K H O A HOC C O N G N G H £
M O T SO Y E U TO AIMH HUDIVG Dg|\| V I KHUAIM Lactabadllus plantarum S 5 CO K H A IMAIVG LEIM SVIEIM
SAlM X U A T A X I T LACTIC T U X E L O B I O Z A
Ngd Thi Anh VdnS Trdn Uln HdS Phi Thanh Mai^'
TX^MTAT
Biomass (smh khdO thuc vilt bao g6m ba thai ndng nghiep, ldm nghidp cQng nhu chat fliai smh boat vd san xudt Biomass la nguyen lieu dl kilm, re tiln. Sii dung biomass dl sdn xudt cdc nguyen lieu phuc vu con ngudi la each tiet Idem nguon nang lupng, giai quylt van de ve m6i tnrdng la xu huong hien nay. Trong nhung nam gan day nhieu nghien ciiu su dung biomass dl san xuat axit lactic va ddy cung dang Id budng pbdt tiiln moi va duoe quan tdm. Chinh vi vay da tiln hanh ngbien ciiu phdn lap, tuyen chpn va khao sat mot sd ylu td anh huong din kha ning len men chiing vi khuan thu axit lactic. Tii 4 mdu san pham len men truyin thdng va 1 mdu ba thai dong gilng da phan lap duoe 23 chiing vi khu^n vd chon chung S5 sinh tdng hgp axit lactic cao nhat 5,13 g/1. Bdng cac phuong pbdp siifii ly, smh h6a va 16S rARN kit qua dinh ten chung 85 CO d6 tirong ddng l6n din 99% voi chung Lactobacibusplantarum strain 87 (GU195646). Da lim dirge dilu kien len men Hnh ddi vol chung L platarum S5: t' = ZTC, pH 6, dirong 10,00 g/1, ty le gidng 10%, cao ndm men 3,00 g/1 sau 120 gio thu duoe axit lactic ndng do 6,66 gfl.
Tb kh6a: Sinh khdi, xelobioza, xerduloza, axit lactic, Lactobacibus plantarum.
1. MffD^U
Ngdy nay, ca thi gidi dang thilu hut nang lirpng, vile tim ra ngudn ndng lirpng tai tJio Id mpt xu hudng mdi. Biomass la ngudn nguyin lilu dl Idlm, re tien tuy nhien nlu khdng dupe su dung nd gdy 6 nhilm m6i tnrdng day Id ngudn tdi tao ndng lupng mdi cho tirong lai. Vilt Nam la mdt nude ndng nghilp cho nln lupng biomass tii than ngd, loi ng6, hau, ram da,... rat ddi dao va ngudn biomass nay cdn chua duge xu ly, gdy 6 nhilm moi trudng. Trong nhung nam gdn day sdn xuat len men axit lactic tii sinh khdi Id hudng di da dupe nhieu nhd nghien ciiu tien hanh [2]. Hang ndm, Viet Nam cd khoang 60 trieu tan biomass tii phi pham ndng nghidp, hong dd chi 40%
dugc su dung dap iing nhu cdu ndng lupng cho h6 gia dinfa vd san xudt dien. Thdnfa phan ehinh cua biomass la linoxeluloza (ehilm khoang 90% chat kho) [11]. Cau tao cila hnoxenluloza gdm xenluloza (30-50%), hembcenluloza (2CW0%) vd hnhm (10-30%) [13]. Trong dd xenluloza la ngudn chmh tbily phan tiiu xelobioza [5]. Xelobioza la disacarit cd Uln kit gUeozit gidng nfau xenluloza va duoe su dung ring rai nfau Id mpt mo hmh cho cdu tnie xenluloza [1].
Viee sii diuig biomass la ngudn vdt heu sinh hoc thu
Vien C6ng nghe Sinh lioc va Cong nghe thirc phdm, Truong D91 hoc Bach khoa Ha Noi
^ Hgc viSn Hau cSn
xelobioza rdi tii dd lln men axit lactic dd bat dau dirge chu y den [7]. Tuy nhiln, d- Vidt Nam thi hudng nghien ciiu nay vdn edn kha mdi me vd ed nhilu triln vpng. N\ vay chiing t6i tiln hdnh nghiln ciiu: "Phin lap, tuydn chpn, dinh tdn vi khio sit mot sdydu td inh huong ddn chung vi khuan len men axit lactic".
2. VAT UEU VA PHUDNG PHAP NGHBU CUU 2.1.Vatli^u
Nguydn bdu: 4 mdu san pham lln men truyin thdng: ed mudi chua, bdp cai mudi, sung mudi, dua mudi chua vd 1 mdu bd thai dong gieng dirpc dung lam ngudn mdu phan ldp.
Hda chit Tris HCl, EDTA SDS (d6dexyl sunphat natii), p-meeaptoetfaanol, proteaza K, CTAB (xefylmetyl amdn bromua), clorofom, isoamyl alcohol, isopropanol, etiianol, TE, dNTP, MgCla, dim PCR, Taq polymeraza, agaroza (Sigma-My), ethidium bromide (EtBr, BioMerieux, Phdp), cao nam men, pepton (An Dd), xelobioza (Trung Qudc), aga (Viet Nam).
Mdi trudng: Mdi hirdng YE - xelobioza aga (g/1): Cao nam men: 3, KH2PO4: 1,36, MgSO4.7H20:
0,2, xelobioza: 10, (NHJ2SO4: 1, FeS0^.7H20: 0,01, NaCl: 2, aga: 15, CaCOai 3, pH = 5,5. M61 tuong YE - nude xuyt xelobioza (g/D: gdm cdc thdnh phan neu hen tni CaCOg vd aga. Moi trudng LB (g/n- Q^Q nam men: 5, pepton: 10, NaCl: 5, xelobioza: 10 nH =
' \
NONG NGHIEP VA PHAT TRIEN NONG THON - KY 1 - THANG 10/20,5KHOA HOC C 6 N G N G H |
7. Tat ca m6i trudng tiianh trimg d 110''C, dp suat 0,5 atm vd trong 30 phiit.
2.2. Phuong phdp nghiln cihi
Phuong phip phan lip vi tuyen chpn chiing vi khuan lactic
Phuong phap phin lip: Sii dung mdi trudng YE - xelobioza aga [3].
Phuang phip tuydn chpn su dung eic phuang phip sau: Dinh tinh axit lactic bdng thudc thu Uffelmann [6], do pH eua dich, dijra vdo kha nang pfaan giai CaCOj (due Id tfaaeh vd cdy cham dilm), dinh lupng axit [Emanuel et al., 2005].
Phuangphip dinh tdn
Xde dinh cdc ddc dilm sinh h, sinh hda [13]: xac dinh ho?it tinh catalaza, kha nang axit hda moi trudng, gram, di ddng,...
Phdn loai dua trin giai tiinh tir 16S rARN cua cac chung vi khudn, vdi cdp mdi 27F:
AGAGTTTGATCCTGGCTCAG; 1492R: TACGGYT ACCTTGTTACGACTT [4]. Chu hinh nhilt: bude 1:
gS-C: 5 phiit; bude 2: 95°C:45 gidy; bude 3; 52°C: 1 phut; bude 4: 72''C: 1 phut 30 giay, ldp l^i tii buoc 2 den 4: 35 cfau ky; bude 5: 72''C: 5 pfaut. San pfaam PCR dirge xde dinh trinh tu hen may ABI PRISM®3100(My).Ket qua dpc hinh tir duoe xu ly trin phdn mem Clustal X va so sdnh vdi trinh tir 16S rARN cua cdc loai da cing bd tCr du lieu ciia DDBJ, EMBL va GenBank.
Phuongphap khio sit ydu to inh hudng Chung L plantarum S5 dupe nu6i trong moi t£vibng DB - xelobioza d 37X, hong 48 fa va canfa trudng dupe su dung lam gidng cho tat ca eae thi nghilm.
Anh hudng eua nhidt do: M6i tnrdng YE - xelobioza dirpe cap 10% gidng, pH ban ddu 5,5 va nuoi dcac nhiet dp SOX, 35X, 37°C, 40X, 45°C.
Bdng 1. Dtic dilm ciia 5 chiing vi khudn dirpc tuyIn chgn
Anh hudng ciia pH: Nudi d nhiet dp da lua chpn mdi trudng YE - xelobioza dupe cdp gidng 10% vd pH ban ddu d cdc gid hi; 4,4,5,5,5,5,6,6,5,7,7,5.
Anh hudng ciia ndng do dudng: Nhiet dp vd pH da dupe lua chpn, cap gidng 10% vao mdi trudng YE - xelobioza. Tfaay ddi hdm lupng xelobioza d* cdc miic:
5,10,15,20,25,30 g/1.
Anh hudng cua ty Id cip gidng: Cac dilu kiln nhilt dp, pH, hdm lupng xelobioza dupe lira chpn tii eac k i t qua h l n . TTiay ddi ty 1§ cap gidng: 5%, 10%, 15%, 20%, 25%, 30% vdo moi tirudng YE - xelobioza.
Anh hmng eua eao nim me/J." Vdi cac dilu ki^n nhi|t dd, pH, ham lupng xelobioza, ty le cap gidng chgn d hen. Tiln hdnh khao sat vdi ham luong cao nam men thay doi: 0,3,6,9,12,15 g/1.
Canh trudng sau khi nudi, mdu dugc Idy sau mdi 24 gid dem dinfa lugng axit lactic bdng NaOH 0,05 N.
3 . KFT QUA VA THAO LUAN
3.1. Phdn ldp vd tuyIn chgn chung vi khu& cd kha ndng lln men axit lactic tii xelobioza
TIT 4 mdu san phdm lln men truyin thdng vd 1 mdu ba thai dong gilng, da phan lap dupe 23 ehiing vi khuan hen moi trudng YE - xelobioza aga. Chon 5 chung: CI, C2, SI. S4, S5 ed vdng phan giai D/dl > 4.
Cac chiing vi khuan dupe nu6i hong moi trudng YE - xelobioza, dem thu bang Uffermann, kit qua cho thdy eanh trudng 5 chung vi khuan lam cho thudc thir chuyin tir mdu tim den sang mau vang rom chiing to 5 ehiing vi khuan cd kha nang sinh axit lactic (hinh 1). Diing 3 phuang phdp tuyen chgn: eay cham dilm, diic 16 thach va dinh luong canh trudng bang NaOH 0,05 N; ta chgn diroe chung S5 smh axit lactic cao nhat la 5,13 g/1, D/dl = 5,5, D/d2 = 2,1 (bang 1). Mpt sd dac tinh smh ly, sinh hda chiing S5 dem dinh ten dupe trinh bdy d bang 2.
TT 1 2 3 4 5
Kihieu chuns CI C2 SI S4 S5
Hinh dang te bao VI khuan Que, dai Que chu6i dai Que, chu6i ngan
Que, ngan Que, dai
Dac di^m hinh thai khuan lac Khuan lac to, tron, tring due K h u ^ lac to, vien rang ctia, trang
Tron deu, trang due, nhSn Tring due, khuan lac to, tron deu
Khuan lac to, trSng due, tron, nhan, CO tam
D/dl 5,5 5 4,8 4,6 5,5
D/d2 2,1 1,9 1,7 1,7 2,1
Luong axit sinh ra sau 120 gio (g/1)
4,95 4,5 4,02 3,68 5,13
NONG NGHIEP VA PHAT TRIEN NONG THON - KY 1 - THANG 10/2015 121
KHOA HOC C 6 N 0 N 6 H I (D-duongkinh vdngph
Mdu S5
Hinh thai t l bdo True khuan
in giii, dl-duang kinh khuin l$c, d2-dudng kinh Id th$eh) Bdng 2. Bdc tinh sinh ly sinh hda chiing S5 Gram
+
Tao bdo tii
-
Kha nang axit hda moi trudng
+
Catalaza
-
Smfa khi
-
Di dpng
to
Hinh 1. Kit qua thii Uffermann-
3.2. Dinh ten b^ng phuong p h ^ sinh hgc phdn tu
ADN he gien ciia ehung S5 dupe tach chiet va doan gien ma hda eho 168 rARN dupe khulch dai nhd phan iing PCR sii dung cap mdi phd biln 27F/149R Kit qua giai tiinh tir doan gien 16S rARN cua S5 cho thay doan gien gdm 1447 bp va trinh tir
2 1
nay duoe so sdnh vdi cde gien 16S rARN vi khuan h l n Genbank vdi phan mem Blastn. Kit qua cho thay doan gien 16S rARN ciia chung S5 cd dp tirong ddng lln den 99% so vdi trinh tu gien cua chung Lactobacdlus plantarum stram S7 (GU195646).
Chung S5 dupe gpi Id Lactobacibus plantarum S5 (hinh 2). Den ndm 2007 tiieo Mukund A et, all, ddi vdi vi khuan lln men axit lactic tii xelobioza cd nhieu nghien ciiu nhung chua cd nghiln ciiu nao vl Lactobacibus hong tir nhiln, chi cd chiing tai td hgp Lactobacibus delbrueekumutant Uc-3 [9]. Tuy nhiln hong cimg nhdm nghiln eiiu, theo Phi Thanh Mai va cdng su dd phdn ldp dupe chiing L plantarum [12].
Hinh 2. Kit qud dinh tin chiing S5
(A Kdt qua didn di, 1: marker (chi tin), 2: san phim PCR eiia gien 16S rARN chung S5, B: Cay phin loai ehing S5)
3.3. Khao sdt mdt sd ylu td anh hudng din kha ndng lln men chung L plantarum S5
Is i
s
1
JiijlJ
25 30 35 37 40 45 Nhiet 46 fC)
124 gioi 148 giir 172961 196 gid I120gicf
• 144 gio'
Hinh 3. Anh huong Cila nhi^ do
Anh huong cua nhi^t do: K^t qua b hinh 3 cho thay ring a nhi$t d6 la 37"C, pH ban dau la 5,5 va sau 120 gia nu6i cay thi ham lirong axit lactic cao nhat la 5,13 g/1. 0 miic nhiet do 14 35*C ta thiy ham luong axit lactic smh ra 4,50 g/1 thap hon so voi miic nhijt d6 la 37°C. So sanh voi chung tai td hpp L plantarum (AldhLl) nuoi a 37'C [10], Nhiet do nudi 37X dupe chon cho cac thi nghidm sau.
Anh huong cua pH: Ket qua cho thay o pH ban dau la 6 ham lupng axit lactic cao nhat la 5,67 g/1 sau 120 gio nuoi cay (hinh 4). Khi pH thap xudng 4,5 mdi trudng axit lupng axit lactic sinh ra 2,97 g/1. Khi mdi tnrdng ki^m tai pH 7,5 lupng axit lactic smh ra chi dat 3,15 g / l Tuy nhien theo IMohamed A. Abdel- Eahman thi pH chung Enterococcus mu,. .'^' g y 25
KHOA HOC CdNG NGHE ten men vdi pH 7 [8]. pH ban ddu 6 dupe sir dung cho
nghien cmi tiep theo.
^ M t •24gi&
1"! • A • J J """""
4.5 5,0 5,5 6,0 6,5 7,0 7.5 mi„g,t, pH
Hinh 4. Anh hudng cila pH Anh hirong cua ham luong dudng: K^t qua cho thay nong dd dudng la ydu td anh hirdng ddn qua trinh sinh tdng hop axit lactic. Khi ham lupng dupng 5 g/1 va 10 g/1 thi lupng axit lactic sinh ra 14 cao hon gap 2 lan tuong iing 3,06 g/1 va 6,21 g/1. Ham lupng dudng la 15 g/1 thi ham lupng axit kctic 7,11 g/1 chi cao hon la 0,9 g/1 (hinh 5). Vi vay chpn ham lupng dudng la 10 g/1 cho cac thi nghiem hdp theo.
5 10 15 20 25 30 Ham lirong dirjmg (g/1)
• 2 4 %K<3
• 48eier
• 72 gia
• 96 gior
• 120 gi&
• 144gicf
Hinh 5. Anh hudrng cua hdm lupng dudng Anh hudng eua ty Id cip gidng: O hinh 6 cho thay ^ le cap gidng la 10% sau 120 gid nuoi cay thi ham lupng axit lactic dat dupe eao nhdt la 6,48 g/1 gdp 3,4 lan so vdi hdm luong axit laehc sinh ra vdi ty 1| cap gidng Id 5%. Lupng cdp gidng la 10% dupe su dyng cho cac thi nghiem sau.
^ 6
5 10 15 20 25 30 Ty le giong (%)
Tuy nhien a h t a lupng cao nam men 3 g/1 lupng axit lactic la 6,66 g/1, khi cao nam men tang 9 g/1 thi lupng axit cung chi tang 1,08 g/1 (hinh 7), Do vjy ndng dd cao nam men 3 g/1 dupe su dung dd Idn men thu axit lactic tit chOng S5.
Hinh 6, Anh hudng cfia t^ Id c4p gidng Anh huong cua cao ndm men: Khi tang ham lupng cao nam men tii 0 ddn 9 g/1 thi ham luong axit lactic sinh ra tang tuong iing 1,26 g/1 ddn 7,74 g/1.
NONG NGHIEP VA PHAT TRIEN NONG THON - KY 1 - THANG 10/2015
Hdm lirgng cao
Hinh 7. Anh hu6ng cua cao n ^ mem Dd cd mpt sd nghiln cuu vl cdc chiing len men axit lactic hi xelobioza:
Vdi chung Enterococcus mundtu Q\] 25 lln men thu axit lactic dd cd thi chuyin hda mgt fain hpp ciia glucoza vd xelobioza ciing liic trong mdi trudng MRS, tdc do lac 200 vdng/phuL Kit qua cho tiiay ham lupng axit lactic thu dupe la 20,4 g/1 d' cdc dilu kien len men tai nhiet dp 43°C, pH 7, ham lupng dudng la 20 g/1, ty 16 cdp gidng la 109fi, hdm lupng cao nam men 5 g/1 [8]. Su dung vi khuan Baclbus subtbis CHI (chung ddt biln) ham luong axit lactic tiiu duoe 13 g/1 tai 37°C va pH 7, tdc d l lde 120 vdng/phut. Gdn day, Okano et al. (2010) da xay dvtng hai ehung biln ddi gien eua L plantarum NCIMB 8826, PCU / AldhLl va PCU-Cela / AldhLl, hong dd san xuat 2,13 vd 1,99 g / 1 axit D-lactic, 2,69 va 2,41 g/1 xelobioza d 37''C vdi tdc dp lde 150 vdng/phiit [10].
4.KErLU0N
Tif 4 san pham \tn men truyen thdng va 1 mdu ba thai dong gilng phdn lap dupe 23 chung vi khudn cd kha nang Mn men axit lactic. Cfagn 5 cfating CI, C2, 81, S4, 85 cd vdng pfaan giai D/dl > 4. Qua cdc bude tuyen chgn tfai chung 85 cho ham luong axit tfau dupe la 5,13 g/1. 85 la true khuan gram (+), khong tao bao tu, catalaza dm tinh, khong smh khi va khdng di dpng. Dinh tin theo phuong phap smh 1;^, sinh hda va smh hpc phdn tu 16S rARN chung 85 cd dd tuong ddng 99% vdi chung Lactobacillus plantarum strain S7 (GU195646), chung 85 gpi Id Lactobacibus plantarum ^b. Dilu kiln d l null chiing S5 thu axit lactic tai nhilt dd 37°C, pH 6, hdm lupng dudng la 10 g/1, ty ll cap gidng la 10% vd hdm lugng 123
KHOA HOC C O N G NGHE
cao nam men la 3,00 g/1 sau 120 gid thi ham lupng axit thu dupe 6,66 g/1
TAHlUlTIUMKIUD
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Inverting GH8 Endoglucanases: A QM/MM
EFFECT OF SOME CONDITIONS ON LACTIC ACID PRODUCTIGN OF LactobacUblsplantarum 85 BY USING CEIIOBIOSE
Ngo Tlii Anh Van, Tran Lien Ha, FhiTlianh Mai Summary
Biomass includes agricultural residues, forestry and domestic waste, industry waste. Tbe use of biomass for useful products is the current trend because it save energy and the same time solve enviromental problem. One of the applications of biomass produce lactic acid is also the new development and interest Therefore, the aim of research is isolating, selecting and survey some elements affect the ability of fennentanon bacteria for lactic acid producUon. From 4 traditional fermentation products and 1 arrowroot residues 23 bactena strams were isolated and stram S5 showing highest lactic acid product of 5.13 g/1, was selected. The 16S rRNA of sb-ain S5 has 99% homologous with Lactobacillus plantarum SU-ain 87 (GU195646). So the name of S5 is Lactobacillus plantarum S5. In fermentahon conditions for strain S5 t° = 37'C, pH 6, celiobiose concentration of 10 g/1, die moculum 10%, yeast extract concenbalion of 3 g/1, after 120 hours of culture and maximum lactic acid of 6.66 g/1 was obtained.
Keywwords: Biomass, celiobiose, cellulose, lactic acid, Lactobacillus plantarum.
Ngudi phSn bidn: TS. Tr4n Thi Mai Ngiy nhln ba: 25/8/2015 Ngiy Ihdng qua phan bidn: 25/9/2015 Ngiy duydt ding: 02/10/2015 124
