KHOA HOC CONG NGHE

PHAIV LAP VA TUYEIM CHOIV DOIXIG IXIAIVI MEIXI {SACCHAROMYCES SP. ) LEIXI MEIXI RUOU VAIXIG

TRAI TRAM [SYZYEiUM CUMtlMii n-nfrih Ng(?c Thanh Tain\ Dao Thanh T&nx^, Nguyin IH Minh TrSm^

van Thi H6ng Hu6=', Duong Thj Mai TliSo'

T6MTAT Nghi&n ciru dij(?c thuc hien nham phan lap va tuyen chpn dupc dong nam men co hoat tinh len men cao tit trai tram, tir do ling dung l n men mau vang trai tram, mpt loai thiic uong giau dinh duong va tot cho siic khoe ngum sii dung. Tir nguon qua trai tram ban d^u dmrc thu tai 6 dia diem gom: Nmh Ki u, Th6t Ndt (thanh pho Can Tho'), Thoai Son (An Giang), Lai Vung. Sa Dec ff)6ng Thap) va Binh Tan (Vinh Long) da phan lap dupc 40 dong nam men. Dua vao khoa phan loai nam men (hinh thai, sinh ly, sinh h6a) cac dong n ^ men dirpc chia thanh ba giong gom: Saccbaromyces, Pichiawa Hanseniaspora. Qua thi nghidm l6n men dupc thitc hi^n trong dng nghiem chira chuong Durham va binh tam giac, ddng nam men TN4 (ditgfc phSn Igp tir dich qua tram thu tai Thdt Ndt, (thanh phd Can Tha) thuoc gidng Saccbaromyces co kha nang len men nhanh va cho dp rupu cao nhat trong cac ddng nam men. Voi dieu kien l n men ban dau pH 4,0, 20''Bnx vA mat sd nim men W te bao/mL, ddng nam men TN4 da len men dich trai tram cho dp rupu dgt 7,53% v/v sau 10 ngay l6n men. Tir ket qua dinh danh bang phucmg phap giai trinh tu gen da xac dinh dong nam men TN4 thupc lo^ Saccbaromyces cerevisiae. Tir kh6a: N^ men, phan lap, rupu vang, Saccbaromyces cerevisiae, trai tram.

1 . DAT VAN B^

TrSin mdc (Syzygium cumlnlL.) (Hinh 1) thuoc hp Myrtaceae, phat ti-i n tir nhifen tren dat set nau cr cac viing nhiet doi cQng nhir cac viing can nhiet doi ciia dong bang [4], dugx; trong nhifiu cr An Do va cac nucrc khac nhu Malaysia, Bangladesh, Mien Dien, Nepal, Pakistan, Sri Lanka, Indonesia va Viet Nam.

Syzygium cumini la mot loai duac li^u tr6n th^ gidi, dinrc sii dung nhu thao dugc do co tinh chdng l^i c^c roi loan tim mach, ha duong huy^t, ha lipit, chong viem, hoat tinh chong ung thu va chdng oxy hoa [IJ. Ngoai ra, trai tram con c6 huong vi chua ngpt h6a quyen, hoi chat va co mau tim dat trung, thich hgp cho viec len men rupu vang.

Gidng nam men Saccbaromyces thudng duoc sii dung trong san xuat rugu vang, phan bd rgng rai

' Vi n NC&PT Cdng nghe Sinh hpc. Trudng Dai hoc Can Tha - Sinh vien Vi?n NC&PT Cong nghe Sinh hpc, Tnidng Dai hpc Can Tha Email: hnttam@ctu.edu.vn

Hinh 1. Cay tram, hoa tram va trai tram dugc thu trong tu nhien nhit la trong dat, tr^n b^ mat nhi^u loai trai cay va cac loai cay luong thuc, thuc pham khac [10]. NhiSu loai trong nhom nay co kha nang len men ruou dugc ap dung trong san xuat rugu, bia, rugu vang va lam banh mi. Te bao nhn men giau protein, vitamin co the diing de che bien mgt so dang thuc pham cho nguoi va bo sung dinh duong vao thirc an gia siic [3].

NONG NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANG 9/2019 59

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Xhieu nghien cuu da cho thay \iec su dung nguon naim men dugc phan tap tu chinh nguon nguyen li^u sii dung l&n men ruou vang se lam tang mili VI va chat lugng cua san ph in . Vi vay. nghien cim duoc thuc hien nham muc dich tuyen chgn dugc ddng nam men co hoat luc cao ung dung vao len men rupu vang trai tram, lam tien d^ khao sat cac nhan t6 anh hudng d^n qua tnnh len men va tim ra nghiem thlic tdi uu giup qua trinh len men dat hieu qua cao. Dong thdi, nghien ciiu ciing nang cao gia tri ciia trai tram va gdp phan da dang hda cac san pham len men.

2. PHUONG PHAP NGHBI CUU

2.1. V&t l i ^ Mau trai tram (Syzygium cuniim) dugc thu hai

lai 4 tinh/thanh: thanh phd Can Tlia (quan Xiiib Kieu va huyfen Thdt Ndt), tinh An Giang (huy?n Thogi Son), tinh Dong Thap (huydn Lai Vung, thanh phd Sa Dec) va tinh Vinh Long (huyen Binh Tan). Trai Iram dugc ddng gdi, bao quan trong tung bao PE (polyetylen) rieng bidt va trir lanh trong cac thiing xdp dc- tranh anh sang mat trdi true tidp. Cudi cimg la van chuydn ngay den phdng thi nghidm de giam thidu tdi da su thay ddi tinh chat, trang thai ban diiu ctia mau.

N^m men ddi chiing Saccbaromyces cercvisiai-(Hoa Ky)- ki hidu DC, dugc luu giii b 4'C tai Xten Nghien ciiu va Phat trien Cong nghe Sinh hpc. Trudng Dai hgc Can TTICT.

2.2. Phuong phAp nghifin cuu 2.2.1. Phan lap va dinh danh sa bo cac dong nam

men tti trai tram Cho 2-3 trai tram da dugc loai bo hat va de

nguydn khdng nghidn vao binh tam giac 100 niL co moi tnrdng YPD (Yeast extract-Peptone-D-glucose). tang sinh b nhitt do phdng trong 24 gid. Tien hanh phan lap tren mdi trudng " TDA {Yeast extract-Peptone-D-glucose-Agar) den khi cd dugc nhung ddng n ^ i men thuan chung va dinh danh sa bd cac ddng nsim men nay bang phuong phap hinh thai hpc dua vao khda phan loai nam men ciia Kurtzman and Fell 13], Luong Due Pham (2009) (3] va X^uyrn Diic Lugng (2006) [5] kdt hgp vdi phuong phap sinh hda (Idia nang len men dudng glucose va sucrose, kha nang phan giai urea).

2.2.2. Tuyen chon ddng nam men cd hoat luc ldn

men nnpn cao Ldn men tnmg chudng Durham

, .,., j„ng nam Myc tidu thi nghiem: Tuyen chpn .ai a" «

men cd kha nang len men trong nioi tmdng d'chtr^

Cach ti^n hanh: Chuan bj djch nam men va

ua n4t sd te bao nam men bAng phuong phap dem

de cd mat s tiuc tiep bang buong dem hong cau i nam men 10 td bao/mL Tien hanh ep trai tram moc (Svz^'gIum cuminiU d^ lay nuoc Tidp dieo la didu chinh cac thdng sd ve 2(>-Bri.x va pH=4.0 va toh Uung bang NaHSO. (140 nig/D trong 2 gio de Ueu diet vi sinh vat cd trong dich trai, Siu do, cho 1 mL dich nam men vao 9 mL djch phdi che da chuan bi san trong ong nghiem vd triing da chua chuong Durham. D^y nap dng nghidm lai va lac ngugc ong nghidm xudng dd dung dich Iran vao chuong Durham, sao cho khdng cdn bpt khi trong chudng Durham va chim hoan toan trong dung dich. Tien hanh do cdi klii CO^ d cac thdi diem 6 gid, 8 gid, 12 gid. 24gid,36gitf |9I.

Chi tidu theo d6i; Tidn banh theo ddi cdt khi CO;, U-ong chudng Durham b cac thdi diem (6 gid, 8 gid, 12 gid, 21 gid, 36 gid) cho den khi CO. day tiong chudng Durham.

Ldn men tivng binli lam giic Tir thi nghidm ldn men trong chudng Durham,

chgn ra nhting ddng nam men cd kha nSng phan giai duong saccharose tdt de tidn hanh len men trong binh tam giac nham tidl kiem nguyen li u va dung cu thi nghidm.

Muc Udu thi nghiem: Tuydn chpn cac dong nam men co ho^t li^ len men rugu cao.

Cach tien hanh: Chuan bj dich nam men va kiem lra mat sd td bao nam men bang phuong phap dem true tidp bang buong ddm hong cau de cd mat sd nam men 10 td bao/mL. Trai tram (Syzygium cummi) drn^c ep lay nude va thanh triing bang NaHSO., (140 mg/L) trong 2 gid de tieu diet vi sinh vat cd trong dich trai. Ti^p dieo la dieu chinh cac thdng sd vd 20"Bnx, pH = 4,0. Sau dd, cho 1 mL dich nam men da tang smh vao 99 ml dich qua phdi che da chuan bi san trong bmh tam giac, ldc d&u binh tam giac dd te bao nam men phan bo ddu trong dich phdi che. Cudi cimg la dem u b nhiet dd phong Sau 10 ngay, tien hanh chung cat de thu cdn. Do c6n thu hdi dugc do bang cdn ke (9|.

Chi tieu theo doi: Su thay ddi pH, "Bnx dd c6n thu hoi a 20"C sau thdi gian Idn men 10 ngay '

2.2.3 Dinh danh bSngphuong phap g.u uinh la

NONG NGHIEP VA PHATTRIEN NONG THON KY 2-THANG 9/2019

KHOA HOC CONG NGHE

Qua thi nghiem d muc 2^.2 da xac dinh duoc ddng nam men cd kha nang len men rugu trai tram tdt nhat Ddng nam men nay dugc chgn de dinh danh d^n miic dg loai bang phucmg phap smh hoc phan tu, ket hgp vdi dac diem hinh thai va thi nghiem sinh hda. Ddng nam men dugc chgn giai trinh tu doan gen bang phan img PCR vdi cap moi ITSl va rrS4 [6] va sii dung chuong trinh Nucleotide Blast de so sanh miic dg tuong dong ciia trinh tu dugc giai vdi trinh tu cua cac ddng nam men trong ngan hang gen tren NCBI vdi phdn mem BLASTN.

2.3. Cdc chi ti6u phan tich vk xir ly thdng kd Phan mem Microsof Excel 2010 dugc sii dung

de xu ly sd lieu thd, tinh cac sd lieu thdng ke nhu sd trung binh, he sd bidn thien (CV96), dp lech chuan (SD). Phdn mem Statgraphics XVI.I dupc dimg de phan tich phuong sai va kiem dinh LSD cac trung binh nghiem thiic.

3 . KET QUA VA THAO LUAN

3.1. Phan lap va dinh danh sa bO cAc ddng nim men tu trii tram

Quan sat hinh dang te bao ciia cac ddng nam men:

Qua qua trinh phan Igp cac ddng nam men tir trai tram tai huyen Thoai Son (An Giang), thanh phd Sa Dec va huyen Lai Vung (Ddng Thap), huydn Binh Tan (Vinh Long), quan Thdt Ndt va quan Cai Rang (tiianh phd Cdn Tha), da thu dugc 40 ddng nam men vdi 7 nhdm hinh dang td bao (cau ldn. cau nhd, oval ldn, oval nhd, elip ngan, elip dai va elip nhgn) (Hinh 2 va bang 1). Quan sat hinh thiic nay chdi dudi kinh hien vi, cho thaiy 40 ddng nam men ddu cd kha nang nay chdi, vdi 3 hinh thiic gdm don cue, luong cue va da cue. Nhdm te bao hinh cau ldn, cau nhd, oval ldn, oval nhd, elip ngdn va ebp dai la nay choi da cue hoae don cue, nhdm te bao hinh elip nhgn la nay choi ludng cue (Hinh 3). Kdt qua cac thi nghidm sinh hda nhu kha nang phan giai dudng glucose, saccharose va kha nang phan giai urea dugc the hidn qua bang 2 va hinh 4.

Dia diem

rhoai Son SaDec Lai Vung Binh Tan rhot Ndt Cai Rang Tong so

Bangl So dong nam men

5 9 6 8 6 6 40

.Sd lugrng Phan tram

(%) 12,5 22.5 15 20 15 15

1000

v^ hinh d^ing c ^ nh6m n ^ men dupc phan 14p tgi c4c dia Siim Hinh dang n ^ men

Cau Idn

1 1

Cau nhd

3

3

Oval ldn

3

2

2 7

Oval nhd

1

3 3 2 9

Elip dai

2 1 4 1 2

10

Elip ngan

2 1 2 2 1 1 9

EUp nhpn

1

1

Cbu thich: (-) khdng xuat hien

ftlH4s\_

'-ii*-

%imt> iM

Hinh 2. Hinh dang t^ bao cua c4c ddng nam men

phan lap duoc tu tr^i trSm

(a) Ciuldn-CR3. (b) Cau nhd-SDl (c) Oval Idn-

SD4. (d) Ovalnhd-TSl, (e) EUp ngan-CR6, W Elip

dai-TS4, (g) EUp nhon-SDd.

(1) IW It)

Hinh 3. Nay chdi da cue (a), nay chdi lu&ng cue (b)

va nay chdi don cue (c)

(a) Hinh oval Idn (TN4), (b) Hinh elip dai (TS4},

(c) Hmh oval Idn

NONG NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANG 9/2019

KHOA HOC CbNOliiwif -

Bang 2. Kha nang phan giai glucose ra saccharose ciia c ^ ddnf; n ^ mei .^ Dong nam mrn

\ , i l lm i

>,,lnli(.

li|) liK'.nl

ll l i d,u

ll]) niKin _

L\; l \ i 1 \ 1

1,\"". Sl)h

'~mpmv •'• 1 A

iN,i n . . SI),<.TL2.CR1.CR2

1, n-:,TL4.TL6.TSl.CR4.CR5 !. lb:;

IL7.TL8.CR6 IN-'. sn9.TL5.TS4.TSS

Glucose

Ket qua phan lap dua vao hinh dang, sinh hoa cua nam men va can cii vao khda phan loai nam men cua Kurtzman va Fell (1998) 121. Luong Biic Pham (2009) 131 va Nguydn Rlic Luong (2006) 151: 40 ddng nam men phan lap tii ngudn U-ai fram ban dau lhudc cac chi &iccliaroniyces. Pichla va Hanseniapora (Bang 3). .'Saccbaromyces. Picliia va Hanseniapora cung dupc Nguydn Minh ThiJy va ctv. (2013) 16]. Nguydn Van Thanh v:i civ. (2013) [81 va NguySn Thi

Hinh 4. Kdt qui phan giai urea ciia cac ddng n i n men Nidmva iVi', (2018) |71 phan lap tir khdm. sim rimg.

A: Ddng L V2. B: Ddng T.\2. C: Ddng SD9. D: DdngCR3

Bing 3. K^t qua djnh danh sgbd ciia cac dong n ^ men phan Igp tir trii l i to

Ddng nam men

Dac diem hinh thai

Hinh dang te bao

Dac di^m nay chdi

(iidiii sinh hda

Saccharose

Phan loai so bd (chi)

SDI, SD2. SDS SD4,SD7,SD8,TL2, CR1,CR2.TN4

Da cue/ don cue

TN1.TN3.TL1,TL3. TU.TL6,TS1,CR4. CR5

Oval nhd Da cue/ don cue

Saccbaromyces

LVl. LV4.TS2.TS3 Elip ngan LV2, LV3.TN5.SD5, TI.7.TL8, CRli

Elip ngan

I.\T). LV6. TNli. TN2, SD9.TL5.TS4.TS5

Elip dai

Elip nhon Luong cue

Pichla

Hanseniaspo,

Chu Ihich: l+j phan giai hoac len men: (-) khdng phan giai hoac khdng len men

3.2. Khao sit hoat tinh ldn men ciia cic ddng Theo Lu<mg Diic Pham (2009) 13| cac ddn nfai men phan lap men thudc chi Hanseniaspora, Pichla se lim m-™

chat luong ruou trai cay. 24 dong nam m, „ ! mf.c'chi

NONG NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANG 9/201?

KHOA HOC C 6 N G NGHE

Saccbaromyces dugc lua chgn cho cac thi nghiem tidp theo.

3.2.1. Tuyen chon cac ddng nam men trong chudng Durham

Chi^u cao cdt khi CO2 trong dng nghiem chiia chudng Durham trinh bay d bang 4 cho thay cudng dg ldn men cua cac ddng nam men khac nhau d timg thdi di^m ldn men 6 gid, 8 gid, 12 gid, 24 gid va 36 gid. Ddng ddi chiing (DC) cd thdi gian day het khi CO3 trong dng Durham sdm nhat (8 gid), ke den la cac ddng SDS, CRI va CR2 day hdt cot khi CO, trong

12 gid. Ddng TN3, TN4 va SD2 day het cgt khi trong 24 gid. Ddng TSS va CR3 khdng xuat hien bgt khi CO, trong 36 gid quan sat.

Qua tiinh len men rugu se tao ra hai san pham chinh la rugu ethylic va CO2, vi vay cd the dua vao thdi gian day het dng Durham sdm nhdt de xac dmh cd hoat luc len men manh nhdt [5]. Tuy nhidn. do thdi gian len men trong chudng Durham ngdn, vi vay, phuong phap do chidu cao cdt khi CO^ bang dng Durham chi la ca sd ban ddu de xac dinh kha nSng len men cua cac ddng ndm men.

STT

1 2 3 4 5 6 7 8 9 10 11 12 13

Ddng n ^ men

LVl LV4 TNI TN3 TN4 SDI SD2 SD3 SD4 SD7 SDS TLl TL2

Bang 4. Chidu cao cdt khi CO? tmng binh cua Thdi gian quan sat (gid)

6

0,1

0,43

8

0,57 0,13

0.27 0,10 0,10 0,10 0,07

0,55 1,17

12

1,13 0,67 0,10 1,37 2,83 0,7 0,63 0.10 0.10 1,21 3,00

0,10

24

2,23 1,9 0,43 3,00 3,00 2,50 3,00 0,60 0,57 2,23 3,00 1,27 0,77

36

3,00 3,00 1,10 3,00 3,00 2,90 3,00 0,78 0,82 2,84 3,00 2,73 1,12

STT

14 15 16 17 18 19 20 21 22 23 24 25

cic ddng Ddng nam men

TL3 TL4 TL6 TSl TS2 TSS CRI CR2 CR3 CR4 CR5 DC

Tlim men (don vi: cm) Thdi gian quan sit (gid)

6

0,03

0,07

0,67 0,30

1,47

8

0,17 0,10

0,10

2,83 2,67

3,00

12

1,SS 1,47

0,13

3,00 3,00

0,40

3,00

24

2,17 2,67 0,17 0,56 0,10

-3,00 3,00

-1,SS 0,37 3,00

36

3,00 3,00 0,77 2,73 0,50

3,00 3,00

2,74 0,89 3,00

(Ghi chu: -: 0 cm, chidu cao cdt kbi CO2 tdi da la 3

3.2.2. Tuyen chon cic ddng nam men trong buih tam giic

Tii ket qua len men trong dng nghiem chiia chudng Durham a muc 3.2.1, chgn 15 ddng ndm men Sacchromyces cd kha nang len men tdt va len men nhanh dd tidn hanh thi nghiem len men trong binh tam giac. Ket qua dugc trinh bay trong bang 5.

Dua vao do cdn cua ddng nam men ddi chiing (6,12% v/v), sau 10 ngay len men ruou, cac ddng nam men da tuyen chpn tir binh tam giac duoc chia thanh hai nhdm: (1) cac ddng ndm men len men manh, cho san pham cd do cdn cao hon do cdn cua ddng ndm men ddi chiing, gom cd 2 ddng TN3 (6,19% v/v) va TN4 (7,53% v/v); (2) cac ddng nam men len men ydu, cho san pham cd do cdn thap hon do cdn ciia ddng nam men ddi chiing, gdm 13 dong (LVl, LV4. TNI, SDI, SD2. SD3, SD4. SD7, SDS. TLl, TL2, TL3. TU,

cm vi sdheu li tmng binh cua 3 Ian lap Iai). TL6, TSl, TS2, TSS, CRI, CR2, CR3, CR4, CR5), khdc biet cd y nghia thdng kd b miic 5%. Kdt qua cho thdy ddng ndm men TN4 cd dd cdn trung binh cao nhdt trong sd cac ddng, ke den la ddng ndm men TN3 va ddng ddi chiing, khac biet khdng cd y nghia thdng ke. Ddng TSl cho san pham cd do con thdp nhdt (1,25% v/v) trong sd cac ddng. Cac ddng nam men cdn lai cho san pham cd do cdn kha thap. Ddng nam men TN4 cd kha nang len men nhanh va cho san pham cd do cdn cao (7,53% v/v) va "Brix dugc do bang khuc xa ke giam (8,17 "Brix). Do dd, ddng ndm men TN4 dugc chgn dinh danh bang phuong phap giai trinh tu.

Ket qua len men rugu trai tram ciia ddng nam men TN4 cho dd con tuong ddng vdi kha nang Idn men rupu ca na cua ddng nam men R2B trong nghien ciiu ciia Nguydn Thi Nidm (2018) [7] vdi dd con la 7,51% v/v. Tuy nhien, ket qua nay thdp han kdt

NONG NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANG 9/2019 63

KHOA HOC C 6 N e N6HE'

qua nghien ciiu ciia Nguyen Vin VQ va Nguyen Van Thanh (2018) 191 khi su dung ddng nam men CBl.l

duvc phan lap lu trii dau de len men Ha Chiu. cho dd cdn cao nhat dat 1 -'.'

mv" vang dau

STT

1

2 3 4 5 6

7 8 9 10 11

12

13 14

15 16

Bang 5. Ket qua pi Ddng nam men

I,\'l

l.\'4 TN3

TN4 SDI SD2 SD7

SD8 TLl TL3 T U TSl CRI CR2 CR4

DC

., 'Brixvidd pH 3,44 3,51 3,58

3,55 3.51 S.51 3.4S

3.63 3.53 3.39 3.41 3.47 3.66 3.68

3.63 3.54

cdn trung bmh cua cic dd "Bnx

11.50""" ±0.23

9.8.1'" ±1.26 8.17'+ 1.04 8,17" ±0,76 16.00" ±5.2

12.67°''"± 1.53 14.67" ±3.79

10.60'~±0.53 14.0'" ±5.29

14.67* ±0.58

12.3.T'""± 1.53 14.67" ±0.58 j 2.40"*" ±0.53 13.(10"'"±0.00

14..'i.1'"±1.15 9.87'" ±0.81

!e5?SLS>Si - r r 7 / v ) D 6 e d n l ' i ; i . l i i ^

j 7 8 ^ ^ ± 1 0 0 _

— — r Q ? ' ± j j j 4

35?^^±Wi5

Tiy°±^-6i

ll

f k i " l ' '

iMli-=:-l'!l'

l ' ''

L\ 'fj m

,,;,.,

'.;!

'il'

•'"

n

'i!

i ,M'

fh

J:;V

1 •ll

ii!

,1 l l l l III'

ll IH';:-' 11'j' • '

"::"

J'' l'l'

1

1 , ' i ' , kiij

Sdlidu la tivng binh cua .llan lap lai. cac chirsd mang cac chir cai khac nhau tiong cung mdt cot khac

bi$t cdy nghia thdngkd dmuc 5% (P<0,05).

3.3. Dinh danh bang phuong phip giii Irinh tv Ddng nSm men TN4 duoc dinh danh bang gene phuong phap giai va phan dch ti-inh tu gen 28S rRNA

vol cap mdi r rSl ; 5'-(iCATATCAATAAGCGGAGGAAAAG-3' va 1TS4: 5'-GGTCCGTGTTrCAAGACGG-3' do Cdng ty TNHH MTV Sinh Hda Phu Sa (Thanh phd Can Tho) thuc hien. Kdt qua trinh lu gen 28S rRNA cua ddng nam men TN4 duoc phan tich bang phin mdm NTSYS thd hien qua hinh 5 (IVIoi dinh tU'ong ting vdi mot nucleotide trong trinh tu).

Trinh tu doan gen duoc giai gdm 586 bases va duoc so sanh vdi cac gen 28S rRNA cua nam men trong ngan hang gen tren website NCBI bang cdng cu BLASTN. Kdl qua phan tich cho thay doan gen 28S rRNA ciia ddng nam men TN4 cd dp tuong ddng den 99,48% so vdi trinh tu gen 28S cua ddng nam men Saccbaromyces cerevisiae IMAU (Accession; GU138458.1) (Hinh 6).

Ket qua dinh danh da xac dinh dong nam men TN4 thudc loai Saccbaromyces cerevisiae, day la loai nam men huu dung ti-ong cdng nghiep san xuat ruou, nd khdng chi len men nuoi qua hay mdi truong chiia ham luong dudng cao ma con tao ra san

Hinh 5. Kdt qui trinh ti; gen 28S rRNA ciia ddng nam pham len men vdi huong vi dac tiimg 13].

men TN4 dupc phin tich bang phdn mdm NTSYS

64 NONG NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANG 9/2019

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@Download v GenBank Graphics

Saccharomyces cerevisiae IMAU:Y1056 26S ribosomal RNA gene, partial sequence

Sequence ID: G U 1 3 8 4 5 8 1 Lens th : 611 N u m b e r o f Matches: 1

Range 1 : 7 t o 587 GenBank Graphics Score

1053 bits(570) Expect

0.0

Identities

578/581(99%) Gaps St rand

3/581(0%) Plus/Plus

Query 6 GGG-TTG-CTTAGTMCGGCGAQTGAAGCGGCAAAGCTC-AATTTGAAATaGCTACCn 52 , m HI i i i i i i i i i i i i i i i i i i i i i i i in i i i i i i i i i i inii i i i i i iMii 7 6GGAnCCCTTA6TAACGGCQAGT6AAQC6QCAA4GaCAAATn6AAATaGGTACCTT 66 S b j c t

Q j e r y 63 CGGTGCCCGAGTTGTAATnGGAGAGGGCAACTnGGGGCCCTTCCrTSTaATGTTCa 122

, . , ,, imiini i i i i i i i i i i i i i i i i iMii i i i i i i i i i i iMii i i i i ini i i i i i i i S b j c t 67 CGGTQCCCGAGTTGTAATnGGAGAGGGCAACTnGGGGCCGnCCrTGTaATGnca 126

Hinh 6. So sinh trinh ty gen 28S rRNA cua d6ng n^m men

4. KFT LUAN

Tir Irai tram thu lai 6 dia diem gom Ninh Kieu, Thdt Ndt (thanh phd Can Tha), Thoai Son (An Giang), Lai Vung, Sa Dec (Ddng Thap) va Binh Tan (Vinh Long) da phan lap dupc 40 ddng nam men. C3n Cli dac diem hinh thai va sinh hoa so bo da xac dinh dugc cac ddng nam men thuoc 3 chi Saccbaromyces, Pichla va Hanseniaspora. Qua qua trinh l6n men trong dng nghiem chua chuong Durham tuy^n chpn dupc 15 ddng nSm men co kha nang len men nhanh. Tir 15 dong nam men nay, tien hanh I6n men trong binh tam giac va chon dupc ddng nam men TN4 phan lap trai tram d-Thdt Ndt, thanh phd Can Tho la ddng nam men len men tdt va cho dp con cao nhat dat 7,53% v/v. Ket qua dinh danh bang phuong phap giai trinh tu gen da xac dinh dong nam men TN4 thuoc loai Saccharomyces cerevisiae.

TAI UEU THAM KHAO

1. Chagas. V. T, L M. Franca, S. Malik, and A. M. de. A Paes, 2015. Syzygium cumini (L.) skeels: a prominent source of bioactive molecules against cardio metabolic diseases. Front Pharmacol. (6): 259.

2. Kurtzman, C. P. and FeU, J. W, 1998. The

Yeast: A Taxonomic study. 4"' ed. Elsevier Science.

1076.

3. Luang Due Pham. 2009. Nam men cong

nghiep. Nha xual ban Khoa hoc va Ky thuat Ha Npi.

(6): 188-217.

^ 4 va ddng nam men Saccharomyces ceren'sae IMAU

4. Maran., J. P, B. Priya, and S. Manikandan, 2014. Modeling and optimization of supercritical fluid extraction of anthocyanin and phenolic compounds from Syzygium cuminitnxit pulp. J Food Sci Technol. 51(9): 1938-1946.

5. Nguyen Due Luong, 2006. Thi nghifim c6ng nghe sinh hoc. Tap 2. Nha xuat ban Dai hpc Qudc gia thanh phd Hd Chi Minh.

6. Nguyen Minh Thuy, Nguyin Van Thanh va Pham Tlii Ngoc Anh, 2013. Phan lap va tuy^n chpn nam men tir sim rimg 6' Phu Qudc (Ki^n Giang) va Mang Den (Kon Tum). Ky yeu Hpi thao Cong ngh^ sinh hpc vimg ddng bang song Cuu Long. 47-53.

7. Nguy&n Thi Ni&m, 2017. Phan lap va tuy^n chon cac ddng nam men (Saccharomyces spp.) I6n men ruou ca na (Canarium album). Tap chi Khoa hpc -Trudng Dai hpc CSn Tho. (54) IB: 44-49.

8. Nguyin Van Thanh, Nguyin Minh Thiiy, Tran Thi Que' va Nguyen Thi My Tuyfen, 2013. Phan lap, tuyen chpn va dinh danh nam men trong len men rupu vang khdm. Tap chi Khoa hpc - Truang Dai hpc CanTha. (25): 27-35.

9. Nguyin Van Vu va Nguyin Van Thanh, 2018. Phan lap, tuyen chon va dinh danh nam men trong len men nrou vang dau Ha Chau (Baccaurea ramiflora L) . Tap chi Khoa hpc - Tnrong Dai hpc can Tho. (54) 7B: 22-32.

10. Truong Anh Thai, 2010. Nghien cihi chi biln nude giai khat len men tir trai mang can xilm. Dd an tdt nghiep. Trudng Dai hpc Ky thuat Cong nghiep thanh phd Ho Chi Minh.

N 6 N G NGHIEP V A PHATTRIEN NONG THON - KY 2 - THANG 9/2019 65

KHOA HOC C 6 N 0 HimE

qua nghien ciiu cua Njniyi'n Van \'u va Nf,aiyen Van dm?c phan lap tir trai dau d^ l n men ruv"

Thanh (2018) [9] khi su dung dong nSm men CBl.l Ha Chau. cho do con cao nhal dat 1-' '**

Bang 5. Itft quA pH, °Brfe vi dp cAn tnmg binh cua cAc d6ng afai nien_

r dau

STT

15

16

Dong nam men LVl

TN4

SD8

TL3

TSl

CR2 CR4 DC

pH 3.44

3.51

°Brix 11..10"~''±0.23

IJ.67'""±.1.7')

10.60'"'±0.5-1

]4.0"'±5.2y

12..ir'""± 1.5.1

14.67"'±0.58 : .40""" ±0.5.1

i.i.(«r*±o.oo 3.(vl 14..1.1"'±I.15

3..'i4 9.87-"'±0.8]

Sdlieu li tiung binh cua 3 lan lap lai, cac t liu sd mang cae chir cai khac nhau tiong cung mot cdt khic bi(tcdf nghia thdngkd dmuc 5% (P<O.OS).

3.3. Djnh danh being phuong phip giai trinh tv

gene

mmm ; ^ n i i i p i i P !t''iii'j;

Hinh 5. K t qua trinh trf gen 28S rRNA cua ddng nim men TN4 dupc phan tich bang phin mim NTSYS

Dong nam men TN4 diroc dinh danh bang phuong phap giai va phan hch h'inh ttr gen 28S rRNA vcri cap m6i ITSl; 5'-GCATATCAATAAGCGGAGGAAAAG-S' va ITS4: 5'-GGTCCGTGTTTCAAGACGGS' do Cong ty TNHH MTV Sinh H6a Phil Sa (Thanh pho Cin Tho) thuc hii-n. Ket qua trinh tir gen 28S rRNA cua dong nam men TN4 dugc phan tich bang phan m^m NTSYS the hien qua hinh 5 (Moi dinh tuong ung voi mgt nucleotide U'ong trinh tu).

Trinh lu doan gen duoc giai gom 586 bases va duoc so sanh voi cac gen 28S rRNA cOa nam men h'ong ngan hang gen tren website NCBI bang cong cu BLASTN. Ket qua phan tich cho thay doan gen 28S rRNA cua dong nim men TN4 co do tuong d6ng den 99.48% so voi trinh tu gen 28S cua dong nam men Saccharomyces cerevisiae IMAU (Accession: GU138458.1)(Hinh6).

Ket qua dinh danh da xac dinh dong nam men TN4 thuoc loai Saccharomyces cerevisiae, day la loai nam men huu dung trong cong nghiep san xuSt ruou. no khong chi len men nuoc qua hay moi tnrong chua ham luong duong cao ma • ',n tao ra san phSra len men voi huong vi dac trung 131.

64 NONG NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANG 9/2019

KHOA HOC CONG NGHE

SDownload » GenBank Graphics

Saccharomyces cerevisiae ltMU.Y1056 26S ribosonoal RNA gene, partial sequence

Sequence ID: GU138458-1 Length: 611 Number of Matches: 1

Range 1 ; 7 t o 587 GenBank Graphics Score

1053 bits(570)

Expect

0.0

Identities

578/581(99''/o) Gaps

3/581(0%)

strand

Plus/PI US

Query 6 6GG-nG-CTTAGTAACGGCGAGTGAAGCGGCAAAGaC-MTrTGAAATaGGTACCn 62 I I I I I I l l l l l l l l l l l i l l l l l l l l l l l l l i l l l l l 1IIIIIIIII1IIIIIIIII

Sb]Ct 7 CGGAnGCCTTAGTAACGGCGAGTGAAGCGGCAMeaCAAATnrOAAATaGGTACCn 66

Q j e r y 63 CGGTGCCCGAGnGTAATnGGAGAGGGCAACrnGGGGCCGnCCTTGTCTATGTTCCT 122

l l ! I I I I I I I M I I ! 1 l l l l i l l l l l l l i n i l l l M I I I I M I I I i l i ; i ' I I I I M i l Sb3ct 67 CGGTCCCCGAGTTGTAATnGGAGAGGGCAACrrTGGGGCCGTTCCTTGTar TGTTCCT 126

Hinh 6. So s^nh trinh tii gen 28S rRNA cua d6ng n ^ men

4. KEf LUAN

Tu trai tram thu tai 6 dia diem g6m Ninh Ki^u, Thflt Not (thanh pho C^n Tha), Thoai San (An Giang), Lai Vung, Sa Dec (D6ng Thap) va Binh Tan (Vinh Long) da phan lap dirge 40 dong nam men. Can cir dac di^m hinh thai va sinh hoa so bo da xac dinh dugt: cac dong nam men thugc 3 chi Saccharomyces, Pichia va Hanseniaspora. Qua qua trinh len men trong ong nghiem chua chuong Durham tuyen chgn dugc 15 dong nam men co kha nang len men nhanh. Tir 15 dong nam men nay, hen hanh len men trong binh tam giac va chpn duoc dong nam men TN4 phan lap trai tram b Th6t Ndt, thanh ph6 Can Tha la dong nam men len men tot va cho do con cao nhat dat 7,53% v/v. Ket qua dinh danh bang phuong phap giai trinh tu gen da xac dinh dong nam men TN4 thuoc loai Saccharomyces cerevisiae.

TAI UEU THAM KHAO

1. Chagas. V. T, L M. Franca, S. Malik, and A. M. de. A Paes. 2015. Syzygium cummi (L.) skeels: a prominent source of bioachve molecules against cardio metabolic diseases. Front Phannacol. (6): 259.

2. Kurtzman, C. P. and FeU, J. W, 1998. The Yeast: A Taxonomic study. 4"' ed. Elsevier Science. 1076.

3. Lirong Diic Phara, 2009. Njtm men cong nghiep. Nha xuat ban Khoa hoc va Ky thuat Ha Ngi. (6): 188-217.

TN4 v^ dong n^m men Saccharomyces censvisiaelMMJ

4. Maran., J. P, B. Priya, and S. Manikandan, 2014. Modeling and optimization of supercritical fluid extraction of anthocyanin and phenolic compounds from Syzygium cumini truit pulp. J Food Sci Technol. 51(9): 1938-1946.

5. Nguyin Due Lugng, 2006. Thi nghi6m c6ng nghe sinh hgc. Tap 2. Nha xual ban Dai hpc Qudc gia thanh phd Ho Chi Minh.

6. Nguyin Minh Thuy, Nguydn Van Thanh va Pham Thi Ngpc Anh, 2013. Phan lap va Uiy^n chpn nam men tu sim rung a Phii Qudc (Kidn Giang) va Mang Den (Kon Tum). Ky yeu Hpi thao Cdng ngh^ sinh hgc viing ddng bang sdng Cuu Long. 47-53.

7. Nguyin Thi Nidm. 2017. Phan lap va tuyen chpn cac ddng nam men (Saccharomyces spp.) len men rugu ca na (Canarium album). Tap chi Khoa hgc - Trudng Dai hgc Can Tha. (54) IB: 44-49.

8. Nguydn Van Thanh, Nguydn Minh Thuy, Tran Thi Que va Nguydn Thi My Tuydn, 2013. Phan lap, tuyen chon va dinh danh nam men trong ldn men nrgu vang khdm. Tap chi Khoa hgc - Trudng Dai hpc can Tha. (25): 27-35.

9. Nguydn Van Vu va Nguydn Van Thanh, 2018. Phan lap, tuyen chgn va dinh danh nam men trong len men nrgu vang dau Ha Chau (Baccaurea ramiflora L) . Tap chi Khoa hoc - Truong Dai hgc CanTha. (54) 7B: 22-32.

10. Trucmg Anh Thai, 2010. Nghien ciJru che bidn nude giai khat len men tii trai mang cau xiem. Do an tdt nghiep. Tnrdng Dai hgc Ky thuat Cong nghidp thanh phd Hd Chi Minh.

NONG NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANG 9/2019 65

KHOA HOC C6NG HOnfJ

qua nghien cuu cua NY^yt'n Van Vu va N^^uycn Van dupc phSn Igp tu trai dau di len men rut": Thanh (2018) [9] khi sir dung dong nam men CBl.l Ha Chau. cho dp cdn cao nhal dat 1-..' > > '

vang dau

15 16

Bang 5. Kit qtia pH. "Brix va dp c6n trung binh cua cAc ddng nfei roen^

— - — ^ ' ; i i . i . ! Dong nam men pH

L\l 11.50""'±0.23

8.17" ±1.04

16.00'±.1.2 12.67~'"± 1.5.1

10.60""' ±0.5.1

J 3JH_

CR4 DC

1 : . . i . t ^ " ± 1 .." .t

3.47

3.63

3.54

1.1.00~"±(1.00

14..1.T"

9.S7"'±0.81

Sd li^u li Irung binh cua 3 lin lip lai. cac chir sii mang cic chir cii kh.u'nhau tiong ciing mot cdt khic

bi^tedy ngbia thdng ke dmuc 5% (P<0.t)5).

3.3. Dinh danh b^ng phutmg phap giai trinh ti; gene

jr l i l

I I I . l . ' I , , 1 ) 1 l l l l ' ; ' 1 I l l I

'11

Jii ijijiiM'iti'iiiii iiii,i,]i|||-'.hii.j

i i ' i ' l '

Hinh 5. K^t qua trinh ty gen 28S rRNA ciia dong nim men •IN4 dupc phan tich bang phin m^m NTSYS

Dong nam men TN4 duijc dinh danh bang phuong phap giai va phan dch trinh tir gen 28S rRNA v(n c§p m6i ITSl: 5'-t;CATATCAATAAGCGGAGGAAAAG-3' va ITS4: 5'-GtilCCGTGTrrCAAGACGG-S' do Cong ty TNHH M'lV Sinh Hcia Phu Sa (Thanh phd Can Thff) thtrc hien. K6t qua tnnh tu gen 28S rRNA cua dong nam men TN4 dugc phan tich b^ng phan mem NTSYS the hien qua hinh 5 (M6i dinh tuong ung vtn mpt nucleotide trong trinh tu).

Trinh tu doan gen dupc giai gom 586 bases va duoc so sanh vol cac gen 28S iRNA ciia nam men Irong ngan hang gen Uen website NCBI bang cong cu BLASTN. KSl qua phan tich cho thay doan gen 28S rRNA ciia dong nam men TN4 co dp tuong dong den 99,48% so voi Mnh tu gen 28S cua dong nam men Saccharomyces cerevisiae IMAU (Accession: GU138458.1)(Hinh6).

Ket qua dinh danh da xac dinh dong nam men 1N4 thupc loai Saccharomyces cerevisiae, day la Ipai nam men huu dung h-png cpng nghiep sjn xuat rupu. no khong chi ten men nuoc qua hay mpi truong chua ham luong duong cap ma con t^o ra san pham 1™ men voi hupng vi dac trung 131,

NONG NGHIEP VA PHAT TRIEN NONG THON - KY 2 - TH^ > IG 9/2019

KHOA HOC CdNG NGHE

SDownload v GenBank Graphics

Saccharomyces cetevisiae IMAU:Y1056 26S nbosomal RNA gene, partial sequence Sequence ID: GU1384581 tength:61l Number of Matches: 1

Range 1; 7 to 587 GenBank ;

Score

1053 bits(570)

Expect

0.0

Identities

578/581(99%)

Gaps

3/581(0%)

strand

Pius/Plus

Query 6 GuG-TTG-CTTieTAACQCCGAGTG;.iGCGQCiAA6aC-AATn"6AAATa6GTACCTT 62

Ml III I I M I I ' l l l ' l l | l | l 1 l l i l l : ^ l l l l ! l l l l l l l l l l l l l l l l l l l l S b j c t 7 GGG.iTTGCCTTAGTA.ACGeCG;GTG;/:'Gi:GGCiA;GaCAAATrT6AAATCT6GTACCrT 66

CGGTGCCCGAGTTGTAATnG6AGAGG6CAACTTTGGGGCCQncaTGTaAT6nCCT 122

i i i i i i i i i i i iM i i i i i i i i i i i i im i i i i i i i i i i i i i n i iM i i iM i i i i i i CGGTGCCCGAGTTGTAATnGGAGAGGeCAACTTTGCGGCCGnCOTGTaATGTTCa 126

Query 63

S b j c t 57

Hinh 6. So s^nh trinh tu g e n 28S rRNA cua dbng n & i m e n

4. KET LUAN

Tir trai t r am thu tai 6 dia d iem g 6 m Ninh Kieu,

Thot N6t ( thanh p h o Can T h a ) , Thoa i Son (An

Giang), Lai Vung, Sa D e c CDong Thap) va Binh Tan

(VTnh Long) da phan lap dirpc 40 d o n g n a m men .

Can cu dac d iem h i n h thai va s inh h o a so bo da xac

dinh dupc cac d o n g n a m m e n thugc 3 chi

Saccbaromyces, Pichia va Hanseniaspora. Qua q u a

trinh Ifin m e n t r o n g ong n g h i e m chua c h u o n g

Durham tuyen c h o n dugc 15 d o n g nam men c6 k h a

nang len m e n n h a n h . Tir 15 d o n g n a m m e n nay, tien

hanh l&n m e n t r o n g b inh tam giac va chpn duoc

dong nam m e n T N 4 phan lap trai t r am a T h d t Ndt,

thanh phd Can T h o la d o n g n^ni m e n len m e n tdt va

cho dp con cao n h a t dat 7,53% v / v . Kdt qua dinh

danh bang p h m m g p h a p giai t r inh tu g e n da xac dinh

dong n a m m e n T N 4 thupc loai Saccharomyces

cerevisiae.

TAI UEU THAM KHAO

1. Chagas . V. T , L . M. Franca , S. Malik, and A

M. de . A Paes , 2015. Syzygium cumini (L.) skeels : a

prominent sou rce of b ioachve molecu les against

cardio metabol ic d i seases . F ron t Phannaco l . (6): 259.

2. Kur tzman, C. P. and FeU, J. W. 1998. T h e

Yeast: A T a x o n o m i c s tudy. 4"" ed . Elsevier Science.

1076.

3. L u a n g Diic P h a m . 2009. N a m m e n c o n g

nghiep. N h a xua t b a n Khoa hpc va Ky thua t Ha Noi.

(6): 188-217.

T^A va dong n a m m e n Saccharomyces cereviaaeTtAMJ

4. Maran. , J. P, B. Priya, and S. Manikandan ,

2014. Mode l ing and ophmizahon of supercri t ical fluid

extract ion of anthocyanin and phenol ic c o m p o u n d s

from Syzygium cuminifrui\ pulp. J Food Sci Techno l .

51(9): 1938-1946.

5. Nguyen Due Lupng, 2006. Thi ngh igm cong

n g h e s inh hpc . T a p 2. Nha xuat ban Dai hgc Q u d c

gia thanh phd H o Chi Minh.

6. N g u y i n Minh Thuy , Nguy6n Van T h a n h va

P h a m T h i N g p c Anh . 2013. Phan lap va tuy^n chpn

n a m m e n tir sim r img b Phu Qudc (Ki6n Giang) va

M a n g Den (Kon T u m ) . Ky yeu Hpi thao Cong n g h e

sinh hgc v img d d n g b a n g song Cim Long. 47-53.

7. Nguyfen Th i Ni6m. 2017. Phan lap va tuyen

chpn cac d o n g n a m m e n (Saccharomyces spp.) len

men rugu ca na (Canarium album). T a p chi Khoa

hpc - T r u o n g Dai hgc Cdn Tho , (54) I B : 44-49.

8. N g u y i n Van T h a n h . Nguyen Minh Thuy , T r a n

Th i Qud va Nguydn Th i My Tuydn. 2013. P h a n lap.

tuyen chpn va dinh danh n a m m e n t rong len m e n

rupu vang k h o m . T a p chi Khoa hpc - T r u d n g Dai hpc

C a n T h a . (25): 27-35.

9. N g u y i n Van Vu va Nguydn Van T h a n h , 2018.

Phan lap, tuyen chgn va dinh danh nam m e n t rong

len m e n rugu vang dau Ha Chau (Baccaurea

ramiflora L ) . T a p chi Khoa hgc - T r u o n g Dai hoc

C a n T h a . ( 54 )73 :22 -32 .

10. T r u a n g Anh Thai , 2010. Nghien cuu che bien

nude giai kha t len m e n tir trai m a n g cau xiem. Dd an

tdt ngh iep . Trucmg Dai hpc Ky thua t Cong ngh iep

t hanh phd Hd Chi M m h .

NONG NGHIEP VA PHATTRIEN NONG THON - KY 2 - THANG 9/2019 65

KHOA HOC C 6 N C £:e^s j>^

ISOLATION, SELECTION AND IDENTmCATION OF YEAST STRAIN (Saccbaromyces sp>

FOR Syzygium cumini WINE FERMENTATION

Huynh Ngoc Thanh Tam, Dao Thanh Tam, Nguyen Thi Minh Tl™' Van Thi Hong Hue, Duong Thi Mai IT'S"

Summary The recent study attempts to isolate and select yeast strains with high fermentation from S}7ygi"^ * From the selected yeast strains, the study then considers uUbsing those yeast strains in iemien produce Syzygium cummi wine - a nutntious and healthy drink for the consumers. The pnmary -from Syzygium cumw/collected in were from six different locations, namely Nmh Kieu. Thot Ni>' I city). Thoai Son (An Giang provmce), Lai Vung. Sa Dec (Dong Thap provmce) va Binli Tan O inh Long province) and there were 40 yeasts strains selected collected. After the process of yeast strains divi based on morphology, physiology and biochemistry. -10 yeasls strains were identified belonging W the s ii y obtamed three separate types of yeast stiams: Saccharomyces, Pichia and Hanseniaspora. The results o the fermentaUon experiment conducted in a Durham tube and an Erienmeyer flask revealed tha Saccharomyces strain TN4 yeast strain, isolated from Syzyinum cumini in Thot Not, (Can Tho city;, belonged to Saccharomyces strain and possessed the highest s|>eed of femienlaUon amongst those examined sU-ains. Besides, this strain also engendered wme with the highest alcohol level In the optimal fermentation parameters including pH 3.5. 20''Bnx and 10' ccIl/mL of yeast cell density. TN4 fermented Syzygium cumini producing wine with 7.53% alcohol after ten days of fermentation. Additionally, the result from the identificabon of yeast by Based on DN •\ sequencing indicated that the Saccharomyces strain TN4 yeast strain TN4 is belonged to Saccbaromyces t erevisiae. Keywords:Saccharoniyct'sct're\isiae, selecUon, nalerberry, wine.yr.ist

Ngu6i phAn bi^n: GS.TS. Ph^m Van Toan

Ng^ynh^nb^: 3/5/2019

Ngdy thdng qua phSn bi^n: 5/6/2019

Ngdy duy^t ding: 12/6/2019

66 NONGNGHIEPVA PHAT TRIEN NONG THON- ^ - Tl NG9/2019