T C . D D & T P 9 ( 1 ) - 2 0 1 3
NGHIEN CUtJ JSNG DUNG CHE PHAM ENZYME CUA CHUNG TRICHODERMA HAMATUM TRONG
CHE BIEN TIEU SO
Nguyen Thj Hodi Trdm', Luu Thi ^? Thdy, Le Thi ^9 Phuang, Vo Tan Hgu, To Lan Phuang Nghien ciru de irng dyng che pham enzyme tir chimg nam moc Trichoderma hamatum trong qua trinh che bien lieu sp lir hat tieu den. D6i tugng, phuang phap va ket qua: chung nam moc Trichoderma hamatum dirpc nuoi cay tren moi trudng ban ran phii hpp de sinh long hpp cac en- zyme c6 kha nang phan huy va lam nicm vo lieu. Che pham enzyme co cac hoat tinh dugc khao sat gom xylanase va cellulase deu co pH toi thich d 4,5. Che pham co ho^t tinh cellulase tuong doi ben nhi^t trong thdi gian 48 gid. Trong dieu ki?n ngam ti phii hpp, hieu sual boc vo tieu dat 97% khi bo sung 15% che pham enzyme sau 48 gid d 45°C, pH=4,5. Ket lu^n: Chiing nam moc Trichoderma hamatum co kha nSng sinh long hpp cac enzyme co kha nang hoc vo hgt lieu den trong qua trinh che bien tieu sp. Kel qua nghien ciru khang djnh viec sii dung enzyme trong che bien thirc pham mang lai nhieu Ipi ich so vdi phuang phap san xuat truyen thong nhu rut ngan thdi gian san xual, san pham co mau s5c va min vi hSp din, du lugng vi sinh vat tren san pham thap, giam thieu sir 6 nhiem miii va 6 nhiem nudc thai tai nai san xuat.
Tir khoa: cellulase, xylanase. tieu sg, bdc vo hgt lieu, Trichoderma hamatum.
\. DAT VAN DE
Tieu sp la san pham dugc che bien tir hat tieu bang each Ioai bo ldp vo ngoai.
O nudc ta, hien nay viec san xuat tieu trang chii yeu la san xuat thu cong va ban cong nghiep di tir tieu den theo phuang phap ngam u tu nhien. Qua trinh ngam va thay nudc dai ngay den khi vo tieu miin ra mdi dem xat vd. Vi vay sau khi xat vo, chat Iugng cam quan cua san pham kem hap dan do sg lieu co mau sam va co miii hoi rat khd chiu, du lugng vi sinh vat tap nhiem rat ldn [4].
Qua trinh boc tach vo ngoai do nhieu lac nhan nhung hieu qua nhat la su dung ket hgp tac nhan vat Iy va sinh hpc. Qua trinh ngam nudc dai ngay Iam truong nd va lam mem Idp vo ngoai ciia hat lieu den cd su tham gia cua cac vi khuan hoac qua
trinh ii am hat tieu vdi viec bo sung che pham nam moc deu co nhiing ban che nhat dinh la chai Iugng san pham kem hap dan va khong an loan ve mat ve sinh thuc pham [ 2 , 4 , 5 ] .
Bai bao nay de cap den viec nghien cim bo sung che pham enzyme sinh long hgp tir chiing Trichoderma hamatum vao qua trinh ngam ii va tao cac di^u kien thich hgp cho enzyme phan hiiy v6 hat lieu de che bien tieu sg vdi chdt Iugng t6t va an loan v8 m5t ve sinh thuc phdm.
IL DOI T U O N G VA PHU-ONG PHAP N G H I E N C U t I
\. Doi tirgng:
Tieu den thupc giong tieu Vinh Linh dugc mua d huyen Lgc Ninh, tinh Binh Phudc. Hal tieu co dp i m 10 - 13%, 'PGS.TS. Vien Cong nghiep Thuc pham HN
DT: 0904310544 Email: [email protected]
Phan bien 1: PGS. TS. Vu Thj Dao- Vien Cong nghe thuc pham Phan bien 2; PGS. TS. Ha Thi Anh Dao-
Vien Dinh dudng Ngay nhan bai: 11/2012 Ngay dang bai: 15/04/2013 48
TC.DD&TP9(1)-2013 dudng kinh d > 5mm.
Chung nam moc
Chimg Trichoderma hamatum ihupc Bp Sim tap giong ciia Phan Vien Cong nghiep thuc pham tai Tp. Ho Chi Minh.
Phuang phap nuoi cay
Moi trudng phan lap va bao quan: moi trudng PGA gdm khoai tay 200g, glucose 20g, agar 20g, nudc ck 1000 ml; pH 6,5).
Hap khir triing d 12rc/20 phut.
Moi trudng nhan giong sinh tong hgp enzyme c6 thanh phan gom cam mi :trau - 7:3, b6 sung dung djch NaNGj 0,5%.
Do am moi trudng la 60%. Hap khu triing di2rC/30phut.
Phuang phap chuan bi che pham en- zyme
Nuoi cay nam moc thu che pham:
Chung nam moc Trichoderma hamatum dugc nuoi cay trong ong nghiem thach nghieng chiia moi trudng PGA d nhiet do phong trong thdi gian 3 ngay. Cay bao tir vao cac binh tam giac chua moi trudng dat mat dp 107CFU/g co chat, nuoi u d d nhiet dp phong 3 ngay. Trich ly enzyme bang each bo sung nudc cat vdi ty le 1:4, lac 150v/p trong 10 phiit, ly tam vdi toe do 5.000v/p thu dugc che pham enzyme.
Phuang phap phan tich hoat tinh xy- lanase [6]
Dyng dudng chuan xylose tir dung dich xylose ndng do lOmM: hoa tan 150mg xylose trong dem Na-citrale 0,05MpH-5,3.
xac dinh boat tinh xylanase theo phuong phap EDC (Enzyme develop- ment corporation): 1,8 ml xylan 1% va 0,2ml enzyme phan img trong thdi gian 5 phiit d 50°C. Them 3ml dung dich DNS 1 % vao lac deu rdi dun each thuy trong thdi gian chinh xac 5 phiit. Sau d6, lam mat. Do dp hip thu d budc song 540 nm va so vdi dudng chuan xylose da dung tren may quang pho UV-Vis.
Tinh toan ket qua: Mgt don vi boat linh xylanase (BXU) la lugng enzyme thuy phan xylan sinh ra Iugng dudng khu tuong ling vdi I nmol xylose trong 1 giay d cac dieu kien phan iing.
So dan vi hoat tinh (BXU) - XxK Trong do:
X: so pmol xylose suy ra lir dudng chuan
K: he so pha loang
v: the tich enzymee cho vao hon hgp phan ung enzyme
t: thdi gian phan ung
Phuong phap phan tich boat tinh cel- lulase [1,3]
Xac dinh boat tinh enzyme cellulase theo phuang phap so mau vdi thuoc thir DNS: 1 ml enzyme cho vao ong thir that, d ong thu khong cho vao 1 ml enzyme da dun each thiiy 15 phiit. Them 1ml CMC 1% cho vao cac ong nghiem va lac deu.
Tien hanh phan iing d 40°C trong 10 phut.
Them 2ml DNS va lac deu de ngirng phan ling. Dun soi each thiiy trong 15 phiit roi lam lanh, so mau d budc song 540nm tren may quang pho.
Cach tinh ket qua: mpt don vi hoat tinh cellulase dugc tinh bang so mg glu- cose tao ra bdi Iml dich enzyme trong thdi gian 10 phiit d 40°C.
So don vi hoat tinh (UI) = C.n.V Trong do:
C: Nong dp dudng trong dung dich mau.
n: He so pha loang V: The tich ban dau m: Trpng lugng mau phan tich.
Phirong phap xir ly nguyen lieu [4]
Tieu den dugc loai bd dat, bui va rira nudc. Ngam tieu vao nudc vdi ty le 1:3 49
T C . D D & T P 9 ( I ) - 2 Q I 3 trong 15 philt dk lam truong nudc ldp vo
ngoai. Thanh triing tieu d 90°C Irong 10 phiit.
Nguyen Ii?u dugc iam ngugi, bo sung nudc vdi ty Ie 1 nguyen lipu : 1,5 nudc r6i bd sung enzyme, li d nhict dg thich
hgp trong thdi gian 48 gid.
Phu-ffng p h a p tinh hi^u suat boc vo x a t v6 bang may xat true ngang co canh xai dan h6i, Iyc xat 0,008 0,023kg/cm-, t6c dg xat 0,2m/s, thdi gian xat la 2 p h u t . D D
I liC'ii suat boc vo (%) = -
X So hat b6c vo hoan loan x 100%
S So hat cd trong mlu
boat ciia enzyme, vi vay vi?c nghien cim anh hudng ciia pH doi vdi hoat linh cua enzyme la rat can thiet.
DiSu chinh pH d cac mau dat cac mite khac nhau: 3,0; 3,5; 4,0; 4,5; 5,0; 5,5; 6,0;
6,5. Khao sat anh hudng ciia pH doi vdi hoat tinh ciia CPE tren co chit xylan 1%
va CO chit CMC 1%, d nhiet dp 45''C.
IIL K E T QUA VA BAN LUAN 3.1. Khao Siit cae yeu to anh h u o n g den hoat tinh cua che p h a m enzyme thu tir chung T r i c h o d e r m a h a m a t u m tren moi t r u d n g ban ran
3.1.1. Anh hu&ng cua pH doi v&i hogt tinh enzyme xylanase vd cellulase cua che phdm enzyme
pH la mpt yeu to anh hudng Idn den
Bang 1: Hogt tinh xylanase, cellulase cda CPE & cdc pH khdc nhau
^~pH HoirtinhXylanas7(BXU7mr)"" ~ Hoai tiinrCeiiuTase (Ul/ml)
" ' 3 " 41.73 "" 5,59 '"
3,5 4 4,5
5 5,5
6 6,5
46,52 50,44 51,58 50,14 44,24 40,91 37,69
7,26 8,73 9,57 8,87 8,54 8.21
Ci pH=4,5 hoai tinh xylanase (51,58BXU/mI) va cellulase (9,57UI/ml) dat cao nhat. Nhu vay, pH thich hgp cho boat dgng ciia che pham enzyme la pH=
4,5.
3.1.2. Khdo sdt dnh hu&ng cda nhiit dp doi v&i hogt tinh enzyme xylanase vd cellulase ciia CPE
Tien hanh khao sat su anh hudng ciia nhiet dp den boat linh ciia che pham en- zyme trong moi trudng dem citrate phot- phat pH=4.5 vdi CO chat la xylan 1 % hoac CMC 1%. Cac mau dugc thi nghiem d cac nhiet dp 3 5 T , 4 0 ^ , 4 5 X , SOT.
55"C, e C C . 65°C- 70''C. Sau 10 phut, xac dinh boat tinh xylanase va cellulase.
Bdng 2: Hogt tinh xylanase, cellulase ciia CPE a cdc diiu kien nhiet do khdc nhau Nhift aoc^Q" Hoat tinh Xylanase (BXU/ml) Hoat tinh Cellulase (Ul/mO
35 40 45 50 55 60 65 70
32,96 42,29 51,50 65,32 69,70 79,01 75,41 67,95
6,44 7,6 9,54 10,6 11,58 12,5 13,06 12,85
TC.DD&TP9(1)-2Q13 Hoat linh enzyme xylanase tang din
theo nhiet dp va dat cao nhat d 60"C (1316,7BXU/mI) sau do giam din. Hoat tinh enzyme cellulase dat cao nhit d 65"C (13,06Ul/ml). Nhuvay, nhiet do toi thich cho boat dpng ciia enzyme xylanase la 60*'C va enzyme cellulase la 65''C.
3.1.3. Khdo sdt dp ben hogt tinh cel- lulase doi v&i nhiet dg
Khi xir Iy nhiet theo thdi gian thi hoat tinh enzyme xylanase va cellulase se giam dan. Vi vay, can klmo sat dp ben
Bdng 3: Ket qud khdo sdt dp ben nhiet cua khdc nhau.
nliiet ciia cellulase theo nlilet dg va thdi gian xir Iy de iing dung vao trong qua trinh san xuat.
Che pham enzyme dugc ngam vao nguyen lieu trong thdi gian dii lau de phan hiiy va iam mem ldp vo ngoai hat lieu, vi vay can lien hanh khao sat do ben hoat tinh cellulase trong dieu kien xir ly nhiet lien tuc trong thdi gian la 48 gid.
Cac nhiel dp can khao sat: 45°C, SO^C, 5 5 r . 60"Cva65"C.
hogt tinh cellulase & nhiet dp vd th&igian
\ Nhiet dp
\ CC)
\ \
Thdi aian\
(h) 0 2 4 6 8 10 12 18 24 30 36 42 48
65°C Hoat
tinh (Ul/m 12,43 2,52 1,15 0,64 0,37 0,15 0 0 0 0 0 0 0
Hoat tinh con
60°C Hoat tlnh ) lai(%) (Ul/ml)
100,0 20,2 9,3 5,2 3,0 1,2 0,0 0,0 0,0 0,0 0,0 0,0 0,0
11,92 7,99 5,71 4,07 3.81 3,60 2,88 2,61 2 52 2.16 1.88 1,54 1.36
Hoat tinh con
55°C Hoat tinh lai(%) (Ul/ml)
100,0 67,0 47,9 34,1 32,0 30,2 24,2 21,9 21.1 18.1 15.8 12.9 11.4
11,03 9,93 7,93 7,39 7,16 6,61 5,86 5.06 4.63 4.42 3.75 3.51 3.29
Hoat tinh con
50"C Hoat tinh lal(%) (Ul/ml)
100,0 90,0 71,9 67,0 64,9 59,9 53,2 45,9 42.0 40,1 34,0 31.8 29,8
10,13 9,65 8,81 7,60 7,08 6,76 6,27 5,88 5.15 4.75 4.35 3,97 3,65
Hoat tinh con
45°C Hoat tinh lai(%) (Ul/ml)
100,0 95,3 87,0 75,0 69,9 66,7 62,0 58,0 50,9 46.9 43.0 39.2 36.1
9,08 9,00 8,90 8,82 8,73 8,63 8,54 8,47 8.34 8.26 8.17 8.07 7.98
Hoat tinh con
lai (%) 100,0 99,1 98,0 97,1 96,1 95,0 94,0 93,3 91,8 90,9 89,9 88.9 87.9
D3 thi 1. Anh hu&ng nhiet dp din dp ben hogt dp cua cellulase cda chi phdm
T C . D D & T P 9 ( 1 ) - 2 0 1 3 Thdi gian xu !y nhipt cang lau thi boat
linh cellulase ciia CPE cang giam. Nhiel dp cang cao thi hoat tinh cellulase ciia CPE giam cang nhanh.
- 6 nhiel dp 65"C CPE cho hoat tinh cellulase cao nhat, song hoat tinh giam rat nhanh. Sau 2 gid gia nhict, hoat ttnh chi con 20,2% so vdi hoai tinh ban dau, sau do gian dan, den gid thu 12 hoat tinh gan nhu da mat hoan loan.
- CJ nhiet dp eO^C, 55"C va 50"C hoat tinh giam cham hem so vdi 65"C. Den gid thu 48 hoat tinh cellulase ciia CPE con lai lin lugt la 11,4%; 29.8% va 36,1% so vdi ban dau.
- C) 45°C, tuy boat linh cellulase thap hon so vdi cac nhiet dp khac nhung boat tinh giam cham. Den gid thii 48, boat tinh cellu- lase con lai xap xi 88% so vdi ban dau.
Bdng 4: Anh huang pH den hi^u suat
Vi vay, khi sir dung ch^ pham enzyme tij chiing Trichoderma hamaham nen chpn nhiet dg 45''C de bdc vd tieu se mang lai hieu qua tdt hon vi hoat tinh cellulase tuang doi ben.
3.2. Nghien cu-u su* d u n g che pham enzyme trong boc bo tieu den
3.2.1. Anh hu&ttg cuapH pH la yeu to rat quan h^gng anh hudng din boat dgng ciia che pham enzyme, vi vay cin tien hanh khao sat anh hudng ciia pH den hieu suat bdc vo.
50g lieu den (d > 5mm) dugc xii ly va ngam nudc vdi ty le 1:1.25. Dieu chinh pH dai 3,0; 3,5; 4,0; 4,5; 5,0; 5,8 vdi acid cittic 2%. Bo sung dung dich che pham enzyme vdi t y l e 10% so vdi nguyen lieu, ud45°C trong 48 gid, xat vd bang may va xac dinh hieu suat bdc v6.
bdc vd tieu den
Khong bo sung CPE Bo sung CPE
pH 3,0
T J le hat boc vo Ty If hat khong Ty le hat boc vo Ty le hat khong (%) b6cv6(%) ' (%) b6cv6(%)
26,3 3,5
4,0 4,5 5,0 5,8
26,3 25,0 24,3 23,0 21,0
73,8 75,0 75,8 77,0 79,0
69,0 66,5 70,5 69,0 67,2
31,0 33,5 29,5 31,0 32,8 pH CO anh hudng den kha nang hoc vo
lieu ciia che pham enzyme, pH cang thap thi hieu suat hoc vo tieu cang cao. Hieu sual bdc vo tieu d pH=3,0 la 73,8% va d pH= 4,5 la 70,5%.
Viec bo sung enzyme gop phan iam mem cau tnic vo va tang hieu suat hoc vo. Tuy nhien, d pH=3,0 ximt hien Idp vang diu cd mill thom tren be mat nudc u tieu. Dieu nay la do d pH thap, cau diic sp deu trd nen long leo nen mgt phan tinh diu trong hat tieu thoat ra ngoai mdi hirdng nudc Iam xuit hien ldp vang diu. Do vay, neu sii dung nudc ii tieu d pH cang thap thi lugng tinh diu ttong tieu
se thoat ra nhieu hon va lam giam chat Iugng ve mill vi ciia tieu. Vi vay, pH= 4,5 la gia tri pH toi uu cho boat dgng cua chi pham.
3.2.2. Khdo sdt dnh hudmg nhiet do ngam u den khd ndng bdc bd tieu den
Hoat tinh cellulase cua CPE dat cao nhat d eS^C, nhung d nhiet dp cang thip thi do ben nhiet ciia che pham cang cao.
Tien hanh khao sat anh hudng cua nhiet do den hieu simt bdc vd tieu den ciia CPE.
50g tieu den dugc xir ly va ngam nudc vdi ty le 1:3 trong 15 gid, thanh trung, bo sung nudc ty le 1:1,25 va dieu chinh pH =4,5.
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T C . D D & T P 9 ( 1 ) - 2 0 1 3 Bo simg CPE voi ty le 10% so vai nguyen 50"C, 60°C trong 48 gicr. Tian hanh xat vo lieu, u a cac nhiet do khac nhau tir 45°C, bang may va xac djnh hieu suit boc vo.
Bdng 5: Anh hw&ng nhiet do ddn hieu sudt bdc vo tieu den Nhiet do ("C)
40 45 50 60
Ty \% h^t boc vo (%) 46,5 70,6 53,9 22,3
Ty I9 hat khong boc vo (%) 53,5 29,4 46,1 77,8 O 45''C hieu suai hoc vo lieu dat
70,6% cao hon so vdi d nhiel dg 40"C, 50''C va 60"C. Tuy nliien, d 45"C hoat tlnh enzyme khong cao nhat. nhung d nhiel dp nay boat tinh enzyme Iai ben ban so vdi cac miic nhiet dp cao hon, nen tac dgng ciia enzyme trong viec lam mem vo ludn dugc tang cucmg trong sudt qua trinh ngam il nen hieu suat boc vo lieu cao hon han.
3.2.3. Khdo sdt dnh hu&ng ti le en- zyme: nguyen lieu den hieu sudt bdc bd tiiu den
De dat hieu suat hoc vo lieu d muc cao hon, can khao sal ty le CPE:NL nhim lim ra ty le enzyme bo sung phii hgp, dam bao vdi yeu cau dat ra ve chat lugng san pham va chi phi san xuat.
lOOg lieu den dugc xii ly va ngam nudc vdi ty le 1:3 trong 15 gid, thanh triing, bo sung nudc ty Ie 1:1,25 va dieu chinh pH ^4,5. Bo sung CPE vdi ty le khac nhau 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, il d45"C trong 48 gid.
Tien hanh xat vo bang may va xac dinh hieu suat hoc vo.
Bdng 6 Anh hu&ng ty Ie CPE/NL din hieu sudt bdc vd tieu Ty le CPE/NL
10 11 12 13 14 15 16
(%)
Ty le hat boc 70,5 75,6 83,5 88,3 92,4 97,2 97,9vo
(%)
Ty ie hat khong boc vo (%) 29,5 24,4 16,5 11,7 7,6 2,8 2,1 17Ty le CPE:NL cang tang thi hieu suat hoc vo tieu cang tang, tang nhanh d ty le CPE:NL tir 10% dSn 15%. Khi tang ty Ie CPE:NL tu 15% h:d len thi hieu suit boc vo tang cham, cu the d ly Ie CPE:NL ta 16% thi hieu suit dat 97,2%, d ly Ie 16%
dat hieu suit la 97,6%, d ty Ie 17% dat hieu suit 98,3%. Vi vay, ly le CPE:NL la 15% dugc chpn vi dam bao yeu cau ve hieu simt hoc vd va chi phi san xuat.
IV.KET LUAN
Chiing nam moc Trichoderma hama- tum CO kha nang sinh long hpp cac en- zyme phan hiiy vo hat tieu den trong qua trinh che bien tieu sp. Che pham enzyme CO hoat tinh xylanase va cellulase toi thich d pH ^4.5. Trong dieu kien ngam ii phii hgp, vdi pH=4,5 va nhiet dp ii la 45''C, hieu suat hoc vo tieu dat den 97% sau 48 gid khi bo sung 15% che pham enzyme.
53
TC. DD & TP 9 ( I ) - 2 0 1 3 1 Ldi cam an: Cac tac gia chan thanh
cam an su tai irg ciia Bg Cdng thuang Vipt Nam cho Nhipm vy Khoa hgc cong ngh? (dh tai s6 DTOI.IO/CNSHCB nam 2010-2011).
T A I L I I U T H A M K H A O
1. Phgm Thj Anh Hong. Ky thugt .sinh hda (2003). NXB Dai Hpc Quoc Gia TP 116 Chi Minh.
2. Nguyin Dire Lugng, (2002), Cong nghp vi sinh vat, tap 2- vi sinh vat hpc cong nghipp, Nha xuat ban Dai hpc qu6c gia, Summary
STUDY ON APPLYING ENZYMES FROM TRICHODERMA HAMATUM IN WHITE PEPPER PROCESSING
The objective of this study was to apply enzymes from Trichoderma hamatum in peeling black pepper skin. Method and Results: Trichoderma hamatum was inoculated in special culture medium to biosynthesizing enzymes, which were capable to soften and hydrolyze black pepper skin. These enzymes were tested including xylanase and cellulase, of which the optimal pH was 4.5. The cel- lulase activity was thermostable within 48 hours. The efficiency reached 97% when adding 15%
enzymes to the amount of black pepper in 48 hours, at 45oC and pH 4.5. Conclusions: Trichoderma hamaruin was capable of biosynthesizing enzymes that were applied in while pepper processing.
The result of this study confirm the benefits of new technology compared to the traditional method, such as reducing production time, improving while pepper quality, reducing contaminating mi- croorganism and environment pollution.
Keywords: cellulase, xylanase, white pepper, peeling black pepper. Trichoderma hamatum.
thanh ph6 Ho Chi Minh
3. Nguyin Van Mui, (2001), Thuc hanh hoa sinh hpc, Nha xuat bao Khoa hpc Ky thual Ha Npi
4. Luu Thi L? Thuy va cpng sy (2009), Nghien ciru img dung enzymee trong che hien tieu trdng tgi cdc ldng nghe, Phan vi?n cong nghidp thyc pham, Tp HCM 5. Namiki Takashi, Nakahara Ryozo, Abe
Maseyuki (2007), Method for Producing White Pepper and White pepper. World Intellectual property, editor.
6. http://www.enzymeedevelopment.com/as- says/xylanase/
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