (04) 37.568.260; Fax: 04.38.363.144; E-mail: quyen@ibt.ac.vn

ABSTRACT
Determination of culture component concentration of coconut fiber, rice bran, soya powder and ur for filamentous strains Aspergillus awamori VTCC-F296, A. niger VTCC-F128 AND A. oryzae MN producing high level of mannanase Three strains A. niger VTCC-F128, A. oryzea MN02 and A. awamori VTCC-F296 screened from 131 filamentous strains Aspergillus, grew and produced a high level of mannanase on carbon and nitrogen sources as agricultural by-product. The strain A. niger VTCC-F128 produces mannanase of 62.4 U/ml in the mineral cultur medium Czapek (0,1% K2HPO4; 0.2% NaNO3; 0.05% MgSO4, 0.05% KCl) supplemented with 2% coconut fiber and 0.1% soya powder. In the replaced cultur medium using 4% rice bran as the carbon source and 0.1% ur as nitrogen, mannanase activity of the strain A. oryzae MN riched 63.7 U/ml; the strain A. awamori VTCC-F296 produced 186.5 U/ml with 3% rice bran and 0.4% ur. In the replaced cultur medium, the strain A. awamori VTCC-F296 produced mannanase three times higher than in the basic culture medium. Key words: coconut fiber, rice bran, soya powder; Aspergillus niger VTCC-F128, Aspergillus oryzae MN, Aspergillus awamori VTCC-F296, culture condition, mannanase.

T VN Endo--1,4-mannanase (EC 3.2.1.78) l enzyme xc tc thy phn ngu nhin cc lin kt 1,4-mannopyranosyl trong chui mannan v rt nhiu polysaccharide khc c thnh phn ch yu l mannose, sn phm thy phn ca mannanase l cc mannobiose, mannotriose v cc oligosaccharide khc. Mannanase c sn xut trn qui m cng nghip v c ng dng trong nhiu ngnh cng nghip nh cng nghip giy (Kansoh, Nagieb, 2004), thc phm, ch bin thc n gia sc (Wu v cs, 2005), dc phm cho n du kh. Hin nay, vi kh nng thy phn b cm da, mannanase cng c s dng phn hy ngun thi ca cc nh my ch bin thc phm t da, sn xut cc oligosaccharide ng dng trong cng nghip dc phm, cc loi thc phm chc nng. Hin nay vic s dng mannanase chuyn ha cc nguyn liu khng c gi tr dinh dng thnh sn phm c gi tr hoc ci thin mt s c tnh k thut ca nguyn liu ang c nhiu nh khoa hc quan tm. -mannosidase v -mannanase c sn xut t chng A. awamori K4 trong mi trng lng vi b c ph 40% v 60% cm la m, ti u pH 5 (Kurakake v Komaki, (2001). Ti Vit Nam, ng Th Thu v cs, (2003) phn lp v nh gi tnh cht endo--1,4-mannanase t A. awamori BK. Chng ny sinh tng hp mannanase cao sau 120 gi nui mi trng lng c thnh phn khong czapek, b c ph (21,3 g/l), guargum (10 g/l), cao nm men (22 g/l) (ng Th Thu v cs, 2003). Trong mt nghin cu trc, ti u iu kin nui cy ban u cho thy thi gian sinh tng hp mannanase cao nht ca chng A. niger VTCC-F128 l 120 gi, nhit 37C, pH 6,0; chng A. oryzae MN l 96 gi, nhit 28C, pH 5,0 v chng A. awamori VTCC-F296 l 168 gi, nhit 37C, pH 7,0 (o Th Tuyt v cs, 2009). Ngun c cht cm ng tt nht cho sinh tng hp mannanase ca c ba chng l guargum. C 3 chng trn u l cc chng c kh
1

VIN CHN NUI - Tp ch Khoa hc Cng ngh Chn nui - S 18-Thng 6-2009

nng pht trin v sinh tng hp mannanase cao trn ngun cacbon v nitrogen l cc sn phm hoc ph ph liu ca sn xut ch bin nng sn, chng A. niger VTCC-F128 (x da, b ma v bt u tng); A. awamori VTCC-F296 v chng A. oryzae MN (cm go v ur) (o Th Tuyt v cs, 2009). Trong nghin cu ny, t l ca cc thnh phn carbon v nitrogen nh x da, cm go, bt u tng v ur tip tc c kho st xy dng cc cng thc mi trng nui cy cc chng Aspergillus awamori VTCC-F296, A. niger VTCC-F128, A. oryzae MN sinh tng hp mannanase cao nhm sn xut cc ch phm enzyme b sung vo thc n trong chn nui. VT LIU V PHNG PHP NGHIN CU Chng ging nm Chng Aspergillus awamori VTCC-F296 v Aspergillus niger VTCC-F128 do Vin Vi sinh vt v Cng ngh sinh hc cung cp; chng Aspergillus oryzae MN02 do Vin Khoa hc K thut min Nam cung cp. Mi trng v phng php nui cy Mi trng nui cy c bn cho sinh tng hp mannanase l mi trng khong (MTK) Czapek (0,1% K2HPO4; 0,2% NaNO3; 0,05% MgSO4, 0,05% KCl); 0,5% cao nm men, 1% peptone; 0,5% guargum. xc nh TP mi trng nui ti u cho cc chng Aspergillus sinh tng hp mannanase cao t cc ngun ph ph liu sn c trn th trng, cao nm men v peptone c thay th bng b ma, b sn, x da v bt u tng cho A. niger VTCCF128; cm go v ur cho A. oryzae MN v A. awamori VTCC-F296. Chng A. awamori VTCC-F296 c nui trong mi trng thay th b sung 0,5% guargum, lc 200 v/ph, 37C, pH 7. Chng A. niger VTCC-F128 c nui trong mi trng thay th b sung 0,3% guargum, lc 200 v/ph, 37C, pH 6. Chng A. oryzae MN02 c nui trong mi trng thay th b sung 0,7% guaragum, lc 200 v/ph, 28C, pH 5. Ha cht: Cc ha cht dng trong th nghim c cung cp t cc hng khc nhau: KH2PO4, MgSO4.7H2O, NaNO3, KCl, guargum, locust bean gum (LBG) (Biochemika), peptone (Merck),3,5-dinitrosalicylic acid (DNS) (Fluka). Ct Microsep 10K Omega (Mexico). Xc nh hot tnh Hot tnh endo--1,4-mannanase c nh tnh theo phng php khuch tn enzyme trn a thch m t trc y (Nguyn S L Thanh v cs, 2006) c ci tin. Thi gian trong t lnh 4C cho enzyme khuch tn l 12 gi v thi gian cho phn ng enzyme c cht xy ra l 6 gi 37C. Hot tnh endo--1,4-mannanase c nh lng chnh xc theo phng php quang ph (Miller, 1959) vi nhit phn ng l 40C. Mt n v hot tnh mannanase c nh ngha l lng enzyme xc tc thy phn gii phng 1mol mannose trong 1 pht iu kin thch hp. KT QU V THO LUN Mi trng thay th nui chng A. awamori VTCC-F296 Chng A. awamori VTCC-F296 c nui trong mi trng MTK; 0,7% guargum; 0,1%

O TH TUYT Xc nh nng cc thnh phn x da ..

peptone, t l cy chuyn ging l 3% vi ngun carbon l cm go (1-5%). Sinh tng hp mannanase ca chng A. awamori VTCC-F296 tng ln 20% mi trng cha 3% cm go (78,8 U/ml) so vi mi trng i chng MTK cha cao nm men (65,8 U/ml). Trong mi trng b sung nhiu cm go (5%) kh nng sinh tng hp mannanase ca chng ny km i nhiu ch cn 50,1 U/ml (Bng 1) Bng 1. nh hng ca nng cm go v ur ln kh nng sinh tng hp mannanase ca chng A. awamori VTCC-F296. Cm go (%) 1 2 3 4 5 C Hot tnh (U/ml) (%) 71,5 109 73,3 111 78,8 120 70,2 107 50,1 76 65,8 100 Hot tnh Ur (%) 0,1 0,2 0,3 0,4 0,5 C (U/ml) 44,6 49,6 51,7 64,5 60,0 52,6 (%) 85 94 98 123 114 100

Chng A. awamori VTCC-F296 c nui trong mi trng MTK; 0,7% guargum; 3% cm go, t l cy chuyn ging l 3% vi ngun nitrogen l ur (0,1-0,5%). Sinh tng hp mannanase ca chng A. awamori VTCC-F296 tng ln 23% (64,5U/ml) khi b sung 0,4% ur (Bng 1). (Alan v cs,1977) nghin cu kh nng sinh trng v sinh tng hp mannanase ngoi bo ca chng A. niger v chng ny sinh tng hp mannanase cao trong mi trng MTK b sung 1,4 g/l ur (Alan v cs, 1977). Nh vy, thnh phn mi trng thay th ti u chng A. awamori VTCC-F296 sinh tng hp mannanase cao l MTK Czapek (0,1% K2HPO4; 0,2% NaNO3; 0,05% MgSO4, 0,05% KCl); 3% cm go v 0,4% ur). Mi trng thay th nui chng A. niger VTCC-F128 Kho st t l (%) b ma, b sn v x da Bng 2. nh hng ca t l (%) b ma, b sn, x da ln kh nng sinh tng hp mannanase ca A. niger VTCC-F128. B ma (%) 1 2 3 4 B sn (%) 1 2 3 4 Hot tnh (U/ml) (%) 53,7 101 69,3 130 61,5 116 49,4 93 54,0 102 48,5 91 50,4 95 35,9 68 X da (%) 0,5 1,0 1,5 2,0 2,5 3,0 Cao nm men (%) 0,5 Hot tnh (U/ml) (%) 48,4 53,3 69,8 77,1 75,3 55,7 53,2 -

91 100 131 145 142 105 100 -

Chng A. niger VTCC-F128 c nui trong mi trng MTK cha 0,3% guargum v 0,1% peptone, t l cy chuyn ging l 2,5% vi cc ngun carbon l b ma, b sn, cao nm men v x da. X da v b ma l nhng ngun carbon c th thay th cao nm men vi mc

VIN CHN NUI - Tp ch Khoa hc Cng ngh Chn nui - S 18-Thng 6-2009

sinh tng hp mannanase cao hn 30% (o Th Tuyt v cs, 2009). Sinh tng hp mannanase tng ln 45% trong mi trng nui cy cha 2% x da (77,1 U/ml) v 30% trong mi trng nui cy cha 2% b ma (69,3 U/ml) so vi mi trng cha 0,5% cao nm men (53,2 U/ml) (Bng 2). Sinh tng hp mannanase gn nh gi nguyn trong mi trng cha 1% b sn (54,0 U/ml) so vi mi trng cha 0,5% cao nm men. Kho st t l (%) bt u tng
140 120
Hot tnh (U/ml)

100 80 60 40 20 0 0 0.2 0.4 0.6 0.8 1 1.2 Bt u tng (%)

Mc sinh tng hp mannanase ca chng A. niger VTCC-F128 tng ln 33% khi gim t l bt u tng t 1% (57,6 U/ml) xung 0,1% (77,6 U/ml) v tng ln 30% so vi mi trng i chng cha 0,1% peptone (59,6 U/ml) (Hnh 1). Nh vy, thnh phn mi trng thay th ph hp cho chng A. niger VTCC-F128 sinh tng hp mannanase cao l MTK Czapek (0,1% K2HPO4; 0,2% NaNO3; 0,05% MgSO4, 0,05% KCl); 0,3% guargum; 0,1% bt u tng; 2% x da (2% b ma hoc 1% b sn).

Hnh 1. nh hng ca t l (%) bt u tng ln kh nng sinh tng hp mannanase ca A. niger VTCCF128.

Mi trng thay th nui chng A. oryzae MN02 Chng A. oryzae MN02 c nui trong MTK; 0,7% guargum; 0,1% peptone, t l cy chuyn ging l 3% vi ngun carbon l cm go (1-5%) (Bng 3). Vi ngun carbon 4% cm go sinh tng hp mannanase (58 U/ml) tng ln 16% so vi ngun carbon 0,5% cao nm men (50 U/ml). Nhng c th s dng 2% cm go (52 U/ml) thay th 0,5% cao nm men (50 U/ml) trong mi trng nui cy chng A. oryzae MN02. Chng A. oryzae MN02 c nui trong MTK; 0,7% guargum; 4% cm go, t l cy chuyn ging l 3% vi ngun nitrogen l ur (0,1-0,5%) (Bng 3). Bng 3. nh hng ca t l (%) cm go v ur ln kh nng sinh tng hp mannanase ca A. oryzae MN02. Hot tnh Cm go (%) Ur (%) (U/ml) (%) 1 48,9 98 0,1 2 51,6 103 0,2 3 54,4 109 0,3 4 58,0 116 0,4 5 48,0 96 0,5 i chng (0,5% cao nm men v 0,1% peptone) Hot tnh (U/ml) 20,2 62,1 33,2 24,6 14,6 50,0 (%) 40 124 66 49 29 100

Mc sinh tng hp mannanase ca chng A. oryzae MN02 tng ln 24% trong mi trng MTK cha 0,2% ur (62,1 U/ml) v 4% cm go so vi trong mi trng i chng MTK cha 0,5% cao nm men v 0,1% peptone (50 U/ml). Nh vy, thnh phn mi trng thay th cho chng A. oryzae MN02 sinh tng hp mannanase cao l MTK Czapek (0,1% K2HPO4; 0,2% NaNO3; 0,05% MgSO4, 0,05% KCl); 4% go cm v 0,2% ur.
4

O TH TUYT Xc nh nng cc thnh phn x da ..

Kh nng sinh tng hp mannanase theo thi gian trong mi trng thay th ca cc chng nghin cu Cc kt qu thc nghim trn cho thy kh nng sinh tng hp mannanase ca chng A. awamori VTCC-F296 (186 U/ml) sau 7 ngy nui cy cao gp 3 ln so vi hot tnh ca chng ny khi nui trong mi trng c bn (66 U/ml). Chng A. niger VTCC-F128 v chng A. oryzae MN sinh tng hp mannanase cao nht ln lt sau 5 ngy v 4 ngy nui cy (64 U/ml v 62 U/ml) (Hnh 2 v Bng 4).

200

160
Hot tnh (U/ml)

120

80

40

0 0 2 4 6 8 10 12 14 Thi gian (ngy)

Hnh 2. nh hng ca thi gian nui n sinh tng hp mannanase ca A. awamori VTCC-F296 (), A. niger VTCC-F128 (); v A. oryzae MN () trong mi trng thay th.

T nhng kt qu trn c th kt lun thnh phn mi trng nui thch hp cc chng nghin cu sinh tng hp mananase cao (Bng 4). Bng 4. Thnh phn mi trng thay th nui cc chng nghin cu Hot tnh U/ml U/mg A. awamori VTCC-F296 MTK + 0,5% guargum + 3% cm go + 0,4% ur 186,4 453,5 A. niger VTCC-F128 MTK + 0,3% guargum+ 2% x da +0,1% bt u 62,4 148,4 A. oryae MN02 MTK + 0,7% guargum + 4% cm go + 0,2 ur 63,7 165,6 Tn chng ng Th Thu v cs, (2003) ti u mt s iu kin sinh tng hp mannanase ca chng A. awamori BK phn lp ti Vit Nam trong mi trng lng c thnh phn khong Czapek, b c ph (21,3 g/l), guargum (10 g/l), cao nm men (22 g/l). Chng A. awamori BK sinh tng hp mannanase t 120 U/ml sau 4 ngy nui cy. Vi mi trng nui cy c bn, thay th cao nm men v peptone ln lt bi 3% cm go v 0,4% ur, chng A. awamori VTCCF296 sinh tng hp mannanase t 186,4 U/ml, nu thay th bi 4% cm go v 0,2% ur, chng A. oryzae MN02 sinh tng hp mannanase cng t 63,7 U/ml (Bng 4). Kt qu nghin cu ny cng ph hp vi cng b ca Kurakake v cs. (2001) khi nghin cu mannosidase v -mannanase t chng A. awamori K4, chng ny sinh tng hp mannanase cao trong mi trng lng b sung 60% cm la m ; Alan v cs. (1977) khi nghin cu kh nng sinh trng v sinh tng hp mannanase ngoi bo ca chng A.niger s dng MTK c b sung 1,4 g/l ur. Cc ngun carbon (x da cho chng A. niger VTCC-F128; cm go cho A. oryzae MN02 v A. awamori VTCC-F296) v ngun nitrogen (bt u tng cho A. niger VTCC-F128; ur cho A. oryzae MN02 v A. awamori VTCC-F296) u l nhng ngun nguyn liu r tin, sn c trn th trng rt thch hp cho vic sn xut mannanase trn quy m cng nghip phc v mc ch to ra mt lng ln enzyme b sung vo thc n cho gia sc. Thnh phn mi trng

VIN CHN NUI - Tp ch Khoa hc Cng ngh Chn nui - S 18-Thng 6-2009

Trong mi trng c ti u ha, chng A. awamori BK sinh tng hp mannanase cao gp 2 ln so vi trong mi trng c bn ban u (ng Th Thu v cs, 2003). Nhng kt qu trong nghin cu ny cng khng nh cc chng nghin cu khi c nui trong mi trng thay th sinh tng hp mannanase cao hn khi nui mi trng c bn, c bit chng A. awamori VTCC-F296 sinh tng hp mannanase (186 U/ml) cao gp ba ln so vi mi trng c bn (66 U/ml). KT LUN Chng A. awamori VTCC-F296 c kh nng sinh tng hp mannanase tt hn chng A. niger VTCC-F128 v chng A. oryzae MN02. Mi trng thay th cho cc chng nghin cu sinh tng hp mannanase cao l MTK (0,1% K2HPO4; 0,2% NaNO3; 0,05% MgSO4, 0,05% KCl), 3% cm go, 0,4% ur, 0,5% guargum (chng A. awamori VTCC-F296); MTK, 2% x da, 0,1% bt u tng, 0,3% guargum (A. niger VTCC-F128); MTK, 4% cm go, 0,2% ur, 0,7% guargum (A. oryzae MN02).
LI CM N Cng trnh c s h tr kinh ph ca Chng trnh trng im pht trin v ng dng CNSH trong lnh vc nng nghip v PTNT n nm 2020, ca B Nng nghip v Pht trin Nng thn, ti: Nghin cu sn xut v ng dng ch phm a enzyme c cht lng t vi sinh vt ti t hp nhm nng cao hiu qu s dng thc n chn nui 2007-2010. Nhm tc gi chn thnh cm n TS Phm Vn Hp(Vin Vi sinh vt v Cng ngh sinh hc (i hc Quc Gia H Ni) cung cp chng A. awamori VTCC-F296 v A. niger VTCC-F128 ; TS L Vn Knh (Vin Khoa hc k thut Nng nghp Min Nam) cung cp chng A. oryzae MN02.

TI LIU THAM KHO


Alan DE, Surekha A and Yuan CL, (1977). Purification and properties of a b-mannosidase from Aspergillus niger. J Biol Chem 6: p.2026 - 2031. ng Th Thu, Nguyn Th Xun Sm v Li Th Ngc H (2003). Nghin cu thu nhn v mt s c tnh ca mannanase t Aspergillus awamori BK. In: Bo co KH, Hi ngh CNSH Ton quc 2003 H Ni 1617/12/2003, p 546 - 549. o Th Tuyt, Anh V v Quyn nh Thi, (2009). Ti u mt s iu kin nui cy chng nm mc Aspergillus awamori VTCC-F296, A. niger VTCC-F128 v A. oryzae MN02 sinh tng hp mannanase. Tp ch NN v PTNT ( gi bi) Kansoh Al and Nagieb ZA, (2004). Xylanase and mannanase enzymes from Streptomyces galbus NR and their use in biobleaching of softwood kraft pulp. Antonie Van Leeuwenhoek 85: p.103 - 114. Kurakake M and Komaki T, (2001). Production of b-mannanase and b-mannosidase from Aspergillus awamori K4 and their properties. Cur Microbiol 42: p.377 - 380. Laemmli UK, (1970). Cleavage of structur proteins during the assembly of the head of bacteriophage T4. Natur 227: p.680 - 685. Miller GL, (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31: p.426 - 428. Nguyn S L Thanh, Quyn nh Thi, Phm Vit Cng v ng Th Thu, (2006). Endo-b-1,4-mannanse t B. subtilis G1: Chn chng, xc nh ng thi sinh trng, nhn dng v phn tch trnh t gene m ha. Tp ch Cng ngh sinh hc 4: p.327 - 334. Wu G, Bryant MM, Voitle RA and Roland DAS, (2005). Effects of beta-mannanase in corn-soy diets on commercial leghorns in second-cycle hens. Poult Sci 84: p.894 - 897. *Ngi phn bin : GS.TS. V Duy Ging; GS. TS. L Vn Lin 6

You might also like