Vi Thi Doan Chinh vd < Tap chi KHOA HOC & CONG NGHE 73(1 1): 1 15 - 119
ANH HUOTNG CUA DIEU KIEN LEN MEN DEN K H A N A N G SINH TONG HOP
C H A T K H A N G S I N H C U A H.4I CHUNG XA K H U A N K 4 va HT 28
PHAN LAP TU DAT THAI NGUYEN
Vi Thj Doan Chinh*, Duong Thj Tinh, Trjnh Ngoc Hoang Trirdng Dai hoc Khoa hoc - DH Thdi Sgii\-cn TOM T A T
Chiing tdi su dung phuong phap thuc nghiem de nghien cuu hai chiing xa khuan K4 va HT 28 phan lap tir dat Thai Nguyen. Hai chung nay cd hoat tinh khang sinh (HTKS) cao. dac biet hi cd kha nang khang dugc mdt sd chiing Pseudomonas aeruginosa gay benh tren ngudi. Chiing tdi da xac djnh dugc mot sd dieu kien len men thich hgp cho sinh tdng hgp CKS cua 2 chiing nav: chung K4 len men thich hgp trong nidi trudng (MT) A-4H cd pH ban dciu = 7. ngudn cacbon thich hgp la glucose va tinh bdt. ngudn nito thich hop la bdt dau tuong va cao nam men. Chung HT 28 len men thich hgp trong MT Gause I cd pH ban dau = 7, ngudn cacbon thich hop la glucose va lactose, ngudn nito thich hgp la mudi (NH4)2S04 va cao nam men.
Til' khoa: xg khudn. dieu kien ten men, hoal linh khdng sinh. ngudn cacbon. ngudn nito
DAT VAN DE
Chat khang sinh (CKS) la mdt nhdm thudc ihiet yen dung trong y hgc. Ddi vdi cac nude ngheo, thudc khang sinh (KS) giu mdt v j tri dcic biet quan trgng. Nhd ed thudc khang sinh, nhieu loai benh tcit, nhat la cac benh nhiem khuan dci bj dciy lui. Tuy nhien, ngay niiy do viec su dung thudc KS khdng hgp li da kim xual hien ngay cang nhieu cac chung vi sinh vat gay benh klicing thudc. Pseudomonas aeruginosa ciing Li 1 trong sd 5 vi kliucin diing dcau ed kba khang thudc [7]. Diing Irirdc thuc trang nhu vay. mdt yeu can cap thiet dat ra hicn nay la tim ra cac chcit KS mdi dc phuc vu eho nhu can dieu tri cac benh nhiem khuan Vci Cling la de bd sung Vcio kho thudc KS du phdng dang ngciy cang thieu but.
Trong sd cac vi sinh vat cd kha nang sinh elicit KS, .xa khuan la nhdm cd nhieu tiem ncing nhal, chiem 80% Ccic elicit KS dci dugc md ta.
Day la ngudn tcii nguyen quy va day trien vgng de phcit hicn Vci tim kiem ra cac chat KS mdi.
Trong bai bao nay. chung tdi thdng bao ket qua nghien cuu anh hiicVng ciia dieu kien nudi cay den khci nang sinh tdng hgp chat KS eiia 2 chiins xa khucin K4 va IIT28 - ki 2 chunsi xa
Tel: 09S-123606
khuan cd HTKS cao dci dugc tuyen chgn trong sd cac cbiing xa khucin phan lap tir dat
fhai Nguyen, de tu dd Xcic dinh dircTc dieu kien len men thich hgp nhal cho qua trinh sinh tdng hgp KS ciia 2 chung nghien ciiu.
VAT LIEU vA PHUONG P H A P
NGHIEN CUU Vat lieu
.Vg khudn: 2 cliiiiig xa kliUcin K4 va I IT 28 cd HTKS cao, cd boat phd rdng, khang dugc ca 2 nhdm vi khuan Gram duong va Gram am, dac biet la cd boat tinh khang dugc mdt sd chung Pseudomonas aeruginosa gay benh.
Cac chiing xa khuan nay dugc phan lap tir dat Thai Nguyen, dugc giu va bao quan trong mdi trudng Gause 1.
Vi sinh vdt kiem dinh la 3 chung Pseuchmionas aeruginosa do Khoa Vi sinh Veit- Benh vien DKTU Thai Nguyen cung ciip.
Cac chung nay phan lap tir benh nhan bj nhiem triiiig. dugc giii gidng va bao qiiein trong mdi trudng MPA.
Phuo'ng phap
- .Xdc dinh HTKS theo phuong phap khdi thach (de so' tuyen xa khuan). phuong phap due Id (de Xeic djnh HTKS trong djcb the).
- Lira chgn mdi tru-dng (MT) len men thich hap: Len men XCT khuan trong Ceic MT co' bcin
ViThi Doan Chinh Tap chi KHOA HOC & CONG NGHE 73(11): 115- 119 tren may lac trdn vdi tdc do 220 vdng'phut d
28"C. Sau 120 gid, xac dinh HTKS cua dich len men theo phuong phap due Id. ' ' ' - Nghien cim anh huong cua ngudn cacbon:
Len men xa khuan trong dung djch mudi A cd bd sung (NH4):S04 0.2%o va cac ngudn cacbon khac nhau. Sau 120 gia nudi lac. xac djnh HTKS cua dich len men theo phuo'ng phap due Id.
- .\ghieii cim anh hiremg cua ngudn iiita: Len men xa khuan trong dung djcb mudi A cd bd sung glucose 1.5% va cac ngudn nito' khac nhau. Sau 120 gid nudi lac, xac djnh HTKS cua djch len men theo phuong phap due Id.
- Nghien cim anh hinnig cuapH ban ddu: Len men xa khuan trong mdi trudng thich hgp da dieu chinh pH ve cac inirc 5, 6. 7. 8. 9. Sau 120 gid nudi lac, xac djnh HTKS cua dich len men theo phuong phap due Id.
KET QUA v A T H A O LUAN
Lua chon moi truo'ng len men thich hop MT len men ddng vai trd I'cit quan trgng trong cdng nghe san xuat CKS. Mdt MT len men tot phai vua thich hgp cho chung xa khuan sinh trudng tdt lai ddng tbdi cho bieu su^l sinh KS cao. De lira chgn dugc MT len men dap ung dugc ca 2 dieu kien tren. chiing tdi lien hanh len men xa khuan trong 4 MT len men co' sa va xac djnh HTKS ciia djcb len men. Kit qua dugc the hien tren bang 1 va binh 1 eho thay:
Chung K4 deu cd HTKS trong ca 4 mdi tru'dng len men co ban. nhung HTKS bilu hien manh nhat trong 2 mdi trudng A-4H va A-4. Chdng HT 28 chi ed HTKS^d trong 2 mdi tru'dng Gause 1 va Gause 2. cdn trong 2 mdi trudng A-4H v a A-4. mac du dd la nhirng mdi tru'dng rat giau dinh dudng nhung lai khdng cd HTKS. Cac ket qua nay cua chung tdi cung hoan loan phu hgp vdi mot sd kit qua da cdng bd trudc day, kbi nghien cuu kha nang sinh tdng hgp CKS cua mdt so chung xa khuan khac nhau [4], [6].
Nhir vay. trong 4 mdi trudng len men da dugc sii dung. MT A - 4H td ra thich hgp hon vdi chung K4. cdn MT Gause 1 lai thich hop hon vdi chiing HT 28. Dich len men cua cac MT nay cd HTKS manh nhat dugc the bien being dud'ng kinh cua vdng vd khuan (VVK) la Idn 116
nhat (hinh 2). Can cii tir ket qua nay. chiing tdi su dung 2 MT tren cho cac thi nghiem tiep theo.
Hinh 1. Anh hudng ciia MT len men den HTKS ciia 2 chiins K4 va HT 28
Chung K4 Chung HT 28 Chiing xa khuan
Bang 1. HTKS cua 2 chiing K4 va HT 28 tren cac MT len men khac nhau
TT MTIen men
Hoat tinh khang sinh (D - d, mm) Chiins K4 Chung HT 1 A-4H 23,6 ±0,9 0 2 A - 4 21.1 ± 0,5 0 3 Gause 1 18,8^0,3 22,2 ± 0,7 4 Gause 2 17.3 ± 0.5 20.2 ± 0.6
Hinh 2. VVK cua 2 chiing xa khu^n K4 va HT 28 trong cac MTlen men
I..A-4 2. .44H 3. Gause 1 4. Gause 2 Anh huong cua pH ban dau den kha nang sinh tong hop CKS
pH cua MT khdng chi anh hudng den sir sinh tru'dng. phat trien cua xa khuan ma cdn cd anh hudng sau sac den cac hoat dgng trao ddi chat, den boat luc cua cac enzym, vi vay ciing se cd anh hudng tdi qua trinh sinh long hgp CKS. De xac dinh pH cua MT thich hgp cho sir sinh tdng hop CKS. chung tdi tiln hanh len men xa khuan trong MT cd pH khac nhau va
Vi Thi Doan Chinh vd es Tap chi KHOA HOC & CONG NGHE 73(1 1 ): 1 15 - 1 19
xac djnh HTKS cua djch len men. Ket qua dugc the hien tren cac bang 2 va hinh 3 cho thay: pi I ban dau ciia MT cd anh hudng rat rd ret den sir sinh tdng licip CKS cua xa khuan.
HTKS cua ca 2 chung deu bieu bien manh nhat trong MT ed pH = 7, giam chut it trong MT cd pH = 6 va cd xu hirdng giam manh trong cac MTcd pH = 5 va MT kiem.
- ^ K4 — - HT28
7 8 9 ' p H m o i triPOTig
Hinh 3, Anh lumng cua pH dau den H IKS cua chirng K4 Vci I \J 28
Bang 2. IffKS cua chung K4 va HT 28 trong MT cd pi I ban dau khac nhau
T T
pH ban dau ciia
M T 5 6 7 8 9
Hoat tinh khang sinh (D - d, mm)
Chiing K4 15,0 i- 0.5 25.5 -t 0,2 28,2 ± 0,6 17.2 1 0.5 9.1 ± 0.4
Chiing H T 28 13.3 + 0.6 23.2 + 0.4 27,5 ± 0,3 14.0 J 0.7 10.0 i 0.9
Hinh 4. V V K cua chung K4 va H T 28 o Ccic pll khac nhau
Nhu vav .pi 1 cua MT = 7 la thich hgp nhcit cho sinh long hop CKS cua ca 2 chung xa khuan K4 va Iff 28. Djcb len men trong MT cd pH
= 7 cd VVK Idn nbeit (hinh 4). Ket i|iia nghien cuu nav cua cluing ldi hoan loan plui hgp viVi
diic diem sinh bgc cua xa kliuaii ndi chung, ddng tbdi ciing pbii hgp vdi kel qua cua mdt sd tcic gia da cdng bd trud'c [2], [31. [5].
.Anh hudng cua nguon cacbon den kha nang sinh tong hop CKS
De nghien cuu anb luroiig ciia ngudn cacbon den kha nang sinh tdng iigp CKS cua 2 chung xa khuan nghien ciiu. ma vdi muc dich la lira chgn du'cTC ngudn cacbon thich hgp. chimg tdi tien hanh len men xa khuan trong mdi trudng len men cd bd sung cac ngudn cacbon khac nhau va xac djnh HTKS cua djcb ien men.
Ket qua the hicn tren bang 3 va hinh 5 da khang djnh: ngudn cacbon cd anh hudng nhieu den qua trinh sinh tdng hop CKS cua 2 chung nghien ciiu va kba niing ddng lioei Ceic ngudn cacbon cua 2 chung K4 va \V\ 28 cd su khac nhau.
Bang 3. HTKS ciia chirng K4 va HT 28 trong MT CO Ciic nszudn cacbon khac nhau
1 T
1 2 3 4 5 6
Nguon cacbon
(%)
Tinh hot tan 1.0 Glucose 1.5
Lactose 1.5 Saccarose1.5 .Maltose 1,5 D-fructose
1.5
lloiit tinh khiing sinh ( D - d , mm) Chiing K4 20,6 ± 0,6 21,2 ± 0,7 13.3 I 0.4 7.3 t 0.2 17,4 ± 0.3 13.2 ± 0,5
Chiing HT 28 17.6 i 0.7 20,0 ± 0,2 19,3 ± 0,2
0 15,4 + 0,5
0
K4 ^^ 23 D Tinri boi tan 1 0 • Glucose 1,5 a Lactose 1 5 D Saccarosel.5 • Maltose 1 5 O D-fructose 1 5
Hinh 5. Anh hudng ciia ngudn cacbon den HTKS ciia chung K4 va I IT 28
Chung K4 cd kba nang ddng boa dugc ca 6 ngudn cacbon va tat ca cac djch Ic-n men deu cd HTKS. iiliuim HTKS bieu hicai manh nhal
Vi Thi Doan Chinh Tap chi KHOA HOC & CONG NGHE 73(11): 115- 1I«
trong MT cd 2 ngudn Ccacbon la glucose va tinh bdt tan. Trong khi dd. chung HT28, HTKS bieu bien manh nhat trong MT cd 2 ngudn cacbon la glucose va lactose. Dac biet.
trong 2 MT cd saccarose va D-fructose, djch len men ciia chung nay hoan loan khdng cd HTKS, bieu bien la khdng cd vdng vd khuan (hinh 6).
Nhu vay, glucose la ngudn cacbon thich hgp cho qua trinh sinh tdng hgp CKS cua ca 2 chung K4 va HT 28 cung nhu nhieu chung xa khuan sinh CKS da dugc nghien cuu trudc day [2],[3], Ket qua nay cua chiing tdi khdng phai la ngoai le
Bang 4. HTKS cua chung K4 va HT 28 trong M T | cd cac noudn nito khac nhau
Hinh 6. VVK cua chung K4 Vei HT 28 trong MT cd ngudn cacbon khac nhau
/. Pinh hot tan: 2. Glucose: 3. Lactose:
4. Saccarose: 5. Maltose: 6. D - Fructose
Anh hudng ciia nguon nito' den kha nang sinh tong hop CKS
Cling nhu cacbon, nito' la ngudn dinh dudng khdng the thieu ciia cac VSV ndi chung va ,xa khucin ndi rieng. De nghien ciiu anh hirdng cua ngudn nito den kha nang tdng hgp CKS va lira chgn dugc ngudn nito' thich hgp cho 2 chung nghien ciiu. chimg tdi tien bcinh len men xa khuan trong mdi trudng cd bd sung cac ngudn nito' khac nhau va xac djnh HTKS ciia dich len men.
Ket quci the bien tren bang 4 va hinh 7 eho thay: ngudn nito' cd anh hudng nhieu din qua trinh tdng hgp CKS cua 2 chung nghien ciiu va ca 2 cluing deu cd kha ining sir dung dugc nhieu ngudn nito' khac nhau.
Trong 5 ngudn nito' dirge su dung trong tbi nghiem. HTKS cua mdi chung deu khdng cd su khac nhau nhieu giua cac ngudn nito'.
118
TT
1 2 3 4 5
Nguon nito' (%) (NH4)2S04
0,2 K N O T 0,1
Bdt dau tuong 1,0
Cao nam men 1,0
Pepton 1,0
Hoat tinh khang sinh ( D - d , mm) Chiing K4
17.1 ± 0.6 8.0 ± 0.3 19,6 ± 0,5 18,4 ± 0,2 17,3 ± 0,3
Chiing HT 28 18,2 ± 0,3 17,2 ± 0,4 6,7 ± 0,2 17,7 ± 0,2 13,7 ± 0.4
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Ctiung K4 Chung HT 28
i
QlNHd|2S0d 0,2 D K N 0 3 0.1 D B6I dau ti/ong J ,0 SCao Hammer, 1,0 • Pepton 1 0
Hinh 7. Anh hudng ciia ngudn nito den HTKS cua j 2 chung K4 va HT 28
Tuy nhien. vdi chung K4. HTKS bieu hien manh ban trong MTcd ngudn nita la bdt dau tuong va cao nam men. Tren cac ngudn nito cdn lai, HTKS cua dich len men bieu hien yeu|
hon, dac biet tren MT mudi KNO3 - HTKS rat thap (hinh 8).
Hinh 8. VVK ciia 2 chirng K4 va HT 28 trong MT cd cac ngudn nito' khac nhau /. (iSHjySOp 2. K.\'()p 3. Bdt ddu tuong:
4. Cao ndm men: 5. Pepton.
Ket quci nay eiing hoan loan de hilu vi bot dau tuang va cao nam men la nhung nguon nito hiiu co'. ngoai cung cap nita cho tl bao.
Vi Thi Doan Chinh vd cs Tap chi KHOA HOC & CONG NGHE 73(1 19 cd the cdn chiia mdt sd chat can cho qua trinh
sinh tdng hgp C K S va chi cd the giai thich rd rcing khi biet ro cac thanh phan hoa hgc cua 2 ngudn nito' nay. Trong khi dd, chung HT28, ngudn nito uu the lai thudc ve mudi (NH4):S04 va cao Ucim men, H T K S yen nhat trong MT bdt dau t u o n g (hinh 8). Trong cdng nghc san xuat C K S . day la mdt Igi the. De kinh te. cd the thay the ngudn cao nam men bang mudi (NH4)2S04 Tuy nhien. vdi cac ngudn iiito' vd co'. nhieu cluing xa khuan khac lai eho HTKS cao hon trong MT mudi NH4NO:, [1] hoac KNO3 hay N H ^ ' l [2]. D i l u nay chi ed the giai thich dugc khi biet rd co' che hinh thanh C K S .
KET LUAN
1. Mdi tru'dng len men thich hgp cho sinh tdng hgp CKS ciia chiing xa khuan K4 hi A- 4H va chiing HT 28 hi Gause 1
2. pH ban dau eiia mdi trudng len men thich hgp cho sinh tdng hgp C K S ciia 2 chiing Xei khuan K4 va IIT 28 hi pH = 7
3. Ngudn cacbon thich hgp cho sir sinh tdng hgp CKS ciia chung XCT kliuein K4 hi glucose va tinh bdt, ciia chung I IT 28 la glucose va lactose.
4. Ngudn iiito' tliicli hgp cho su sinh tdng hgp CKS cua chiing xa khuan K4 hi bdt dau tirong Vci cao nam men. cua chimg lEf 28 la (NIfOiSOj Vei cao nam men.
TAI LIEU fHAM K H A O
[IJ.Pham Kim Dung. Le Gia Hy. Ngd Dinh Binh (1992), "Nghien ciiu sinh long hgp Chlotetraxiclin tir chung Streptomvees
aureofaciens KH-30 trong dieu kien nudi cay chim" Tgp chi Sinh Imc, tap 14. sd 4. Tr. 5 2 - 5 5 . [2]. Biii Thi Viet Ha ( 2 0 0 6 ) , Nghien ciru xg khudn sinh chdt khdng sinh chdng ndm gdy benh thuc vdt a Viet Nam. Luan an Tien si Sinh hgc.
[3]. Le Gia Hy (1994). .Nghien cim xg khudn thugc chi Streptomyces sinh chdt khdng sinh chdng ndm gdy benh clcio dn vd thdi cd re phdn lap a Viet Nam. Euan an Tien sT Sinh hgc.
[4] Le Gia Hy. Pham Kim Dung, Ngd Dinh Binh (1992), " N g h i e n ciiu kha nang sinh chat khang sinh c h d n g benh dao dn ciia mdt sd cluing xa khuan ( S t r e p t o m y c e s ) phan lap d Viet Nam"". Tgp chi Sinh hgc. Tap 14 sd 4, t r . 5 9 - 6 3 .
[5] Le Gia Elv, Nguyen Quynh Cheiu, Pham Kim Dung (1992), "Anh hudng cua Ca va Co len kha nang sinh tdng licip clotetraxilin va vitamin B2 bang phuong phap len men be niaf".
Tcip chi Sinh hgc.iap 14. sd 4. Tr. 5 6 - 5 8 . [6]. Dang Van Tien. Nguyen Dinh Tuan. Vi Thj Doa^i Chinh, Ngd Dinh Binh (2009), S'ghien cim xg khudn sinh chdt khdng sinh khdng vi khudn .Xanlhomonas oryzae gdy hi'iili hgc Id. Bao cao Hdi nghj CNSH lOciii quoc n a m 2 0 0 9 , T r . 4 1 4 - 4 1 9 .
| 7 | . Nguyen Thj Vinh Vci Cs (2006), Bdo cdo liogi ddng theo ddi su- de khdng khdng sinh cua vi khudn gdv benh thudng gap ir Viet Nam 6 thdng ddu ndm 2006. Chuo'iig trinh Antibiotic Susceptibity Test Surveilance (ASTS) - Td chiic phat trien Qudc te ciia Thuy Dien (SIDA).
S U M M A R Y
T H E I N F L U E N C E O F F E R M E N T . 4 T I V E C O N D I T I O N S ON A N T I B I O T I C B I O S Y N T H E S I S A B I L I T Y O F T W O S T E P T O M Y C E S S T R A I N S K4 A N D HT 28 I S O L A T E D IN THAI N G U Y E N S O I L
Vi Thi Doan Chinh*, Duong Thi Tinh, Trinh Ngoc Hoang College oj Sciences - Thai Sginen I nlvcrsily Two streptomyces trains K4 and HT28 isolated from the soil in Thai Nguyen Province have antibiotic aclivciv well, especially, ability of resisting sonic pathogenous Pseudomonas aeruginosa strains used for this studv. We have determined some suitable conditions for antibiotic biosynthesis of these streptomvees trains. A-4H medium with pH = 7 is suitable for K4 strain. The source of cacbon apply for this train are glucose and starch, the source of nitrogenous applv are soya tlour and yeast extract. For HT28 strain, the suitable cacbon source are glucose and lactose. Nitrogenous source are (NH4):S04 salt and yeast extract.
Key words: aetinomvcetes. fermentative conditions . antihioiie aelivitv, eaehon source, nilrogeniios source Tel im:123606
