ph^ll: CdNG NGHg VI SINH 2 4 9
PHAN LAP, DINH DANH VA TUYEN CHON CAC CHUNG Enterococcus CO TIEM NANG probiotic
TlT PHAN TRE SO SINH
Ho Van Thao, Hodng Quoc Khanh
Phong Vi Sinh img dung, Vien Sinh hoc Nhiet d&i
MdDAU
Trong mdt ngudi cd khoang 10 ti vi khuin trong 1 gram phan, gom vdi hdm lodi vi khuin tao nen he vi sinh dudng mdt het siic phong phii [14]. Enterococci vd sheptococci nhdm D tao nen mdt phan quan trgng dac biet trong he vi sinh vat ban dia trong mdt ngudi vd mdt sd lodi ddng vat, ma phd biln la E. faecalis vd E. faecium. Ddc biet E. faecalis thudng xuyen dugc tim thiy trong mdt gid ciia ngudi, thinh thoang ciing dugc tim thiy trong mdt gd, nhung it khi tim thiy trong cac dgng vat khdc.
Theo Shleifer vd Kilpper- Balz (1984, 1987) da md td cac ddc dilm vd dugc dp dung cho hau het cac loai thudc gidng Enterococcus. Day la nhdm vi sinh vat ky khi tiiy y, dang cau don hay ket chudi, gram duong, catalase am tinh. Xuat hien khdp noi trong mdi trudng, nhung phd bien trong cac san pham sira vd nhat Id trong mdt ngudi va mdt sd lodi ddng vat cd vu. Enterococci cd the sdng va phat trien dugc trong bien do nhiet kha rong Idn, tir 10°C - 45°C, tdi uu d 35°C - 37°C, sdng sdt it nhit 30 phiit d 60°C. Nhd cd bom Natri-ATPase ndm tren mang te bao chat nen co the chiu dugc ndng do NaCl len den 6,5%. pH boat ddng tir 4,6 - 9,6, tdi uu d khodng 6,5 - 7,5. Hau het cdc lodi thudc Enterococci cd thi thuy phan dugc esculine vdi sy hien dien ciia mudi mat 40%.
VAT Ll£U VA FHirONG PHAP Phan lap va djnh danh [10,11, 13]
Ngudn giong vi khuan: gidng vi khuin dugc phan lap tir phan tre so sinh thu thap tai benh vien Tir Dii vd Benh vjen Gia Dinh thudc Thdnh phd Hd Chi Minh, Viet Nam.
Mau dugc pha loang vd frdi tren mdi tmdng BEA (Bile Esculin Azid), dugc ii d 37°C, [4], [5]. Sau 2 ngdy chgn nhiing khuin lac tach rdi cd dudng kinh tir 0,5 - 1mm, mau tring due hoac hoi trong, ddng thdi tao thdnh quing den xung quanh khuin lac, ldm thuin va giir gidng tren mdi tmdng MRS broth (cd bd sung them 20% glycerol [16].
Cdc chimg vi khuin thu dugc se dugc tiln hanh nhudm gram, test catalase, khdo sat
% tinh len men glucose, khd nang song sdt va phat triln trong mdi hardng MRS broth CO nong do NaCl 6,5% va mdi tmdng cd nong do dich mat 40% [4]. Khd ndng phdt triln
% c d 10°C, 45°C h-ong khoang thdi gian tir 2 - 7 ngdy vd song sdt dugc d nhiet do 60°C h-ong thdi gian 30 phut. Sau dd, kit hgp vdi thuc hien ky thuat PCR dk khulch dai trinh tu viing tmng gian rDNA 16S - 23S (Tyrrel et al., 1997) [1], dugc bdo tdn tdt d
250 HOI nqhj KHOA HQC VA CdNG NGHg 2007
Enterococcus viing ITS dl giup cho viec dinh danh gidng Enterocccus. Sir dung cap mdi pS1490, 5'-TGC GGC TGG ATC CCC TCC TT-3' va pL132, 5'-CCG GGT TTC CCC ATT CGG-3' (Normand et al., 1996)
2,2. Khao sat cac dac diem probiotic
2.2.1. Anh hudng ciia Enzyme va pH thap 2.2.2. Anh hudng cua trypsin d pH 8 2.2.3. Kha ndng ket bam
2.2.4. Khao sat kha nang khang vi khuan gay benh 2.2.5. Khao sat kha nang khang khang sinh
2.2.6. Khao sat dac diem sinh bacteriocin khang khuan KET QUA
Ket qua phan lap
Qua qua trinh phan lap vd lam thuan, da thu dugc tat ca 34 chimg vi khuan, phan giai dugc esculine [2], dang cau chudi, gram duong, catalase am tinh, phan giai arginine.
Trong dd cd 13 chiing tu mau phan tre sinh tai Benh vien Tir Du, ki hieu tir TDI - TDI3, va 21 chiing tir mau phan tre sinh tai Benh vien Gia Dinh ki hieu GDI - GD21.
Ket qua dinh danh
Dac diem sinh ly, sinh hda
Bang 1: K i t qud khdo sdt cdc d^c d l i m sinh ly cua 34 chung thu du'gc
Chung GD1 GD2 GD3 GD4 GD5 GD6 GD7 GD8 GD9 GD10 GD11 GD12 GD13 GD14 GD15 GD16 GD17 GD18 GD19 GD20 GD21
NaCI 6,5%
+ + + + + + + - + + + + + + + - - - - + +
Dich miit '40%
+ + + + + + + + + + + + + + + + + + + + +
pH9,6 + + + + + + + + + + + + + + + + + + + + +
lO'C + + + + + + + - + + + - - + - + + + + + +
45°C + + + + + + + - + + + +
-•
+ - + + + + + +
60°C/30' + + + + + + + + + + + + + + + + + + + + +
L§n men glucose sinh acid *
+ + + + + + + + + + + + + + + + + + + + +
Phltn gi^i arginine
+ + + + + + + + + + + + + + + + + + + + +
Ph^n 11: CdNG NGHg VI SINH 251
Chung Ef TD1 TD2 TD3 TD4 TDS TD6 TD7 TDS TD9 TD10 TD11 TD12 TD13
NaCI 6,5%
+ + + + + + + + + + + + + +
Dich mdt
• 4 0 % •
+ + + + + + + + + + + + + +
pH9,6 + + + + + + + + + + + + + +
10»C
+ + + + + + + + + + + + + +
45''C
+ + + + + + + + + + + + + +
eo'c/ao'
+ + + + + + + + + + + + + +
Len men Glucose, sinh aicd
+ + + + + + + + + + + + + +
Phan giai arginine
+ + + + + + + + + + + + + +
(+): vi khuin song va phat trien dugc;
(-): vi khuan khong ph^t trien dugc.
•: test len men glucose sinh acid lactic, khong sinh hai
Ddc dilm sinh ly: sau hdng loat cac test nhan thay rang: da sd cdc chimg deu cd kha ndng phat triln dugc trong NaCl 6,5%, dich mat 40%, mdi trudng cd pH 9,6, mdi tmdng cd nhiet do 10°C va 45°C, vd 60°C yong thdi gian 30 phiit. Ngoai trir cd mdt sd chiing cho kit qua am tinh nhu: GD8, GDI6, GDI7, GDI8, GDI9 khdng phat triln dugc trong NaCl 6,5%; GD8, GD13,GD15 khdng phat triln dugc trong mdi tmdng cd nhiet do lOV lin 45°C.
Ddc dilm sinh hda: tit cd 34 chimg diu len men dugc glucose sinh acid lactic va khdng sinh hoi, phan giai dugc arginine.
Nhu vay da sd cdc chiing khdo sdt diu cd dac dilm sinh ly phii hgp vdi cdc ddc dilm ciia Enterococcus thudng hien dien d ddng vat. Tuy nhien cd mdt sd chimg cdn chua cd sy khdng dinh nhu GD8, GD13, GD15, GD16, GD17, GD18, GD19. Do dd, can phai kit hgp vdi ky thuat PCR khulch dai viing ITS cd kich thudc tir 280 - 620 bp de cho sy khang dinh chinh xac hon.
Ket qua khao sat cac dac diem probiotic trong dieu kien in vitro
Khd ndng chiu duac pH thap
; 6 pH 3 tit cd cdc chiing diu chiu dugc va khdng bi anh hudng tdi siic sdng sau 180 i phut. C) pH 2 dd cho thiy khd ndng chiu dyng khac nhau ciia cac chung, nhu 30 phut ddi ' vdi TD3, TD4, TD5, TD6, GDIO, GDI 1, GD12, GD20., 60 phut vdi TD2, GD3, GD21, I 90 phut vdi TDI, GD4, GD7, GD8 va 180 phut thi khdng cd chiing ndo sdng dugc.
252 HQI nghi KHOA HQC VA CdNG NGHg 2007
Cac chiing chiu dugc pH 1 sau cac khoang thdi gian la 30 phut ddi vdi TD6, 60 phiit vdi TDS, GD4, GD7, GD21. d thdi dilm 90 din 180 phiit khdng cdn chiing nao sdng sdt dugc.
Ket qud khd ndng chiu duac enzyme trypsin apH kiem
Vdi dilu kien mdi trudng cd chira trypsin 0,01% d pH 8 trong khoang thdi gian tir 0-180 phtit, nhan thiy d ndng do trypsin va pH nay thi khdng lam anh hudng den sire sdng ciia tit ca 31 chimg khao sat. Nhu vay ciing vdi kha nang chiu dyng dugc ndng do mudi mat 40% cho thiy cac chiing khao sat cd thi sdng tdt trong dilu kien mdi tmdng mdt non.
Ddc tinh ket bdm cua 31 chung khdo sdt
Tu hai kit qud khdo sdt ve dgc tinh kit bdm cua 31 chung Enterococcus nhdn thdy rdng cdc chiing cho dgc tinh kit bdm tdt, the hien duac a thi nghiem ket tu trong muoi amonium sulfat vd ket bdm vai dimg moi. Ket qud cho thdy tinh tuang dong khd cao, da giup nhdn thdy duac mot so chiing co tinh ket bdm khd tot: Ef TDIO, TDI2, TDI3,
GDI. GD5. GD6, GD9, GDIO, GDI7. GDI9.
Dgc tinh khdng cdc vi khudn gay benh
Cd 31 chiing khdo sdt diu khdng tot doi vai E. coli, Salmonella vd khdng yeu han doi vai Staphilococcus.
Noi bat CO 13 chung khdng tot vai cd 5 logi vi khudn chi thi: TD3, TDIO, TDU, GD5, GD6, GD7.
Khd ndng khdng khdng sinh
Doi vai khdng sinh vancomycin (Va): da so cdc chung nhay khi tiep xuc vol loai khdng sinh ndy, trit mot so chiing co hogt tinh khdng thuoc la TDU, GD4, GD5, GD6, GD7; vd mot so chiing the hien hogt tinh khdng trung gian TD2, TDI2, GDI, GDM, GDI 7. Nhang chiing ndy co the chica gene khdng vancomycin dang hogt dong co the co tinh chuyen doi khd cao nen rdt nguy hiem vd duac cdc to chiec cUng nhu cdc nghien cim ciia nhieu nhd khoa hoc cdnh bdo la khong duac sic dung.
Ket qud khdo sdt khd ndng sinh bacteriocin ire che cdc vi khudn chi thi
Da so cdc chiing khdo sdt deu the hien hogt tinh bacteriocin khd tdt len E. coli ATCC 25922 vd St. aureus ATCC 25923; nhung co 4 chiing khdng duac Lactobacillus
acidophillus NRRL B-2092: TDI, TDS, GDI. GDIO. ' ^
KET LUAN
Da phan lap, khao sat cac dac diem sinh ly, sinh hda va cac dac dilm probiotic, cudi cimg da chgn dugc 6 chimg: TD3, TDS, TDIO, TD13, GD3, GD19 trong sd 31 chimg thudc gidng Enterococcus cd cac dac tinh tdt kha dong diu nhu kha nang chi^
dyng dugc pH thap trong dudng rudt, kha nang kit bam va dinh cu tren tl bao bilu niS;
mgt, chiu dugc sy tac ddng cua enzyme mdt va pH kilm, dong thdi cd kha nang khdngi dugc mot sd loai vi khuan gay benh, khang dugc mdt so loai khang sinh d mdt l i ^ lugng nhat dinh, va tao dugc bacteriocin khang khuin. ' I
i
Ph^ll: CdNG NGHg VI SINH 253
XAI LIEU THAM KHAO
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2 5 4 H6i nghi KHOA HOC VA C O N G N G H £ 2007
SUIMMARY
Isolation, identification and selection of Enterococcus strains with high potential probiotic from infant faeces
Ho Van Thao and Hoang Quoc Khanh Institute of Tropical Biology
Bacteria of the genus Enterococcus are found in a wide variety of habitats such as soil, water, plants, fermented food, and in the gastrointestinal tracts of humans. From the metabolic point of view, Enterococcus spp. produce lactic acid as the main product of carbohydrate fermentation.Thus, they may be considered to be lactic bacteria (Doming et al. 2003). With the purpose of researching and studying the capacity applying of this group in the field of probiotic, our group carried out isolations and selection the Enterococcus types having applicability to use for probiotic micro-^
organism having the origin from strong infant intestine. We isolated and identified;
Enterococcus spp. by combining physiology tests and biological chemistry tests with^
sequenced amplification of the intermediate ITS - PCR. As a result, we selected 31]
bacteria strains belong to Enterococcus spp. Then we continued to survey probiotic]
properties such as durable capability and survival in the low pH condition, digestive enzyme, adhering to intestinal epithelial cell in in-vitro condition. In addition, we still survey the resistance to pathogenic bacteria in the intestine, the capability of resistance|
to some types of antibiotic products and the produce bacteriocin initially. Through the, research, we affirm that the isolates have potential in producing and applying, through the survival capability in inclement condition and may live well in intestinal condition.]
Besides, they can resist and compete to some pathogenic bacteria strains. Finally, the'- survey initially help us pick out 6 genera belong to Enterococcus that having the most probiotic potential to apply for researching and applying probiotic bacteria
