Tsp chi Khoa hoc DHQGHN, Khoa hpc Tyr nhiai va Cong nghe, Tiip 29, So 3 (2013) 8-16
Toi iru hoa sinh tong hgp lactase tit Aspergillus oryzae sii dving phuong phap dap ting be mat- phucmg an can true c6 tam
N g uyen T huy H u a n g * , M ai T huc D i B6 mon Cong nghe Sinh hoc, truang Dai hpc Bach Khoa Tp.HCM 268 Ly Thtiang Kiel, Phucmg 14. Qudn 10, Thanh pho Ho Chi Mmh
Nhanngay 11 thang 12 nam 2012
Chinh sua ngay 25 ih ^ g 12 nSm 2012; chap nhan dSng ngay 15 th w g 4 2013
Tom t a t Tu 6 chiing nam moc Aspergillus oryzae chung loi da chpn dinyc chiing Aspergillus oiyzae BKO smh lactase cao nhat. L6n men ban ran (SSF) voi co chat la cam gao v l trau dupe sir dung de thuc hien qua ttinh len men. Aspergillus oryzae BKO da dupe t6i ira hda qud trinh nuoi cay de thu dupe sm lupng lactase cue dai. Chung toi thiet ke thi nghipm theo phuong phip b6 mjt (RSM) - phuong m cau true cd tam (CCD) da thuc hiSn va dm ra g ii tri t6i uu cua 5 yeu td lactose (13.3%), (NH,;)2SOj (1.7%), pH (5.94), do dm (61.04%). ti 16 |i6 o g (1.05%) cho san lupng lactase epe dpi theo m6 hinh 4.120 U/g. Chung lot thu nghipm nu6i cay v6i di6u kipn cua md hlnh va kdt qua thu dupe san lupng lactase Ik 3.947 U/g.
7Vr Idwa: lactase, Aspergillus oryzae, RSM - CCD, solid state feirnentation.
1. Mo- dau
Lactase (EC 3.2.1.23) hay con goi Ik lactase phlorizin hydrolase hoac lactose galactohydrolase, Ik mot loai enzyme thuoc ho p - galactosidase. Lactase Ik enzyme xuc tac cho phan ting thuy phan lactose thanh p - D - galactose va a - D - glucose va phan ting galactosyl chuyen hoa cac gdc P - D - galactosyl cua lactose lao ra galactose- oligosaccharide (GOS). Viv |y , viec ting dung lactase la mpt trong nhimg ting dung hiia hen nhat trong ckc enzyme nganb c6ng n ^ e p thuc pham [1-3]. Tren the gioi da san xuat lactase va ling dung rat rpng riii trong thuc phkm [4]. Tuy
Tfic gia lien h?. DT: 84-966008067.
E-mail; [email protected]
nhiSn 6 Vift Nam, lactase van chua dupe san xuat voi qui mo c6ng nghiep, chi bude dlu nghiSn oiu 6 qui mo ph6ng tht nghiem.
Lactase c6 the dupe tao ra tir nhieu chiing nam moc nhu A. oryzae, A. niger, Aspergillus fonsecaeus, Aspergillus alHaceus. Lactase c6n CO the tong hop tir ckc loai nim men nhu Kluyveromyces lactis, Kluyveromyces fragilis, Candida pseudotropicalis. Tuy nhiSn lactase tir nam moc rat on djnh va khdng ckn ion kim loai (cofactor) cho boat dong ciia mlnh [5], ddng then nam moc san xuat enzyme ngoai bko nen de dkng cho v i ^ thu nhan enzyme. Vi vay, trong nghiSn ciiu nky chiing t6i sir dung nam moc Aspergillus oryzae. De giam chi phi san xuat enzyme thi viec lire chon cp chat re tien vk skn CO cung vdi vi sinh v |t san xuat phCi bpp,
N.T Hiftmg, M.T Di /Tap chiKlicm hoc DHQGHN, Khoa hoc Tirtibicn tw Cong nghe, 29, So 3 (2013) 8-16
toi uu hoa cdc dieu ki^n nu6i cay la rat can thiet. T6i an h6a qua tnnh Ifin men de xay dirng mo hlnh nhiim thu dirge san lugng Ion gia tSng quy mo xuSt c6 mgt y nghia quan trgng trong viec umg dung nhCmg nghien ciiu c a ban lactase vao trong cong ngfai^. Cdc nghiSn ciiu toi uu hoa dugc thirc hien bang phuemg phdp toi uu don yeu to khfing phan dnh duoc su tuong tac lln nhau gifta cac yeu to va phuong phap t6i uu n&y khfing hien thi anh huemg thuc cua cic yeu td len ho^it tmh enzyme. De khSc phuc van dh nay, chung toi su dung phuong phap dap ung be mat RSM de toi uu hoa gid tri ede yeu to dang dugc nghien ciiu.
Phuong p h ^ bd mat dap ting (RSM) mgt phuong phSp sir dung tom hoc v i thong ke dugc ap dung rgng rai de x3c djnh anh huong cua cac yeu to v l de toi uu hoa qu4 trinh len men sinh hoc [6, 7]. RSM da dugc 4p dung rgng rai de toi uu thong so qua trinh nudi cay de san xuat lipase [8), tannase [9], a-amylase [10], P-cyclodextrin glucanotransferase [11], lactase [6,12].
2. Nguyen li|u va phuotig phap 2. /. Nguyen lieu
Chung vi sinh vfit. 6 chiing nam mdc thude loai Aspergillus oryzae cua bo mdn Cong nghf Sinh hoc - Dai hgc Bach Khoa Thinh phd Hd Chi' Minh. Gidng dugc nudi tren mdi trudng PGA 6 30“C trong 6 ngay v4 giS gidng 6 4°C.
Mdi tru d n g nudi cay. Mdi trudng giu gidng PGA. Mdi trudng idn men: mdi trudng ban tan vdi ti le c4m/trau la 1/3, lactose, amonium sulphate.
Hoa ch^t
ONPG(o-nitrophenyl-P-D-galactopyranoside) cita Duchefa, H4 Lan.
2.2. Phuemg phap
Phm m g phap vi sinh. Khao sat cac dieu kien nudi cay anh huong den qu4 trinh len men b ^ g phuong phap tdi uu don yeu to, cac kel qua ciia thi nghiem trude 14 tien de cho tlu nghiem tiep theo.
Phuoitg phap hoa sinh
Phuemg phap thu enzyme rhd. Dhng nude cat pha trdn vdi mdi trudng nudi cay vdi ti le 1:
4 ve the ti'ch d nhiet dd phdng. deu (ISOrpm) trong Ih. Dem hdn hgp thu dugc loc qua 2 Idp vai, ly tSm dich sau Igc o 5000 vong/phdt trong 20 phut, phan djeh thu dugc la enzyme thd [3].
Phuemg phap xdc dinh hoar link lactase.
Hoat ti'nh lactase dugc xdc dmh dga tren phan ung vdi CO chit ONPG, khi co chat ndy bi lactase thiiy phSn se tm ra galactose va o- oitrophenol (mau vang). Thanh phan oNP hap thu bude song 420nm. Hoat dg lactase dugc xac dinh 14 lugng enzyme thiiy phan Imicromo!
ONPG thdnh galactose va o-niuophenol uong 1 phiit d 30°C trong mgt don vj the ti'ch (hoac 1 don vi khdi lugng co chSt) [13-15].
2.3. Bo tri thi nghiem
2.3.1. Khao sdtkhd ndng sinh lactase cua 6 cluing Aspergillus oryzae
A.ojyzae BKO, B K l, BK2, BIG, BK4, BK5 duoc nudi tren mdi trudng co ban, ti le cam/trau la 3/1, ham lugng lactose 12.5%, ham lugng amonium sulphate 1.5%, pH 5.5, dg am 65%, ti le gidng 1%. Sau 5 ngay thu v4 xac djnh hoat tinh enzyme lactase.
2.3.2. Thiet ke thi nghiem tiin y iu to & tarn va phuemg phap RSM-CCD
Dd thidi kd thi nghiem tdi uu bdng mo hinh CCD, trude tidn ta cim thi nghiem d tSm ciia 5
1 0 N.T. Humg. M.T. Di /Tap chi Khoa hoc DHQGHN, KJwa hoc Tu nhieti t>a Cong ngitf. Tip 29,So3 (2013)8-16
ySu t5 c i n khao sat la lu g n g lactose, h^m lu g n g am onium sulphate, p H , dp am , ti le g ion g theo b an g 2. D ie m 6 tarn du(?c chpn l i diem m a a dd ch o boat ttnh enzym e cao nhat khi ta khao sat tm ig yeu to rieng le.
S au khi c 6 ca c d iem 6 tim , ta thiet k e thi ngh iem ih eo m o hinh C C D , chpn nhSn phucmg an Ik y6u t6 tim g phan 2* ' vk dirge nghiSn cm i a 5 m u c ( - a , -1, 0, +1, + a ) (B a n g 3), c 6 32 thi ngh igm dirge tien hknh (Veri a = = 2 ) [7]- Hkm dap ling dirge chon la boat tfnh enzyme lactase (Y, U/g). Mo hinh h6a dirge bieu diln bkng phuong trinh bgc 2:
Y = b(3 +b]Xi + b2X2 + b3X3 + b.jX4 + bjXj + hi:XiX: + bisXiXs + b]4XiX4 + bisXiXs + b23X2X3 + b24X:X4 + b25X2Xj + b34X3X4 +b35X3Xs + b4jX4X5 + biX] + b2X2 + b3X3~ + b4X4'" + b5X5
T ro n g do, Y Ik san lirgng lactase (U /g); Xj, X3, X3, X4. X5 Ian lijg t la hkm lirg n g lactose, hkm lu g n g (NH4)2S04, p H , d o am v k ti le gio n g; b j, b i, bs, b4, bs Ik ckc h e s o b ac 1; bi2, bi3, bi4, b is , b:3, b24, b25, b34, b35, b45 Ik ck c hg so Cirong tac ciia tim g c3p y€u to; Xj, X2, X3, X4, X5, X1X2, X1X3, X1X4, X1X5, X2X3, X2X4. X2X5, XsX4, X3X5, X4X5 la ckc bien d o c lap. T inh loan , lap p h u o n g trinh h oi qu y, ch gn diem toi u u cu a ck c ye u to ch o san lu g n g lactase dat cu e dai b ioig ch u o n g trinh
Design-Expert 8.0.7, tir dd kp dgng vao thyc nghiem [7].
2.3.3. £>dnh gid mo hinh thuc nghiem L in men thii nghigm mo hinh toi uu vk so sknh veri ket qua du dokn ciia mo hinh.
3. Ket qua
3.}. Ket qua tuyen chpn chung r hap lactase mqnh
mdc long
TCr 6 chung Aspergillus oryzae ciia phbng thi nghigm bg mon C6ng nghe Sinh hgc Dai hgc Bkch khoa Thknh pho Ho C!hi Minh, ta thu dugc chiing Aspergillus oryzae BKO c6 kha nkng sinh lactase manh nhkt (Bang 1).
Bang 1. Ket qua khao skt kha nkng sinh lactase ciia 6 chung Aspergillus oryzae Chiing Aspergillus Hoat rfnh enzyme
oryzae lactase (U/g)
BKO 3.736
BKl 3.293
BK2 1.325
BK3 0.557
BK4 2.279
BK5 3.276
Bang 2. Muc khao sat ciia hkm lugng lactose, nitg, pH, dg am vk ti le gibng dS dm thi nghigm tai tarn Ckc mijc Ham lugng lactose Ham lugng (NH4):SOj (%w/w) pH D$ km Ti Ig gi6ng (%w/w) _______________ (% w/w)_________________________________________________________
Miic ] t3 o3 4 l 55 o l
Muc 2 10 1.0 5.0 60 0.75
Miic 3 12.5 15 5.5 65 10
M uc4 15 2.0 6.0 70 1.25
M uc5 175 2 5 6.5 75 1.5
Mire 6 20 3.0 7.0 80 2
N T, Huang, M T. Di /Tap chi KJioa hoc DHQCHN, KJaahpc Tif nhiSn va Cong iighe. Tap 29, Sff3 (2013) S-IS
Brag 3. N6ng dp 5 yeu t6 dilng trong RSM - CCD
Bien NMn to Hp so ma h6a \■a tu nhifin tuong ling
-a -1 0 1 a
X| lirpng lactose (% w/w) 10 12.5 15 17.5 20
X2 Ham lupng (NH.).S04 (% w/w) 0,5 1 1.5 2 2,5
X3 pH 4,5 5,0 5,5 6,0 6.5
U Dp am (%) 55 60 65 70 75
Xl Ti le eiong (%) 0.5 0.75 1,0 1,25 1,5
Bang 4 k4 hoach chuc hi^n RSM - CCD de t6i ini hda san luong lactase
Thinghietn - M6i cruong ccrb^ Hoai ti'nh lactase (U/g) - 5 ngay Xj Thvc nghiem Suy tu mo Wnh
1 12.50 1.00 5.00 60.00 1.25 0.662 0.715
2 17.50 1.00 5.00 60.00 0.75 0.127 0.120
3 12.50 2.00 5.00 60.00 0.75 0.129 0.1100
4 17.50 2.00 5.00 60.00 1.25 0.951 0.915
5 12.50 1.00 6 00 60.00 0.75 3.927 3.880
6 17.50 1.00 6.00 60.00 1.25 0.803 0.780
7 12.50 2.00 6 00 60.00 1.25 3.879 3.798
8 17.50 2.00 6.00 60.00 0.75 0.694 0.625
9 12.50 1.00 5.00 70,00 0.75 0.856 0.803
10 17.50 1.00 5.00 70.00 1.25 0.934 0.925
11 12.50 2.00 5.00 70.00 1.25 0.261 0.277
12 17.50 2.00 5.00 70 00 0 75 0.24 0.204
13 12.50 1.00 6,00 70.00 1.25 1.936 1.899
14 17.50 1.00 6.00 70,00 0.75 3.825 3,789
15 12.50 2.00 6.00 70.00 0.75 1.134 1 099
16 17.50 2.00 6.00 70.00 1.25 0.515 0.431
17 10.00 1.50 5.50 65.00 1.00 1.489 1.513
18 20.00 1.50 5.50 65,00 1.00 0.635 0,707
19 15.00 0.50 5.50 65 00 1.00 2 607 2.666
20 15.00 2.50 5 50 65.00 1.00 0 4 4 - 0.514
21 15.00 1.50 4.50 65.00 1.00 0.396 0.480
22 15.00 1.50 6.50 65.00 1.00 2.76 2.810
23 15.00 1.50 5.50 55.00 1,00 2,098 2.160
24 15.00 1.50 5 50 75.00 1.00 1.04 1.112
25 15.00 1.50 5.50 65.00 0.50 0.511 0.532
26 15.00 1.50 5.50 65.00 1.50 0.953 0.987
27 15.00 1.50 5.50 65.00 1 00 3,796 3,721
28 15.00 150 5.50 65 00 1 00 3.779 3.721
29 15.00 1.50 5.50 65 00 1.00 3.721 3 721
30 15.00 1.50 5.50 65.00 1.00 3.727 3.721
31 15.00 1.50 5.50 65,00 1.00 3.798 3.721
32 15.00 1.50 5.50 65,00 1.00 3.716 3 721
12 NT. Htnmg. M.T Di/Tap chifQwa hoc DHQCHN, Khoa hoc Tie nhiin va Cong nghi, 29, So 3 (2013) 8-16
3.2. K it qua the' nghi?m a tarn cua md hinh CCD de toi uu hoa lu m g lactase
Sau khi t6i uu timg y£u t6 ri6ng le bSng phuong pbdp CO dien de dm diem tai cua mo hlnh CCD, ta thu dugc ket qua nhu bang 5.
Cac didm tai tarn la him lugng lactose 15%, h k n lugng amonium sulphate 1.5%, pH 5.5, dp am 65% va ti 1? gidng 1 %. Theo S. Nizamuddin va c^ng su (2008) thi h&m lucmg lactose b6 sung la 12.5%. Trong nghien cuu ciia nhom
cong sir nky, moi tnrong nu6i cay cd bo sung them glucose vi vay c6 sir sai khic v6i ket qua cua chung t6i. Ket qua pH 5.5. phii hgp vdi nghiSn cuu cua S.Mirdamadi va cong svr (1997), theo nhom tac gia n iy pH toi uu ciia A.
oryzae tir 4.5 den 5.4. Dq am ciia m6i truong nudi cay theo nghien cuu ciia S. Nizamuddin va cong su (2008) la 90%, Dieu nSy c6 the gial thfch Vi moi truong b in ran trong nghido ciiu ciia nhom tac gia nay d le cim g w v atriu la 1:1.
Bang 5. Ket qua khio sat c4c diem tai tim ciia m6 hinh CCD Cdc
miic Him luqng lactose (% w/w)
Hoet rinh lactase
(U/g) Hdm lugng ngudn nilo (%w/w)
Hoat rinh lactase
(U/g)
pH Hoat rinh lactase
(U/g) Do am
Hoat tinh lactase
(U/g) T ile gidng (% w/w)
Ho?t tuih lactase
(U/g)
Miic 1 7,5 0.821 0.5 1.206 4.5 0.859 55 1.830 0.5 1.830
Miic 2 10 1.760 1.0 2.732 5.0 1.596 60 2.792 0.75 2.792
Mire 3 12.5 3.717 1.5 3.791 5.5 3.779 65 3 793 1.0 3.793
Miic 4 15 3.794 2.0 2.660 6.0 2.491 70 2 469 1 25 2.469
Mite 5 17.5 2.795 2.5 1.603 6.5 2.428 75 1.589 1.5 1.589
Miic 6 20 1.740 3.0 1.010 7 0 2,296 80 1.101 2 1.101
3.3. Toi ifu hoa gid tri cdc yeu to dnh hu&ng den son lu^mg lactase bM g phuong phdp RSM -CCD
Sau khi cd cic diSm a tarn, thirc hien thi nghiem theo RSM - CCD, xir 1^ bang Design expert 8.0.7. G ii tri him dap ung theo thuc nghiem va tien doan theo m6 hinh duoc trinh bay trong bang 4. Dua vao so lieu thong ke ta phSn tich nhung yeu to anh huong den ket qua toi uu so vdi ket qua d tim phuong an theo tieu chuan Student.
Bang 6. Phan ti'ch thi' nghiem d tarn phuong an
- = 0 .0 0 1 5 ,
No y l y" y - 7
1 3.796 0 040 0.0016
2 3.779 0.023 0.0005
3 3.721 -0.035 0 0012
4 3.727 3.756 -0.029 0.0009
5 3.798 0 042 0.0018
6 3.716 -0.040 0.0016
Vdi Nq l i so thi nghiem d tSm, N l i tdng so thi nghiem
Tra bang Student- vdi p = 0,05;
/ = V o - l = 5 ; ro,o3(5) = 2.015 Tmh y nghTa duoc rinh theo cong thiic
N.T. Hitmg, M T. Di/Tap chiKJioa hoc DHQCHN, Khoahgc Tirnhien tw Congnghi, Tap 29, So 3 (2013) 8-16
B ^ g 7. So s4nh tinh y nghla cua cac h3ng so hoi quy theo lieu chuin Student Cac bien
sd hdi
Cac hkng so hoi quy
Tinhy nghta
Kiem djnhy nahia
*0 3.64 529 583 +
X| -0.267 38.846 +
•0.400 58.196 +
X3 0.720 104.753 +
X4 -0 149 21.678 +
Xs -0.004 0.582
XiX-> -0.082 11.930 +
XIX3 -0.336 48.885 +
XIX4 0.459 66.780 +
X1X5 -0.148 21.532 +
X^X3 •0.204 29 680 +
X.<X4 -0.346 50.339 +
X;X5 0.488 70.999 -I-
XiXj -0.145 21.096 +
X3X5 -0 244 35.500 +
X4X5 -0.239 34.772 +
x,^ -0.559 81.329 *
X^- -0.444 64.597 +
Xs^ -0.430 62.561 +
X4^ -0.433 62.997 -1-
X5^ •0,642 93.404 +
voi 5,7 =-^
S i
Trong do: yi: gia tri do dugc dong thirc nghiem; y, : gia tri tfnh todn theo phucmg trinh.
S a = ~ = 0 .0 0 7 ; F = - ^ = 4.622
VSy he so c6 y nghta li: / = 20 PhuoTig trinh h6i quy c6 dang: Y= 3.64 - 0.267xi - 0.400x2 + 0.720xj - 0.149 X4 0.0 82 xiX :-0.336x1X3 + 0,459x1X4-0.148x1X5 - 0.204x2X3 - 0.346x2X4 + 0.488x2X5 - 0.145x3X4 - 0.244x3X5 - 0.239X4X5 - 0.559X,- - 0.444 x.- - 0.430x3* - 0.433 X4- - 0.642 X5-
He so hoi qui (R*) tmh duqc la 0.9608, Dieu niy thd hien ring c6 96.08% so lieu thuc nghiem tucmg thich vdi s6 Jifu tien doan theo mo hinh.
Kiem tra sir tuonng thich ciia phirong trinh theo deu chuan Fisher
N - l Trabang voi /? = 0,0 5 ;
/■ ^ ,„(12 ,5) = 4.6777
^ do do, phuong trinh tirong thich voi thuc nghiem.
Ket qua phSn tich hoi quy cho thay h ^ lugng lactose, h ^ luqng (NH4)2S04. pH, do am la bon yeu to anh hucrag den qua trinh sinh tong hop lactase, trong do pH duoc xem la ydu to anh huong Ion nhat.
M3t dap ung (Hlnh 1, Hinh 2, Hinh 3, Hinh 4) the hien svr toong tac ciia timg c |p ySu to va tir bieu do n^y c6 the xac dinh dirge gia tri toi ITU ciia timg yeu to lam cho h ^ dap ung circ dai. D6 thi hinh 1, 2, 3, 4 cho thay 3 yeu t6 Ik ham lugng lactose, hkm lugng (NH4)2S04 va do am deu tkc dong Sm con yeu to pH tac dong duong den qua trinh sinh tong hgp lactase. pH cang tang thi san lugng lactase cang tang. Tuy nhien khi ham lugng lactose, ham lugng (NH4)2S04, do am tang den mot muc do nko do thi san lugng lactase b it dSu giam dan.
M6 hinh da du doan san lugng lactase toi da dat dugc 4.120U/g khi ham lugng lactose 13.3%, ham lugng (NH4)2S04 1.7%, pH 5.94, do am 61.04%, ti le giong 1.05%.
D anh gia mo hinh tburc nghiem De kiem tra ket qua ciia mo hinh, tien hanh thf nghiem vdi cac gia tri du doan de thu lactase cue dai. Hoat tfnh lactase nhan dugc tir ket qua thuc nghiem la 3.947U/g, hoat tfnh lactase thu
14 N T. Huang, M.T Di /Tap chi KI^oa hoc DHQCHN, Khoa hoc T{f filtien va CSng nghe, TSp 29, SS3 <2013) 8-16
dugc theo di^r doan ciia mo hinh la 4.120 U/g (Hinh 5). Su tirong quan chSt che giira hai ket qua tinh to in giup k hw g djnh tinh chinh xac ciia mo hinh v& sir t6n tai ciia diem coi uu [16].
Hinh 1. Do th) b€ m|Lt ddp ung bieu diln sir phu thuQc cua boat tfnh lactase (U/g) vao h^m lucmg ciia
lactose (%) va (NHal^SOi (%).
Hinh 2. Do thj m$t dap ling bieu diln sy php thu$c ciia boat tfnh lactase (U/g) vao him lupng ciia
lactose (%) va pH.
Hinh 3. D6 thj be mat dapihig bieu dien sirphp thugc ciia boat tfnh lactase (U/g) vao ham lugng ciia
(NHtlzSOa (%) va pH.
Hinh 4. Do thj b§ mat ddp irng bieu diln sir php thupc ciia boat tfnh lactase (U/g) vao d$ im va pH.
I
Hinh 5. So sanh boat tfnh lactase (U/g) giua kit qua ihuc nghiem va mo hinh.
N T. Hu<mg, M.T. Di /Tap chi Klioa hoc DHQGHN, Khoa hoc Tif ithien vd Cong nghe. Tap 29, So 3 (2073) 8-16
4. Ket luSn
V6i muc tiSu nghien axu san xuat che pham lactase a V ilt Nam, bM b io da chpn dugc chung nam m6c Aspergillus oryzae BKO c6 kha nang sinh tong hop lactase cao nhat trong 6 chung ciia b^ m6n C6ng nghe Sinh hpc - DH Bdch khoa Thanh pho Ho CW Minh.
Cong cu thiet ke thi nghiem toi iru da yeu to theo phuong phip dip ung be mat - phuong in cSu true cd tim (RSM - CCD) theo chdng t6i danh g ii la nhimg c6ng cu manh trong vi§c toi uu hoa g ii trj cic yeu to lam cho ham dip ung cue dai. Viec su dyng cac cong cy nay cCmg v6i phan mem chuySn dung Design expert giam dugc thbi gian tieu ton, giam cic thi nghiem dong thbi cd th€ lya chpn mot trong nhung giai phap tdi iru do phan mSm dg nghi.
Qua ip dyng phuong phip mo hlnh hda RSM - CCD va kiem tra bang thirc nghiem, da xac dinh moi (rubng ISn men b in lin toi uu de nuoi cay Aspergillus oryzae BKO thu lactase eye dai la (4.120 U/g) d g ii tri ham luong lactose 13 3%, him luong (NH4):S04 l i 1.7%, pH la 5.94, dp am l i 61.04°, ti le gidng la 1.05%.
Tai lieu tham khao
[1] Holker U, Lenz J , Solid-stale fermentation- biotechnological advantages. Cuir. 0pm.
Microbiol. 8 (2005), pp 201-306.
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16 N T. Hucng, M T. Di /Top chi Khoa hoc DHQGHN. KJioo hpc Tic nhien va C6ng ngli4, 29, Sff3 (2013) 8-16
Optimization of Lactase Production using Aspergillus oryzae by Response Surface Methodology-Central Composite Design
N g u y in Thiiy H u a n g , M ai T huc D i
Department o f Biotechnology, University o f Technology, VN U -H C M 268 Ly Thuong KietStr., 14 Ward, lODist., Ho Ch(Minh, Vietnam
A bstract: Aspergillus oryzae BKO strain producing lactase was selected from 6 Aspergillus oryzae strams of laboratory. Aspergillus oryzae BKO has the ability of producing the hightest activity of lactase and chosen to study the conditions of fermentation to produce lactase, from soil polluted with milk dairy factory effluents. Optimization of lactase (E.C 3.2.1.23) was carried out in Solid State Fermentation (SSF) with . rice bran and rice husk. Response surface methodology - Central composite design was used to inv^tigate the effects of five fermentation parameters (lactose concentration, (NHi)2S04 concentration, pH, initial moisture and spore ratio) on lactase enzyme production. Results of the statistical analysis showed that the fit of the model was good in all cases. Maximum specific enzyme activity of 4.120 U/g was obtained at the optimum levels of process variables (lactose (13.3%), (NH4)2S04 (1.7%), pH (5.94), moisture (61.04%), spore ratio (1.05%)). The response surface methodology was found to be useful in optimizing and determining the interactions among process variables in lactase enzyme production. Testing the model obtained with lactase activity of 3.947 U/g.
Keywords: Aspergillus oiyzae, lactase RSM - CCD, solid state fermentation.