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HOt NGH! KHOA HOC C O N G N G H $ SINH HQC T O A N Q U 6 C 2013

PHAN LAP VADINH DANH MOT SO DANG VI KHUAN SINH ACID ACETIC TRONG LjEN MEN HAT CACAO B A N G THUNG G O TAI D A K L A K

Phan Thanh Binh', Nguyen Thj Thoa, Nguyin Thj Mai, Pham Van Thao, Tran Thi Tham Ha, Vo Van Thang, Vo Thj Thuy Dung va Trin Thj Hoang Anh

VI$n Khoa hgc Ky thu$t Nong lam nghiep Tiy Nguyin

T 6 M

TAT

Vi khuan sinh agd acetic (VA) co vai tri cue kJ- quan trgng trong qua trinh len men h^t cacao. Chung tham gia vSo qua trinh oxy hoa ruqra etylic, dugc t?o ra bii hoat dpng cia nim men, dS tao thanh acid acetic. Trong qua trinh ISn men hat cacao Acid acetic k6t hgn v6t nhift dO cao dugc l?o thinh do cic phan ung chuySn h6a cac hgp ch§t sinh hgc se l^m chet mim h^t vi phi vd cSc cau true ciia te bio d^ cic hgp chat h6a hgc tac dyng voi nhau lam chuySn hoa mau sSc va chit hrgng cOa hat cacao cu6i cing. Qua trinh ISn men hat cacao li qui trlnh dau tien vi quan trgi^ nha! dS hinh diinh nSn cac hgp chit tiSn huong vj, mill sac va cau tnic dac tnmg ciia hat cacao trong che biSn sB c3 la. Phan % v i xic dinh m5t s6 loii VA trong qui hinh ISn men hjit cacao la budc diu tao co s6 oho v i ^ nhSn djnh eo ch^ cia qui trinh t?o nSn huong, vj s6 co la, la co scr dS nghiSn cim v i xay dyng cic quy hinh len men nham nSng cao vi 6n djnh diit lugng h?t cacao cu6i ciing. Phuong phip djnh danh dugc sir duag h-on^ nghien cuu niy li phuong p h ^ ket hgp djnh danh bing d|c di^m hlnh thii vi phSn tich Binh tv gen ma hoa 16S rRNA cua mgt s6 dang vi khuan da dugc phan l?p. Kel qui thi nghi?m chd thiy s6 lugng tS bao VA biln tiiien tir 1,8 x 10'' dSn 7^2 x 10' Da phan !pp dugc 7 dang khuin lac c6 hinh thai khae nhau vi djnh danh chiing Ihu^ 3 loii, bao g6m: Acetobacter iropicalis. Acetobacterpasteurianus. Acetobacter indonesiensu.

Tit khoa. Aceiobacter, cacao, com ohiy, len men, vi khuin

DATVANpe

Cacao Id mft trong nhOng nguyfin Ji$u cln phdi tral qua qua trinh len men dk chuyin h6a c^c hgp chat sinh hda nhfy vi , sinh v|t (ho^c enzyme), t^o nSh bkn hirang vj, mau sac, cau tnic cua hgt cacao dCing iim nguyfin lifiu ch^ bi^n so co la

(Thompson vi cs, 2001] C6 4 nh6m vl smh vSt chlnh tham gia vao qua trinh len men hgt cacao Id nim men, vi khuln slnh acid lactic, vi khuin sinh add acetic vh Bacillus (Andhana va Fleet 2003). Nim men va vi khuln smh acid lactic chilm uu thi trong giai dogn diu cua qua trlnh Ifin men (tCr 0 - 48h), nim men tham gia vao qu^ trinh chuyin hoa dirdng trong ldp com nhiy thdnh n^gru etylic va qud trlnh phfin cat cSc pectine ISm cho I6p com nhay tan ra. Khi l&p com nhiy giSm din, mfli truing trong khli hgt thflng thoang ho'n t^o dilu kifn cho sy ho^t d^ng cGa VA. VA se sj> dyng rupu etylic tgo thanh tCr ho?it dqng cua nim men d l tgo thfinh acid acetic dong thfi'i tfla nhift cao Tac d^ng cua acid acetic vfi nhi^l dO cao Ifi nguyen nhan lam chit phfli mim cCia hgt va tgo ra hang logt cac phan Crng sinh h<5a bfin trong hgt, lfim thay flli mfiu sic vfi cfic chit tien huong vi trong cacao (Cieenwerck va cs, 2007). Mgt so nghiSn ciru cOa cao tfic gifi tnrdc da cfing b6 Ifi Acetobacterpasfeuriansis la mflt lofii VA ty phat chinh tham gia vfio trong qua trlnh lfin men hgt cacao, chiing cd khfi nSng thich nghi v&l acid va nhifit flO cao ma chimg "tgo thanh, dflng thoi chung cung cd khfi nfing chiim uu thi khi trong kh6l u c6 chi>a acid lactic vfi manltol (Camu vfi cs, 2008). MOt s6 nghign ciru khfic cung da kit lufin Ifi c6 thi b l sung m^t s l lofii VA da dugrc kiem tra bang cac bifin phfip sinh h6a, Sl tham gia vfio trong qua trlnh lfin men (Lagunes vfi cs, 2007). Sy da dgng cCia cfic lofii nam men trong qua trinh len men hgt cacao tgi nhilu vimg trfin the g\d\ dfi duvc xfic djnh, tuy nhifin chi co mflt s6 lofii co lien quan tryc tilp den sy chuyen hoa cac chit trong qufi trlnh ISn men hgt cacao (Camu vi.cs, 2008). Nielsen vfi cfing Sy nghifin ctfu vi sinh vat trong len men ij d6'ng vd trong khay 6 Ghana cho thfiy dc lofii vi khuln san smh Add acetic cfl mfit dfi cao nhu: A. syzygll, A. pasteurianus and A.

Iropicalis dj^p'c xfic dmh Ifi hi?n difin trong sufit qua trlnh lfin men Mfit so loai duoc xfic dinh co sy hign dien vc^i s6 lupng Idn o.giai dogn cu6i cua qufi trlnh Ifin men Ifi Bacillus licheniformis vi Bacillus spp. Mfi't dd v'l khuln san sinh add acetic log cfu/g ti> 3,7 din 7,13 (Nielsen, 2007). Ardhana vfi cgng sy da phan Ifip vfi xfic djnh c^c vl khuln trong lfin men hgt cacao tgi Indonesia v6i kit qufi: Doi vfl'i vi khuln san slnh add acetic ca 2 lofii A. pasteun'anus vfi A. aceti diu thiy xufit hl#n sau 24 giir vfi cfl mgt dfi cyc dgi tu 10^ - 10^cfu/g, lofii A. pasteurianus phfit triln trong su6t qu^ trlnh Ifin men, chiu du'g'c nhifit dO cao. d<l cfln trfin 10% Loai A. aceti chi s6ng sot fl' d$ con 5-7%. 2 loai nay khflng thay cfl lifin quan toi cfic enzyme celluiase, amylase, protease, lipase ( Ardhana, 2003). Tai Ecuado 2oi Papalexandratou da phan Igp aug-c nhlfiu lofii vi khufin trong do cfl loai A. pasteunanus Ifi loai hi|n dign diinh trong qufi trinh chuyin hfla rupu thfinh a xi a xfi fc o giai dogn sau (Papalexandratou, 2011). Nghifin ciru tai C^ng hoa Dominic v6i-44 dgng dugc phfin l^p cfl i.sctofiac(//us pentosus (6 dgng). L plantamm hogc L paraplantamm (24 dang), L. paracasei subsp. paracasei (10 dang) va L. e/w;s (4 dgng). Tfit ca c^c dgng nfiy diu cfl khfi nang tgo acid lactic, acid acetic, nfou va COz (Galvez. 2007).

rlT^ f^^ i'^lT u-^" ^ift'^acao cac logi vi khuln da dug'c chpn lya vol nhung uu dilm: L femientum dugc chgn lya Mil fl,]?!" ^ i " 7 ^""'3 '''^''l ^° "^'^ '^'^'"=' "=^^"3 ""^^ ^P r^^" '^^O'-chiu nhiet, chiu acid lactic; A. Iropicalis fl xy nrA?;7. nht ^^h1 I f • J " -"^'^*- ^'^'^ ^'^^''- '^^'^ ^°''^^^° ^'"'^'"^ *^^ ^^^^ PerBira,-2012). Tai mSi vung. m5i quIc b L ohL^frih h L ^ ' ' i T - "t ^ f ' l ^ w " " ^ ^^"^ "^^''' *'°"S "9^'^" ^'^'^ "^y '^hOng toi da phfin l§p vfi xac dinh bing J i nnhJL rti, =T ' I ^ ""^^'° '°^' ^'^ ''^'"^' ^^^"^ 9'^ "^° "i"^ ^'"^^ 1^" ™n hgt cacao tgi Daklak d l lam ca sd cho cfic nghifin dim sfiu hon v l qui trinh len men hat cacao tgi Vi|t Nam. ' NGuyeN VfiJ L1$U VA PHirCfNG PHAP

Nguyfin v^it lif u V^tll^u

Sm ^ ^ P n p " i l ^ f "r? ^Zf-^ «^ ^'''"''^"° ^"'^'^ '^y '^ ^^"9 ty Trfich nhigm huu hgn Tu vin Diu tu Phfit triln Nflng

Lfim nghifip Eakmat, Bufln Mfi Thu^t. Daklak, nifin vy 2011 - 2012

H O l N G H ! K H O A H Q C C O N G N G H ? S i N H H Q C T O A N Q U 6 C 2 0 1 3

Mdi tru-dng pfiifn0pWl(f]ijanVA~(Drysdalel/^ — YGC Agar: glucose 50g/l (Merk), yeast extract 10g/l (Merk), C a C O j 30g/l (Merk), agar 20g/l , 0 1 % cydoheximide,

50mgfl penicillin (Sigma), p H = 5.6. Nufli trong tii fim d nhiet do 25°C trong tho'i gian ti> 5 - 8 ngfiy.

1^61 trirdng quan sat hinh thai ti bio va kha ning dl ddng (Nielsen, 2007) Mfii toro'ng long YG fl' n h i f t dO 25°C trong thoi gian tip 3 — 4 ngay.

Nhugm gram

Bfing phu-crng phfip n h u f m kep ttieo phuo'ng phap Christian Gram.

Thiit bj sd-dung

Cfin phfin tfch OHAUS, M o d e l P A 214, MJ. Mfiy do pH, Model: Hi 2 1 1 . HANNA, Italia. TCi a m SANYO, MIR 162, x u l t x u Nh§t. T h i l t bi theo doi n h i f t dg cam tay Model: Hi 39, HANhJA, Italia. Kinh h i l n vi quang hoc, Model: B353FL. OPTiKA, Italia. Kinh h i l n vl soi noi, Model- SZM-2, OPTIKA, Italia. Tu c l y , blnh dinh m i i c , binh tam giac, SOa thiiy tinh, dTa petri ...vfi m f t so t h i l t bi khae.

Phu'O'ng p h a p nghien c t r u Phin l$p VA td" khoi d cacao

T l l n hanh dap qua l l y hat, 0 trong thCing g5, thai gian u 6 ngay, 2 ngay dao hgt 1 lan vfi l l y mfiu theo tOng ngfiy, moi l l n l l y khoang 200 g m l u hgt tal 5 \^ tri theo dirfl'ng cheo va cr d g s a u lOcmtrongldidiQ.

Tfich p h l n c a m n h i y , n g h i i n mjn vfi t r f n d i u , pha loang n i n g dfl t u nong dg 10"^ d i n 10"'^ moi nong d f c l y 3 dfa, nuoi c l y 2 - 8 n g ^ trong tu nuoi d i l u nhift 6 n h i f t d f 25''C, d i m vfi xfic djnh mgt d f vi khuan VA. Quan sat bang mat, nhan dgng, chpn vfi' mo t l cac dgng khuan Igc, dfinh d i u vfi d i m s6 l u y n g khuan Igc mgc tren m5i dTa.

Phuang phap lfim t h u i n : Sinh khoi vi k h u l n d u g c nufii trfin mfli tnrcrng YGC Agar, pH-=^5',6; n h i f t d f 25°C. Chgn cfic k h u l n Igc cfl dgc d i l m rieng, dgc trung de c l y t h u i n ; C l y chuyen nhieu lan tren moi tnffi'ng Y G C Agar d d i l u kifin n h u trfin d l logi bfl nhOng tgp k h u l n cho d i n khi chi cfln 1 dgng vi k h u l n da chgn duy nhat, h u y l n phCi hfla khuan Igc trong n y f l c muoi sinh ly vfl triing vfi ria trfin mfli trufl'ng tiigch YGC Agar de lfim I h u i n vi k h u l n , c l y fnang ong nghifm, trfin mfii tru'fl'ng thgch nghieng d l bfio qufin dong ttiuan.

Phuvmg phip xic djnh cic d$c diim hinh thai

Quan sat k h u l n Igc bang mat t h u a n g , quan sat kha nang di dfing bang each lam tieu bfin gigt treo. Lfim tifiu bfin vi k h u l n khflng n h u f m vfi n h u f m Gram theo phuang phap n h u f m vl khuan cfla Christian Gram, soi duo'i kinh h i l n vi difin to Optika B-350 d v f t kinh 100. Xac d|nh cfic dac diem cOa hlnh thai vi k h u l n lam c a sd cho qua trinh phfin loai n h u : hlnh thfii, b l mat, mgt c i t vfi mfiu sac ciJa k h u l n Igc theo tirng dfing da dupe c l y lam t h u l n . d y a trfin dc n g u l n tfil lifiu mo ta cfic dfic diem cua vi sinh v§t cua ( N g u y i n Lan Dung 1976; N g u y i n Di>c Lupng 2006, Use Cieenwerck 2008).

Phirfmg phip dinh danh VA bang sinh hgc phin tir

M l u VI sinh v f l dfi d u p e lam t h u i n , nudi' ciy tr§n moi tnrdng YGC Agar, L l y 1,5 ml d]ch vi k h u l n , ly tam 10.000 vfing/phOt trong 2 phiit, logi bfl dich n l i . hfla tan phan lang bfing 567 pi d f m TE, thfim 30 pi^SDS 10% va 3 pi proteinase- K 2 0 m g / m l , t r f n deu, 0 trong 1 gi& 6 ST^C, them 100 pi NaCI 5M. trgn deu, them 80 pi dung dich CTAB/NaClr trfn^vfi Ci Irong 10 philt cr GS^C, thfim mgt lupng cfin bang chlorofomi/ isoamyl alcohol.trfn d i u vfi ly tfim td 4 d i n 5 phiit (loc dfi 10.000 vfing/phut). C h u y i n dich n l i sang 1 I n g s g c h , thfim mgt l u p n g can bfing phenol/chloroform/isoamyl alcohol trfin deu vfi t i l p tuc ly tfim trong 5 phOt. T i l p tyc chuyen djch n i l sang 1 I n g sgch khae, them 0,6 the tieh isopropanol, ly t f i m de logi bfi dich noi, hfla tan p h l n k i t tua trong 1ml ethanol 70%, ly tfim trong 5 phiit sau dfl logi bo djch noi v f i j f i m khfl nhanh trong mfiy u n h i f t khfl, ta thu d u p e DNA eua \ri khuan. Do nong dg DNA (25- 500ng/ reaction) bang mfiy Biophotometer (Model 6131, Eppendoft, Dire, 2007). L l y 5 pi mau cho vao phan iing PCR d e khuech dgi dgc h i f u doan DNA dfii 500 bp trfin vimg gen 16S rONA ciia vi k h u l n bang h f thong may PCR Thennal C y d e r eua Bio-Rad. D i f n di sfin p h i m PCR trfin gel agarose 2%. chyp hlnh b i n g h f thfing may Gel Doc cOa Bio - R a d . Tmh sgch sfin p h i m P_CR b i n g b f QIA quick PCR Purification Kit cOa QIAGEN. D i f n di san pham dfi tinh sgeh bang may h f thong mfiy Agilent 2100 Bioanalyzer (kJ h i f u 2100, Hfing sfin x u l t Agilent, My, 2005), PCR SEQ san p h i m da tinh t r u d c khi gifii trinh t y tren h f t h i n g mfiy ABI 3130XL (kJ hiSu 3130XL, Aplied Biosystems, M?, 2007). Phfin trch'ket qufi b i n g phfin m i m sequencing analysis 5.3 (Aplied Biosystems, My, 2007) vfi so vfl'i k i t qufi tren ngfin hang gen t h i gifl'i (Genbank), (Phong xfit n g h i f m Nam Khoa B I O T E K - ISO 15189).

K ^ T Q U A v A T H A O L U - f l j g

Kit qua xac djnh m$t do VA trong miu cacao len men

Qiia trlnh ifin men hgt cacao d u p e chia lfim 2 gial dogn chinh: giai dogn 48 gifl d i u thufl'ng la giai dogn len men y i m k h i vdi s y hogt d f n g cOa n i m men e h i l m u u the vfi giai dogn sau 4 8 gifl' la giai dogn ten men h i l u khi vfl'i s u tham gia chinh ciia v A . VA sir dyng c a c h i t nj'pu etilic d l san sinh aeid acetic. K i t qua phfin l | p cho t h i y s y b i l n dfing cua mgt d f VA Iheo h u d n g c h i l m u u ttii tgi thdi d i l m 24 g i d ; 4 8 g i a ; 72 g i d , eao nhat tgi thdi d i l m 72 g i d vdi mfit dfi 7,2 x 1 0 ' CFU/g c h i t khfi, sau do gifim d i n d i n cuoi qufi trlnh lfin m e n . M^t d f k h u l n Igc t h i p d cfic thdi diem 0 g i d ; 120 gi6';144 gid, giao d f n g tir 1,8 x 10^ - 5 , 9 x 10* CFU/g (Bang 1). Nguyfin nhfin cCia s y bien d f n g mgt do VA trong qua trinh len men trfin Ifi do tgi cfic thdi d i l m 2 4 , 4 8 . 72 h hfim l u p n g c o chat trong c a m n h i y d o i dao, moi t r u d n g thong thoang h a n tgi thdi d i l m 0 gid do s y Ifin men vfi thoat dich ra ben ngoai k i t hpp vdi hfim lupng r u p u etiie tao thfinh do hogt d f n g d i a n i m men n h i l u , dfiy lfi d l l u k i f n thich hpp cho s y fiogt dfing cua VA. Bgc bift tgi thdi d i l m 72 g i d sau qufi trinh dao

HOI NGH! KHOA HOC CONG NGHE SINH HQC T O A N Q U O C 2013

. . „ . . - . .A. .. !,.£„ kh.- hon M vi^i cac thin (film khae, k^t hop v o i moi tnrong dinh dirang d6i dSo h , l tai tho, d l S ™ f . " l ' T " ^ ? " f ; S n g thS^g thoang hon nhl-ng lu.ong CO chSl da hSt. bSn c n S d6 phai T " 5 * J " K • ? , I ^ l ? m S aa VA da" oia M l^n n h i t tal thoi d i l m 36 , S 72h, sau dd g l i m san IhM diSm 72h S " * l S r = ' ' 2 ? , T t^ X o » a l ^ * m ? » L n U g c n a a vS cs. (2007) cho k«t qua h i m lu.ynB ethanol cao 6 48 g l 6 «a VA t3l IMS c f i m 48h dat g l i tri cao nhal voi mCte 1,5 X10* CFU/gam c h i t khd.

Wng 1. M9t dp tning b)nh cua VA trong m i u h?t cacao qua cac ngay ten man

T l f t g i m Nhistdsningblnh pH tmng blnh c u . pH tntng blnh c d . h,t S6 I w n g CFU/gam hat

(gB) k>6iuCci com nhiy t i r o i i w ) 22,6

40,1 41.7 45.9 41,9 43,7 40.9

3.96 3,49 3,50 3,61 3,86 4.1 S 4.29

6,59 6.38 5,75 5,73 5.07 5,13

0,0018 4,744 2,6333 7,2000 0,1700,- 0,0433 0,0588

Xic'djnh cie d^c diim blnh thai cua vi khuin

TCr m l u hgt cacao Ifin men, chung tfii da phfin I f p dupe 7 dgng VA khfic nhau v l hlnh th^i k h u l n Igc n h u mfiu s i c , hlnh dgng. m^t d t , bk m$t (Bfing 2). Dfiy lfi m f t trong nhung CP s d d l xfic dinh tfin chi, tfin lofii VA. Bfin cgnh dgc d i f m hlnh thfii k h u l n I4c chCing tfli cl3ng t i l n hfinh xfic dph hlnh thfii t l bfio k h u l n Igc vfi khfi nfing di d f n g cOa chiinp d f cfl thfim cp s d CO v i f c dinh danh (Bfing 3). K i f q u i cho t h i y h l u h i t cfic dgng k h u l n Igc deu hinh trfln, u d t mfiu trang, nfiu nhgt vfi v i n g nhgt (Bfing 2).

Bang 2 Dac d i i m hlnh tM) khuin I^c VA

ST KyhlSu ,., . . ,,u i , BftmStkhu^n Msn,h,.a„i=^ MSu sac khuln M?t cSt ngang khuln T VA ' " ^ ^'^ lac ^ ^ Igc l?c 1

2 3 4 S 6 7

V A l VA2 VA3 VA4 VA5 VA6 VA7

Trtin. d i u Tr6n, diu, tgo vAr^h TiAndiu Titindiu Tr6n. m6p tgo ring ara Tr6n, diu Trtn, diu

Bdng vdt BdnguiM Hoi nhfin. udt Hoi nhfin. udt Bfing uiM Hcri nhan, udt Hoi nhan, udt

PhIng • Bang phang

Gpn sfing Gg'n sfing Bang phIng

Go'O sfing Ggnsfing

Trang sua Trlng sua Trang dgc Trlng sOa Hong s3m Trlng sOa Trang sUa '

L6i

Lt\

Hai det L i i Ldi Cfic dfic diim cho Uily cfi 5^ khdb nhau giua cfic khuln Igc, tuy nhiSn mft sd miu cfi sy khfic nhau khdng rfi rfing, thfim chl gilng nhau v i c^c hlrih deng, djc diim ciia khuin Igc nhu dgng VA6 vfi VA7, VA3 vi VA4...Cfic dang nfiy cd khfi nang cting m f l lofii vl khuin.

U n g 3. D$c diim hlnh t h i l t i t>do khuin Igc VA - - STT Ogng khuin Igc (VA) Efc diim hlnh thdi l i bdo

1 VA 1 Gram fim, hlnhhgl, hlnh try ngin. t i bfio kit thfinh d&i, Ihfinh cgm hof c dung rifing rfi 2 VA 2 Gram fim, hinh hgi. t i bfio k i t ttifinh d6i, thfinh cym hofic «>ng rifing Ig

3 VA 3 Gram fim, hinh hgt. hinh try ngan, te bfio kit thfinh cym hogc dung rifing 18. khflng di dfng.

* VA 4 Gram fim, hinh hgt. t i bfio kit Ihfinh cym hofic dirng rifing le, khong dl dfng.

S VA 5 Gram fim, hlnh que ngfin. que dai. t i bfio kit thfinh cym hole ddng ding B.

°' VA 6 Gram fim, hinh hgt, hlnh tnj t i bfio kit thfinh dfli. thfinh cym hogc dung rifing IS, khdng di dfng.

__J_ V ^ ^ Gram fim. hlnh tty ngin, dung rifing Ifi hay xip thfinh timg doi. tCmg cym, khflng di dfng H l u h i t cfie t l bfio k h u l n Igc deu o- dgng Gram fim, c6 hlnh dgng hgt hofic hlnh que n g i n . h o f c hlnh tn,i v 6 i cfic te bfio kft thfinh dfii, thfinh egm h o f e dirng rifing le, cfic t l bfio it di d f n g hofic khflng di d f n g . M f t so dgng co cac dgc d i l m ' * f 9 ' ' J " S "'^^^ ' ^ g qua trlnh quan sfit nfin khd phfin bift bfing hinh thfii d l xfic ^ n h chlnh xfic d u p e lofii (xem t h f i m

' • • . : . • ' " • • - ' • . . - •• /' •' ^ l '^.i

mmm

HOI NGH! KHOA HOC C O N G NGHg SINH HOC TOAN QUOC 2013

P- _{^•'- t} mmm

Hinh 1. Kinh inh cdc dgng t i bdo khuan lac

Kit qui djnh xic djnh bang slnh hgc phin tu"

D e xfic dinh dupe chinh xac tfin lofii cCia cfic mau da phfin I f p vfi xfic djnh cfic dgc diem dgc trung von c6 cilia c f i c y i khuan chiing tfli da d y a vao k i t qua phfin giai trinh t u gen 16S rRNA vfi ket q u i tra cuu tren BLAST SEARCH. V 6 i m i i c d f tuong ddng ciia cac m l u so vfl'i ngan hang gen t h i gifl'i tCr 99-100% da xae djnh dupe. 3 loai A. iropicalis, A.

pasteurianus, A. indonesiensis, trong do A. indoneslensis lfi loai chi d u p e Vuyst xfie d|nh Ifi cfl sg h i f n d i f n trong qufi trinh lfin men hgt cacao. 2 lofii efln Igi cfl s u hifin dien o-'tlt cfi cfic nghien c i i u v l vi khuan trong len men hgt cacao tren t h i gifl'i.

Bang 4. Trlnh t u gen 16S rRNA va k i t qufi so sfinh vdi Genbank cOa cdc chung vi khuan

K i t qufi gidi trlnh tu gen 16SrRNA TuOTig tu VI

cdc tdc gifi AGTCGCACGAAGGTTTCGGCCTTAGTGGCGGACGGGTGAGTAACGCGTAGGAATCrATC A trOpiCaliS CATGGOTGGGGGATAACTCTGGGAAACTGGACCTAATACCQCATGATACCTGAGGGTCA m(3cdd/d 100%

AAGGCGCAAGTCGCCTGTCCAGGAGCCTGCCTICGATrAGCrrtnTOOrGCGIjrAATGGC CTACCAAGGCGATGATCGATAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAG ACACGGCCCAGACTCCrACGCXIAGGCAGCAGTGGGGAATATTGGACAATGGGGGCAACC CrGATCCAGCAATGCCCCGTGTGTGAAGAAGGTTTTCGGATTGTAAACCACnrCGGCGG GGACGATGATGACGGTACCCGCAGAAGAAGCCCCCGCTAACrrcCTGC

AOTcGcAcaAAOOTrrcGaccTrAQTCQceoACGaGTaAaTAACocoTAcaTATCTATCt A. Iropicalis ATOaOTaOOOOATAACACrOqaAAACrCGTCCTAATACCGCATCACACCTQAdGGTCAA , AOCCCCAACTCaCCTGTCaAGOACCCTCCGTTTCATTACCTACTrOGTOGGGTAAACCC niUC OP IB 99%

CTACCAACGCCATOATCAATACCrGGTrTOAGAGGATCATCACCCACACTOGGACTCAO ACACGGCCCAG ACTCCTACOOO AGGCAGC AOTGGGC A ATATTC OAC AATCG GGGCAACC CTGATCCAGCAATGCCCCGTOTaTGAAGAAGGTCTTCGGATTGTAAACCACnTCGACGC GG ACGATG ATG ACGGTACCCCTAGAAC A AGCCCC GGCTAACTTC<JTCCCAOC AGCCGCO

AACCCTGATCCAOCAATaCCGCCiTOTaTCAAaAAGt

CCQ OOQ ACOATOATOACGOTACCCGC AC AAG AACCCCCC QCTA ACTTCGTGCC AQCAOC

OATCCTOGCrCAGAGCGAACCCTOOCCGCATCdTAACv TTCQOCCTTAOTOGCaO ACGGGTGAOTAACOCGT AGGTi ACACTGGOAAACTCrTrGCTAATACCGCATGACACCrGl

j r a r a g A C a A O C C r r O C C T T T G A T T A C C T A C r i T Q C T C O C G T A A A G G C C T A C C A A O G C O A T a ATT-A AT AOTTGlTrTTCi A f i AGG ATCi ATC A ( ! { T AC ACrTGOr. A r r n A C l ACA CCCiCCTC AGACn CCTACCG a A a t K : 5 G C M T r a B G G A A T A T T G a A C A A T C G ^ a C A A C C C T O A T C C A C e A A T I CCaCCTCTCrOAAGAAGaTCTTCGCATTaTAAAGCACTTTCC

OGTACCCOTAO AAQ AAaCCCCOGCTAACTTCGTGC

(Nicholas Camu, 2007;

Schwa n et al, 2004)

(Nicholas Camu, 2007;

Sctiwan Bt al, 2004)

(Garcia et al, 2010; Camu e( af, 2008; T.- Romero- Cortes. 2011) (Garcia et al, 2010; Camu et al, 2008; T.

Romero- Cortes. 2011)

ACCCGCAOAACAAOCCCCOGCTAACTTCGTCC

ATCCT<KXrrCAOAGCCAACljCTCGCOGCATG<^rTAACACAT(X:AA(jTCGCACCAACCT]-I fl paSfgUfjanUS CrCTTGOOAAACTGCAOCrAATACCaCATGACACCrrOAOGGTCAAAGaCOCAAOTCGCCr mOC (Jl? / d 100%

CAATACCTOCn'CTGAOAGGATaATCAGCCACACTGOGACTGAGA*:^CGGCCCAGACTCC

(Garcia et al.

2010; Camu ef ai, 2008; T.

Romero- Cortes, 2011)

HOI NGH! KHOA HOC G O N G NGHE SINH HQC T O A N Q U O C 2013

Romero- Cortes, 2011) Il^w-«a-i-TT»moccGOAC-occroACTAAcoccTAGGTATtrr fl_ pasteunanus. (Garcia e* al.

sr;Se^::u's\f:Tv^xt^^

S n g lfi n ^ C t o f i r S ^ ^ g ? ^ ^ trong k h l i 0 hgt cacao d dc n u 6 c trfin t h i gidri n h u ; Indonesia. tha«and,

? h X n e s , Ghana Bfiy I f i ' S i g c f m | t d f vfi t i n s l x u l t h i f n khfi cao trong h6. u hgt .^^°-f " " 9 " ^ / ^ ^ ^ ^ ^ ^ k* quan tn?ng d l tgo n l n cfic phan i^ng smh hfla hlnh thfinh ti^n h u o n g v| sfl cfl la. ChT c6 dgng VAS t " u l t h 9n trong S c nghifin c^u t r u * c dfiy. dfiy cfl IS lfi m f t trong. nhOng lofii cfl d i l u k i f n phfit I r l i n kha d f e b i f t nfin chl x u l t hign o m f t s6 viing.

K£T LUAN

Trong qufi Irlnh Ifin men s v b i l n d f n g m f t d f vl k h u l n khfi cao tir 0.18 x 10*- 7.2 x 10^ CFU/gam hgt tifcri, vi M i u i n dgt m f t d f cao n h l l tgi th&l d i l m 72h. 7 dgng vi k h u l n dfi dug^: phfin l | p vfi xfic djnh cac d f e d i l m : t f bfio khufin Igc c6 hlnh Irftn u 6 t mfiu t r l n g , nfiu nhgt vfi vfing nhgt. cfl hlnh hgt, hlnh t n j ngfin hoge hinh que, cdc t f bfio k f t thfinh dOi, thfinh cym hof c dirng rifing Ifi. It hofc khflng di d f n g . K i t qua xfic ^ n h tfin lofii theo sinh hgc phfin tir di^a tren phucrng phfip gifii trlnh t u gen 16S rRNA cho k i t qufi 7 dgng VA t h u f c 3 lofii khfic nhau la A. Iropicalis ( V A I , VA2), Apasteurlanus (VA3, VA4, VA6, VA7), A. Indoneslensis (VA5) vfi'i m i i c d f t u o n g dflng t i r 99-100% so v o l ngfin hfing gen cOa t h i gidi (Genbank)

T A I U ^ U THAM K H A O

Ardhana MM, Fleet HG(2003). The microliial ecology of cocoa bean fernienlations in Indonesia. InL J. Food'll4ihrobiol., 86(1); 87-99.

D.S Nielsen. O. 0 . T. L Ban-Kofli. M. OMUSU, T . S . Aodorsson, W.H. Holzapfel (2007). "The microbiology of Ghanaian cocoa femianlatlons analysed using cuRure^Jependent and culture-independent methods.' Inlemab'onal Joumal of Food Microbiology 114 166-186.

Garcia AT, Papalexandratou Z, Hendryckx H, Camu N, Vraneken G, Vuyst L, Cornells P (2010). Diversity of the total bacterial community associated inrtlh Ghanaian and Brazilian cocoa bean fennentation samples as revealed by a 163 rRNA gene clone library.

App/.MfcroWb/. aotec/?no/.i7{6): 2261-2292.-

Giltierto Vinidus de Melo Pereira, M. G. d C P. M., Clntia Lacerda Ramoa, Rosane Preites Schwan (2012). 'Microbiological and Physicochemical Characterization of Smail-Scale Cocoa Femientations and Screening of Yeast and Bactena Strains for Ihe Developrfient of a Defined Starter Culture * American Society for Mierobidogy.

Use aeenvierck.A'ngel Gonzalez, Nicholas Camu. Katnen Engeltieen, Paul OeVos and Luc De Vuyst, Acetobacter fabarumsp. nov., an acetic add bacterium froiri a Ghanaian cocoa bean heap feimentation, Inlemattonal Joumal of Systematic and Evolutionary Micmbiology (2008), 58.2160-2165.

Luc De Vuyst, Nicholas Camu, Tom De Winter. Katnen Vandemeulebreocke, Vincent Van de pene. Marc Vancanneyt, Paul De Vos, . Ifse Cieenwerck, Velidatfon trf the (GTG)-rep-PCR fingerprinting technique for rapid classification and Identifieahon of acetic acid bactena, with a focus on Isolates from Ghanaian fermented cocoa beens, International Joumal of Food Microbiology 125 (2006) 79-90.

microblal.ecology of cocoa bean fermentations in Indonesia." International Joumal of Food

Nicholas Camu. Tom Oe Winter, Knstof Verbrogghe, Use Cieenwerck. Paler Vandemme. Jemmy S.Takrama. Marc Vancanneyt and Luc Oe Vuyst, Oynamics and biodiversity of populations of lactic add bacteria and acetic add bacteria involved in spontaneous heap fermenlation of cocoa beans in Ghana, Applied and environmental micrcAk^ogy. mar, 2007. p.1609-1624

Nicholas Camu. Angel GonZalet Tom Do Winter. Ann Van Schoor, Katrien De Bniyne, Peler Vandemme. Jemmy S.Takrama. Solomon K Addo and Luc De Vuyst, Influence of turning and envlnanmental contamination on the dynamics of populations of lactic acid and acetic add badena involved in spontaneous cocoa bean heap femientation In Ghana, Applied and environmental microbiology, mar, 2007, p 1803-1824

Sjnjra L ^ i i r » s Gaivei G- L. Jos. Luis PamOss, Mrchel Bard, Joseph-Prerre Guiraad (2007). "Sludy on Die microllora OlU WochBmistfy ot cocoa fBonenlaliwi in Ihe DooAiican Rfipubfrc.' intemalionat Journal of Food Microbiology 114; 12*-130.

L ' J f r . ' : ! ' ^ ^ / " • "'f^!:!:'' ' ^ ' ' " • ^ • J i ^ " ' ana M R.mr»z-L.p,. 2011. Isolation and ctiataoterizaBon of acoBc aoid bactena in cocoa lermentatwn, ^ c a n Journal af UKfobiology Resaarch Vol 6(2), pp. 339-347,16 January, 2012.

ISOLATION AND IDENTIFICATION OF ACETIC ACID BACTERIA IN WOODEN BOX COCOA FERMENTATION IN DAKLAK PROVINCE

Phan T h a n h B l n h Nguyeri ThI Thoa, Nguyen ThI Mai, Pham Van T h a o ; T r a n Thi T h a m H a , V o V a n T h a n g , V o T h I Thuy D u n g , Tran ThI Hoang A n h , '

HOI NGH! KHOA HOC C 6 N G N G H E SINH HpC? T O A N Q U 6 C 2013

The Western Highlands Agriculture Forestry Saentitic addTectmi^tlttstitate

SUMMARY

Acetic acid bacteria is very important role in the cocoa fennentation. They oxidize ettianol, which was produced by tlie yeast, to produce-acetic acid. In the cocoa fennentation, acid acetic aad temperature (which formed by biocheimcal-reaca.on)Jdll germ and break the wall cells, which make flie ways of chemical compounds to contact together and change flie color and quality of cocoa beans. Fennentation of cocoa beans is the first and most impoitant stage to make the piescusor flavor, color and texture characteristics of cocoa beans in chocolate processing. Isolation and identification of some species of accehc bacteria are the first stage of researching the produce of cocoa flavor and quality and to complete the cocoa fennentation process for increasing and stable cocoa jjuality. Morphological charactenstics and sequence analysis of 16S rRNA genes mefliods were used in this study. The results show fliat the number ofCFU from 1.8 x 10''to 7.2 x lO'CFU/gfiesh cocoa bean. 7 different forms of bacteria were isolated and"

identified by iCS rBNA and which are similarities in Acetobaters of Genbank at 99-100%, including Acetobacter tropicalis,

Acetobacter pasteurianus,A cetobacter indoneslensis.

Keywords; Acetobacter, cocoa, fermentation, identification, pulp

•Author for correspondence: Tel: +84 5003 862 845, binhanphuo_chanh@gmail.com