Science & Technology Development, Vol 12, No.

13 - 2009

NGHIN CU ENZYME CELLULASE V PECTINASE T CHNG TRICHODERMA VIRIDE V ASPERGILLUS NIGER NHM X L NHANH V C PH
Trn Th Thanh Thun, Nguyn c Lng Trng i hc Bch Khoa, HQG-HCM TM TT: V c ph l mt ph phm khng th trnh khi trong qu trnh sn xut c ph. Trong thnh phn ca n cha nhiu hp cht kh phn hy t nhin. Bng vic s dng 2 chng nm mc Trichoderma viride v Aspergillus niger sinh tng hp cellulase v pectinase lm cho qu trnh phn hy din ra nhanh hn. Chng ti nghin cu v xc nh cc iu kin nh hng n qu trnh sn xut hai enzyme ny. iu kin ti u sn xut enzyme pectinase ca Aspergillus niger l: 2 ngy, m 62%, ging 8%. iu kin ti u sn xut enzyme cellulase ca Trichoderma viride l: 4 ngy, m 58%, ging 8%. Qua th nghim thc t chng ti nhn thy, v c ph phn hy trong 14 ngy, 60% m, 8% ging. T kho: Trichoderma viride, Aspergillus niger, cellulase, pectinase. 1. GII THIU V c ph chim mt s lng ln trong qu c ph. Nhng nghin cu ch yu tp trung vo vic x l tn dng v c ph nh l cht dinh dng cho gia sc, gia cm, tn thu axit hu c, sn xut hng thm t nhin, cc nghin cu loi b cc c cht caffeine v tanin c trong v c ph, nghin cu ng dng nhit nng, trng nm n v ng dng ln men v c ph to phn bn [4].

Ln men gip loi b nhng cht c hi v sau cng l tn dng lm phn bn. y l mt gii php hu hiu v khai thc trit vic tn dng ph ph liu nng nghip hin nay. y mnh vng chu chuyn vt cht cc ph thi nng nghip trong c v c ph l to phn bn [4]. Trong bi nghin cu ny, chng ti tin hnh cc phng php ln men v c ph bng Trichoderma viride v Aspergillus niger. Qu trnh ln men Trichoderma viride v Aspergillus niger nhm tn dng ngun ph ph phm trong nng nghip, lm gim kh nng nhim mi trng do cc cht thi gy ra nh v c ph, hm bia sau khi ln men bia, mt r ng, cc loi tru ca nh my xay xt ng dng ch yu ca ti ny nhm nghin cu kh nng phn hy pectin v cellulose bng enzyme pectinase v cellulase.
2. VT LIU V PHNG PHP NGHIN CU 2.1. Vt liu v phng php Vt liu: - V c ph c ly t mt nh my xay xt Bun M Thut v Gia Lai, chng thuc ging c ph Robusta, thuc loi coffea canephora. - Chng Trichoderma viride v Aspergillus niger do B mn Cng ngh sinh hc, Trng i hc Bch Khoa Tp.HCM cung cp. Phng php: - Xc nh hot tnh pectinase bng phng php so mu [2] - Xc nh hot ca cellulase da vo ng kh to thnh [1] - Cc phng php khc l cc phng php ln men v nui cy vi sinh vt c trong phng th nghim vi sinh vt, Trng i hc Bch Khoa Thnh ph H Ch Minh.

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2.2. B tr th nghim Nghin cu thnh phn mi trng n sinh tng hp enzyme pectinase v cellulase Chng ti tin hnh b tr th nghim vi cc thnh phn mi trng tng ng nh sau. C nh thnh phn cm 75% v thnh phn (NH4)2SO4 1%, thay i thnh phn tru v bt c rt. Bt c rt l tc nhn gip kch thch sinh tng hp pectinase v tru l tc nhn kch thch sinh tng hp cellulase. Ta c bng thnh phn mi trng th nghim c b tr nh sau. Bng 2.1. Thnh phn mi trng th nghim theo phn trm Mi trng s Cm Tru Bt c rt (NH4)2SO4 1 75 22 2 1 2 75 20 4 1 3 75 18 6 1 4 75 16 8 1 n v tnh phn trm (%) 5 6 75 75 14 12 10 12 1 1

Thm nc iu chnh m 60%. Cy ging vo v nui cy nhit phng khong 28 300C. Thi gian kho st i vi A. niger l 48 gi v T.viride l 72 gi. Sau xc nh hot tnh pectinase (i vi A.niger) v cellulase (i vi T.viride) bng cch o hp thu quang ph bc sng 584 nm cho pectinase v 540 nm cho cellulase. Nghin cu nh hng ca thi gian n sinh tng hp enzyme pectinase v cellulase Kt qu ca th nghim trn c p dng tip tc cho th nghim ny. Sau khi chn c mi trng tt nht cho sinh tng hp enzyme pectinase (i vi A. niger) v cellulase (i vi T. viride), ta tin hnh b tr th nghim nui cy nm mc v ly mu cc thi im khc nhau. Thi gian kho st t 28 32 36 40 44 48 52 gi, gi nguyn m 60%. Nghin cu nh hng ca m n sinh tng hp enzyme pectinase v cellulase Cng ging nh th nghim trn, nhng kt qu kho st c p dng nh thnh phn mi trng v thi gian cho th nghim ny. Thay i m theo cc gi tr nh sau: 50% - 54% 68% - 62% - 66% - 70%. Nghin cu nh hng ca lng ging Trichoderma viride v Aspergillus niger n kh nng phn gii v c ph Cy ging vi sinh vt vo mi trng nhn ging xc nh cc thnh phn ti u nh trn. Nui trong thi gian 7 ngy cho mc sinh bo t cc i. thi im ny ly mt lng ging trong mi trng nhn ging cy vo cc bnh tam gic cha mi trng v c ph theo t l 2% - 4% - 6% - 8% - 10% - 12%. C nh m trong cc bnh nui cy l 65% v thi gian kho st l 7 ngy. Xc nh thnh phn cellulose v pectin thy gii so vi ban u. Nghin cu nh hng ca m n kh nng phn gii v c ph ca Trichoderma viride v Aspergillus niger Sau khi xc nh c lng ging tt nht a vo mi trng v c ph, c nh ch tiu ny. Thay i m nh sau: 50% - 55% - 60% - 65% - 70% - 75%. Nui cy trong 7 ngy, sau em xc nh thnh phn cellulose v pectin thy gii so vi ban u. Nghin cu nh hng ca thi gian n kh nng phn gii v c ph ca Trichoderma viride v Aspergillus niger Cng ging nh trn, sau khi xc nh cc ch tiu ti u c nh lng ging a vo mi trng, m v nhit ti u. Kho st theo thi gian: 7 14 21 28 35. Theo di s thay i hm lng cellulose v pectin.

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Science & Technology Development, Vol 12, No.13 - 2009 3. KT QU THC NGHIM V THO LUN 3.1. nh hng ca thnh phn mi trng n hot tnh cc enzyme pectinase v cellulase Kt qu th nghim c trnh by trong hnh sau:
4.0
610

3.5
Ho t tn h cellu lase (U I/g )

Hot tnh Pectinase (UI/g)

560 510 460 410 360 310 260 0 1 2 3 4 5 6 7 Mi trng A. niger

3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 1 2 3 4 5 6 Mi trng 7 T.viride

Hnh 1. nh hng ca thnh phn mi trng n hot tnh enzyme pectinase

Hnh 2. nh hng ca thnh phn mi trng n hot tnh enzyme cellulase

Trong thnh phn mi trng nhn ging, chng ti c nh hm lng cm v mui sunphat amn ((NH4)2SO4), thay i thnh phn tru v bt c rt. Nh vy, bt c rt l tc nhn kch thch cho nm mc sinh tng hp pectinase. Tru l tc nhn lm cho mi trng thong xp, to iu kin cho nm mc pht trin tt. Ban u nm mc s tn dng nhng cht d tiu trong mi trng nh cc loi ng nhng khi mi trng dinh dng b cn kit th nm mc phi tit ra nhng enzyme phn hy c cht khc thnh ng tip tc tn ti v pht trin. Tuy nhin, cng ty thuc vo nng ca c cht nhiu hay t m nm mc s tit ra lng enzyme khc nhau. Hm lng bt c rt l 8% kch thch cho qu trnh sinh tng hp pectinase c hot tnh cao nht. Tuy nhin, khi ta tng hm lng c rt th hot tnh ca enzyme vn khng tng v s tng hp ca enzyme cn ph thuc vo kh nng tit ti a ca tng loi vi sinh vt c ngha l nu tng hm lng c cht vt qu mt gii hn nht nh th hot tnh enzyme s gim do 2 nguyn nhn, th nht do c cht to p lc lm gim qu trnh sinh tng hp, th hai do c cht tng trong khi nng lc tit ti a khng i lm hot tnh enzyme gim. Cng ging nh th nghim trn, chng ti c nh thnh phn cm v mui sunphat amn, chng ti ch thay i hai thnh phn l tru v bt c rt sao cho tng ca chng chim 24%. Nh vy nu tng lng bt c rt ln bao nhiu th lng tru tng ng s gim by nhiu v ngc li. Bt c rt l tc nhn kch thch cho sinh tng hp pectinase th tru s l tc nhn kch thch cho nm mc tng hp cellulase. Nm mc s u tin s dng nhng cht d tiu trc nhng khi mi trng dinh dng ban u tr nn b thiu ht th chng s u tin s dng nhng cht d phn gii chng c th s dng c. Chng ti nhn thy khi hm lng c rt a vo mi trng l 6% v hm lng tru l 18%. Khi cellulose c trong tru s cao hn lng pectin c trong c rt v mi trng lc ny s lm cho nm mc tit ra nhiu enzyme cellulase hn. 3.2. nh hng ca thi gian n qu trnh tng hp enzyme pectinase v cellulase Kt qu th nghim c trnh by trong hnh sau:

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650.00
H o t tn h P ectin ase (U i/g )

600.00
H o t tn h C ellu la se (U I/g )

4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 1 2 3 4 5 6 Thi gian (ngy) T. viride

550.00 500.00 450.00 400.00 350.00 0 1 2 3 4 5 6 Thi gian (ngy) A. niger

Hnh 3. nh hng ca thi gian n qu trnh tng hp enzyme pectinase

Hnh 4. nh hng ca thi gian n qu trnh tng hp enzyme cellulase

Khi cy nm mc vo mi trng, chng s khng tit ngay ra enzyme m s tit kim nng lng n mc ti a v s dng ngay nhng cht d s dng nht l cc ng n, khong cht c sn. Sau chng s tit ra enzyme ty vo thnh phn cc cht c trong mi trng. Nm mc pht trin c chia ra thnh 4 giai on nh sau: thch nghi, pht trin, cn bng v t vong. giai on thch nghi v pht trin, nm mc s tm cch thch nghi v tit ra loi enzyme no v s lng bao nhiu c th tn ti v pht trin. Theo thi gian th lng enzyme tit ra s tng dn v n mt mc n nh, sau s gim i khi mi trng dinh dng cn kit dn. mi trng th nghim ny, qu trnh sn sinh enzyme pectinase trong khong thi gian 2 ngy l cao nht. Cng tng t nh trng hp sinh tng hp enzyme pectinase, qu trnh sinh tng hp enzyme cellulase cng tng tng ng theo thi gian. T. viride, thi gian tng trng chm hn nhng to ra h enzyme cellulase rt tt. Sn phm ca h enzyme cellulase l cellobiose, mt cht km hm hot tnh ca exo - glucanase. Do m qu trnh sinh tng hp enzyme cellulase T. viride tng trng mnh trong khong thi gian t 4 ngy. 3.3. nh hng ca m n qu trnh tng hp enzyme pectinase v cellulase Kt qu th nghim c trnh by trong hnh sau:
580
Hot tn h petinase (UI/g)
5.0 4.5
H o t tn h ce llu las e (U I/g )

550 520 490 460 430 400 50 54 58 62 66 70 m (%) A. niger

4.0 3.5 3.0 2.5 2.0 1.5 1.0 50 54 58 62 66 70 m (%) T. viride

Hnh 5. nh hng ca m n qu trnh tng hp enzyme pectinase

Hnh 6. nh hng ca m n qu trnh tng hp enzyme cellulase

Cc qu trnh sinh trng v pht trin ca nm mc u c lin quan n m. m l yu t quan trng trong mi trng. Nu m qu thp th xy ra hin tng loi nc ra khi t bo nm mc v lm cho t bo b cht. iu ny s lm hn ch s sinh trng v pht trin ca nm mc. Nu m qu cao th cng khng tt cho s sinh trng v pht trin. mt m nht nh, khi hot ng trao i cht din ra tt nht th s sinh trng v pht trin ca nm mc s din ra tt nht. mi trng th nghim ny, m thch hp cho A. niger pht trin tt nht l 62% v qu trnh sn sinh enzyme pectinase l cao nht.

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Science & Technology Development, Vol 12, No.13 - 2009 Cng tng t nh trng hp trn, chng ti nhn thy m 58% thch hp cho qu trnh sinh tng hp enzyme cellulase c hot tnh cao nht T. viride. T cc kt qu trn, chng ti nhn thy nm mc sinh trng v pht trin tt v sn sinh ra tng loi enzyme cc i th ty thuc vo tng loi ging m chng ta b tr m thch hp. 3.4. nh hng ca hm lng ging n s phn gii pectin v cellulose trn v c ph Kt qu th nghim c trnh by trong hnh sau:
6.0
Hm lng cellulose thy gii (%)

5.5

Hm lng pectin thy gii (%)

5.5

5.0

5.0

4.5 T. viride 4.0

4.5

A. niger

4.0

3.5

3.5

3.0 2 4 6 8 10 12

3.0 2 4 6 8 10 12 Hm lng ging (%)

Hm lng gi ng (%)

Hnh 7. nh hng ca hm lng ging n s phn gii pectin

Hnh 8. nh hng ca hm lng ging n s phn gii cellulose

Qu trnh phn gii pectin ca nm mc cao hay thp ph thuc vo qu trnh sinh tng hp enzyme pectinase nhiu hay t. T l ging ban u l 2%, 4%, 6% gip cho pha n nh v pha sinh trng c hnh thnh. Vi lng ging 8% th hm lng pectin phn hy cao nht.

S phn gii cellulose da vo qu trnh sinh tng hp enzyme cellulase. t l ging 8% th hm lng cellulose b phn hy cao nht. T l gy b ca v c ph cao nht.
3.5. nh hng ca m n s phn gii pectin v cellulose trn v c ph Kt qu th nghim c trnh by trong hnh sau:
9
Hm lng pectin thy gii (%)

Hm lng cellulose thy gii (% )

6 5 5 4 T. viride 4 3 3 2 50 55 60 65 70 m (%)

8 7 6 A. niger 5 4 3 2 50 55 60 65 70 m (%)

Hnh 9. nh hng ca m n s phn gii pectin

Hnh 10. nh hng ca m n s phn gii cellulose

m thay i lm thay i s phn gii pectin. m 60% th s phn gii ca pectin l tt nht. Theo th nghim 3.3 vi m 62% th hm lng pectin t mc cao nht. y c s thay i v s thay i ny khng ln lm. Chng ti ngh rng, m c th lm thay i hnh dng v trong mi trng lm nh hng n kh nng tip cn vi c cht v iu ny lm nh hng n kh nng phn gii v c ph. m 60%, qu trnh phn gii cellulose l tt nht. y l iu kin thch hp cho mi trng v c ph phn hy nhanh v ch phm enzyme pht trin ti u.

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3.6. nh hng ca thi gian n kh nng phn gii v c ph ca Aspergillus niger v Trichoderma viride
24 22 20 18 A. niger 16 14 12 10 7 14 21 28 35 Thi gian (ngy)

14
Hm l ng cellulose thy gii (% )

Hm lng pectin thy gii (%)

13 12 11 10 9 8 7 14 21 28 35 Thi gian (ngy) T. viride

Hnh 11. nh hng ca thi gian n kh nng phn gii v c ph ca Aspergillus niger

Hnh 12. nh hng ca thi gian n kh nng phn gii v c ph ca Trichoderma viride

Chng ti nhn thy, s phn gii pectin xy ra mnh m sau thi gian 7 ngy khi cho ch phm vo mi trng v c ph. Qu trnh phn gii pectin khng xy ra ng thi vi s pht trin ca nm. Chng ti nhn thy, nm mc pht trin kh tt v sinh nhit mnh trong thi gian u l 7 ngy nhng s phn gii ca pectin xy ra mnh vo tun th hai. iu ny chng t nm mc pht trin tt nh nhng cht d phn gii c sn trong v c ph nh ng v cc khong cht. Chng ti nhn thy, sau thi gian 21 ngy tr i, qu trnh gy b ca v c ph tng i chm hn. iu ny c th do lng nm mc trong mi trng t trng thi cn bng. Nh vy qu trnh ln men phn gii v c ph trong vng 14 ngy s gip chng ta tit kim c thi gian v chi ph cho qu trnh ln men. Cng tng t nh trng hp pectinase, chng ti nhn thy s phn gii mnh ca cellulose cng sau 7 ngy khi cho ch phm vo mi trng. Chng cng phn gii kh tt v sau 14 ngy th qu trnh ln men phn gii cellulose l mnh nht. 4. KT LUN Chng ti tm thy iu kin ti u cho sinh tng hp enzyme pectinase ca A.niger l: Thi gian: 2 ngy, m: 62%, hm lng ging: 8%. iu kin ti u cho sinh tng hp enzyme cellulase ca T.viride l: Thi gian: 4 ngy, m: 58%, hm lng ging: 8%. iu kin ti u cho s phn gii trn v c ph l: Thi gian: 14 ngy, m: 60%, hm lng ging: 8%.

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Science & Technology Development, Vol 12, No.13 - 2009

RESEARCH ON CELLULASE AND PECTINASE ENZYME FROM TRICHODERMA VIRIDE AND ASPERGILLUS NIGER IN ORDER TO PROCESS COFFEE PULP QUICKLY
Tran Thi Thanh Thuan, Nguyen Duc Luong University of Technology, VNU-HCM

ABSTRACT: Coffee pulp, which is waste material from coffee production line, has many poor natural degradeble property. Cellulase aillus nigernd pectinase, which were biosynthesized by the fugus races of Trichoderma viride and Asperg, can improve the disintergration process of coffee pulp. In this study, the conditions affecting the production of these enzymes were studied.. The optimum conditions for producing Pectinase enzyme of Aspergillus niger include: 2 days, 62% of humidity, 8% of culture. The optimum conditions for the production of Cellulase enzyme of Trichoderma viride are lasting for 4 days, 58% of humidity, 8% of culture. By carrying out real experiments we found that coffee pulp was disintergrated in the duration of 14 days, 60% of humidity, 8% of culture. Key words: Trichoderma viride, Aspergillus niger, cellulase, pectinase. TI LIU THAM KHO

[1]. Phm Th nh Hng, K thut sinh ha, nh xut bn i hc quc gia thnh ph H Ch
Minh,113 - 114, 2003. [2]. Nguyn c Lng, Cao Cng, Th nghim cng ngh sinh hc, tp 1- Th nghim ha sinh hc, nh xut bn i hc quc gia thnh ph H Ch Minh, 128 - 129, 2003. [3]. L Hng Ph, Nghin cu sinh tng hp Enzyme Pectinase v Cellulase t Aspergillus niger v ng dng x l v c ph trong sn xut phn hu c, Lun vn thc s ngnh sinh hc, Trng i hc Khoa hc T Nhin thnh ph H Ch Minh, 2003. [4]. Trung tm CAI i hc Bch Khoa thnh ph H Ch Minh, Ti liu nhng cng trnh thc hin trn i tng v c ph, Tp.HCM, 2003. [5]. Gary J. Samuels, Trichoderma a guide to identification and biology. Unit States Deparment of Agriculture Agricultural Research service Systermatic Botany and Mycology Laboratory 304, B-011A Beltsville, MD 20705-2350, USA, 2004. [6]. Okada G., The growth of Trichoderma viride on media containing cotton fibres, J. Biochem., trang 591 607, 1963. [7]. Ruy D.D.Y. And Mandels M. Cellulase, Biosynthesis and applications - Enzyme Microbiology Technology, 1980.

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