Tgp chi Khoa hgc Trudng Dgi hgc Cdn Tho Phdn A: Khoa hgc Tv nhien, Cdng nghi vd Mdi trudng: 34 (2014): 8-17
Tap chl Khoa hpc Tri/dng €)ai hoc Can Tha website: sj.ctu.edu.vn
KHAO SAT KHA NANG C H 6 N G CHIU DIEU KIEN PH THAP VA
KHANG THUOC KHANG SINH CUA HE VI KHUAN ACID LACTIC PHAN L ^ Ttr St^A DE VA CHIE P H A M SINH HQC*
Nguyen Phudc Hiln', Le Dlep Thiiy', Trin Tra My- va Nguyin Hiru Hiep^
' Sinh vien ngdnh Cong nghe Sinh hgc khoa 36, Trudng Dgi hgc Cdn Tha
^ Vien Nghien ciru & Phdt triin Cong ngh? Sinh hgc, Trudng Dgi hoc Cdn Tha Thdng tin chung:
Ngdy nhdn: 20/06/2014 Ngdy chdp nhdn: 30/10/2014 Title:
The survey of tolerance to low pH condition and antibiotic resistance of lactic acid bacteria isolated from goat's milk and probiotics
Tirkhda:
Enterococcus. khdng khdng sinh, pH thdp, probiotic, sda de, vi khudn acid lactic Keywords:
Antibiotic resistance,
^ESiierococcus. goat's milk, lactic acid bacteria, low pH, probiotic
ABSTRACT
Sixteen bacterial strains were isolated in MRS medium, including 13 isolates from goat's milk and 3 isolates from probiotics. Most colonies of them were
round, opalescent white color to milky white, raised or convex elevation, lobulated or intact margin. Results of the survey of biological characteristics showed that 4 strains had rod shape (25.0%) and 12 strains had spherical shape (75.0%) and existed as single or double cells. All isolates were Gram- positive, unable to move and gave oxidase-negative test Besides, 7 isolates had catalase-negative test. The results experienced that 7 strains belonging lo lactic acid bacteria group were selected (43.8%). Surveyed results for the resistance to low pH environment illustrated that 6 strains had the resistance to pH 3 for 3 hours. Two strains Biol.2 and G6.1 had the resistance to environmental condition of pH 2 for 2 hours with density of 9.01 log(CFU/ml). Strain Bio2.1 resisted to 4 types of antibiotic as Streptomycin, Cephalexin, Penicillin V (256 mg/l concentration) and Ampicillin (128 mg/l concentration). Three strains Gl.l, G5.6 and G6.1 were resistant to 3 types of antibiotic as Streptomycin, Cephalexin (256 mg/l concentration) and Ampicillin (128 mg/l concentration).
In addition, Probi strain had the resisitance to 3 types of antibiotic as Streptomycin, Cephalexin and Tetracycline (256 m^l concentration).
TOM T A T
Mudi sdu dong vi khudn dugc phdn ldp tren moi trudng MRS. trong do 13 dong phdn lap tir sda de vd 3 ddng tic chi sinh hgc. Phdn ldn cdc ddng vi khudn co khudn lgc tron, mdu sdc trdng due den trdng siia, do noi dgng md hay ldi, bia nguyen hay chia thuy. Kit qud khdo sdt cdc dac tinh sinh hgc cho thdy 4 dong CO hinh que (ty 14 25,0%) vd 12 dong hinh cdu (ty le 75,0%) tdn tgi a trgng thdi dan hay kit doi. Tat cd cdc dong vi khudn phdn ldp diu Gram duang, khong di dgng vd thie nghiem oxidase dm tinh; ben canh 7 dong co thu nghidm catalase dm linh. Kit qud tuyin chgn dugc 7 ddng thuoc nhom vi khudn lactic (ty li 43,8%). Kit qud khdo sdt trong moi trucmg pH thdp cho thdy 6 dong co khd ndng chong chfu moi trucmg pH 3 trong 3 gid. Dgc biit, dong Biol.2 vd G6.1 CO Idid ndng tdn tgi trong diiu kien moi Iru&ng pH 2 trong 2 gid vdi mdt so 9,01 log(CFU/ml). Dong Bio2.1 khdng dugc 4 logi Ididng sinh Streptomycin, Cephalexin vd Penicillin V (256 mg/l) vd Ampicillin (128 mg/l). Ba dong Gl.l, G5.6 vd G6.1 phdn lap tir siia di khdng dugc 3 logi khdng sinh Streptomycin, Cephalexin (256 mg/l) vd Ampicillin (128 mg/l). Ngodi ra, dong Probi co khd ndng khdng 3 logi khdng sinh Streptomycin, Cephalexin vd Tetracycline (256 mg/l).
Tgp chi Khoa hgc Trudng Dgi hgc Cdn Tho Phdn A: Khoa hgc Tunhiin. Congnghi vd Mdi Irudng: 34 (2014): 8-17
1 D.^TVANDE
Hien nay, cic benh truyen nhilm li mdt Uong nhiing vin dl duoc quan tdm hang ddu tren thi gidi vd moi nim cae benh nhiem triing dudng tilu hda li nguyin nhdn chinh gdy ra benh tdt v i td vong Uin todn toi gidi (Culligan et ai, 2009). Bin canh dd, qud Uinh dilu tri benh bang toudc khdng sinh di tieu di^t phin ldn quin toe vi smh vat mdt cich khdng chpn lpc, din din sy mdt cdn bdng he vi sinh vdt va cd toi gay ra nhung rdi lo^n vung mot nhu binh tilu chdy lien quan din khdng sinh (Tambekar vi Bhutada, 2010). Ddng thdi, khi nang khing thuoc khing sinh cua cac loii vi siito vat gay betto hien nay li rit cao, die biet Id ddi vdi cic khing sinh Streptomycin vd Oxytetracycline (Dung et ai, 2008). Nhilu nghien eiiu cho ring probiotic cd lpi Uong dilu tti cdc b?nh do rdi lo?n tilu hda nhu tieu chdy, kilt ly, thuong ban... do vi khuin giy binh dudng nipt gay ra. Bd sung probiotic dupe xem la mot hoEit ddng hieu qui ttong vi?e cai toiln tinh ttang sue khod ciia con ngudi (Fuller, 1989). Sy gia ting boat ddng khing vi sinh vdt gay benh ciia vl khuan probiotic da md ra hudng iing dung Uong phdng va dilu Uj benh, ciing nhu Id mdt lya chpn mdi de toay toe toudc khing sinh (Ahmed, 2003). Hon niia, siia dpng vdt (bao gdm sda ngudi, siia bo, sUa de...) dupe xem la mpt trong nhihig ngudn vi khudn lactic c6 tilm nang probiotic nhimg ehua dupe nghien cihi sdu.
Vi khuin probiotic cd vai Ud vi tilm ning vd cung to ldn. Tuy nhien, nhihig nghiin cim vi khai thdc ling dyng nhOng tilm ning ndy vdn c6n nhilu ban die d Vi?t Nam vd Uen the gidi. Bin canh viec ling dung cdc tilm nang probiotic ciia vi khudn lactic hi^n nay chua that sy phd bien vd mang lai hi?u qui cao, die bi^t li Uen ngudi. Khi nang ehing chju moi Uudng pH thip vi khdng vdi cic loai khdng sinh li cdc d§c tinh rit quan Upng dl vi khudn probiotic cd toi tdn tai ma khdng bi tilu diet nhdm phuc hdi he vi sinh vdt cd lpi d dudng mot (Tambekar vd Bhutada, 2010). Trudc toye U?ng Uen, nghiin ciiu nay dupe toye hiln nhim tiiyln chpn ra cic ddng vi khuin probiotic tir sOa de cd khd ning chdng chiu dupe mdi trudng pH thdp va khing cdc loai khdng sinh phd biln hi$n nay d ndng d0 cao. Kit qud cua de tii la tiln dl cho nhiing nghien cihi chuyin siu tilp theo vl probiotic, sin xuat eae che phim sinh hpc vd iing dung trong bio ve vi ndng cao sue khoe con ngudi trudc vin de vl dich benh.
2 PHlTONG TIEN NGHIEN CUtr Ngudn miu dupe sii dung bao gdm mlu siia de vi cac mlu chl phim sinh hpc Probio vd Bioacimin. Cie lo^i khai^ sinh dupe sd dyng ttong nghien ciiu d dang hot dupe ban d cdc bilu touoc tren dja bin tliinb pho Cin Tho. Trong do, SUeptomycm do Trung Qudc sdn xuit, 4 loai con 1^ la Penicillin V, Cephalexin, Ampicillin vi TeUacycline do cac Cdng ty Dupe d Viet Nam san xuit dya ttin ngudn nguyen liiu nhap khau. Moi trudng nudi ciy, phan lap vi khao sit bao g^m:
mdi Uudng MRS (De Man, Rogosa and Sharpe), dung dich dim PBS (Phosphate buffer saline), moi trudng nude mudi peptone SPW (Saline peptone water). Cie hda chat dung dl phin tich va chudn dd: CaC03, NaOH, HCI... vd cac phuang ti^n cdn thilt khdc trong phdng thi nghiim.
3 PHU'CNG PHAP NGHIEN CUtI 3.1 Thu th^p va xu* ly mau
Ddi vdi mlu s&a de, 10 mlu siia dupe tou toip tir 10 con dl cii dang cho eon bii ttong vong 3 thing diu sau khi sinh dupe tou d huyen Chp Lach, tinh Ben Tre. Mdi mdu siia dupe chiia trong eae tiii nylon vd trung vi trii lanh de mang ve phdng thi nghiim. MIU ehl phim Probio va Bioacimin dupe mua tai cdc cda hdng tiiudc ttIn dja bdn Thdnh pho Cdn Tho. Ddng todi, tiln hinh do pH cdc mdu siiadl.
3.2 Phan l^p vi khuin lactic tir sira dl vd chl pham men tieu boa
Trdn deu mdi mau siia dl va cho vdo moi ttudng MRS Idng vd trung vdi ^ 111 ml mlu/4 ml mdi trudng dl ho?t hda ttong 24 gid dudi dilu kien ky khi d 37°C. Ddi vdi men tieu hda, tiln hdnh hda tan 1 gram chl |)him vao 100 ml moi trudng MRS Idng vd Uiing dl boat hda Uong 24 gid dudi dilu kien ky khi d 37°C. Dilu kiln ky khi dupe tao bing cdch dat mdt ngpn nen dang chay vio ttong mpt binh toiiy tinh day kinh chda mdi trudng nudi cdy vi khuin, khi ngpn nln chiy hit cd the tao dupe dilu kiln ky khi. Sau khi ho^t hda, dung djch mlu chiia vi khuan duac pha loing v i ttii tten mdi trudng MRS agar dl tao cac khudn lac rdi rac. Tiln hanh phan lap cae ddng vi khuin ttIn mdi trudng MRS agar den khi dat dp rdng nhit djnh khi quan sdt dudi kiito hiln vi quang hpc.
Tgp chi Khoa hgc Trudng Dgi hgc Cdn Tha
3 3 Khao sat cic dac tinh sinh hpc va sinh boa 3.3.1 Quart sdt hinh thdi khudn lac, ti bdo, khd ndng chuyen dgng vd nhugm Gram
Quan sat hinh dang, mdu sac, dp ndi khuan lac sau 48 gid li ttong dilu kiln ky khi d 37"C. Hhib dang, kich toudc t l bao va idia nang di ddng cua vi khudn dupe quan sat v i ghi nhin dudi kinb biln vi quang hpc. Sau do, tiln bdnh nhudm Gram t l bao.
3.3.2 Kiem tra cdc dgc tinh sinh hoa Cac tou nghiim sinh hda bao gdm khi nang tdng hop cic enzyme catalase, oxidase vd khd nang phin giai CaCOa. Tir kit qud khao sit cie die tinh sinh hpc va tod nghidm sirih hda sa bd chpn ra cdc dong vi khudn lactic dl thyc hiln thi nghidm dinh gii khi nang chdng chju mdi ttudng pH thip vi khdng toudc khdng sinh.
3.4 So sanh v^ ddnh gia kha nang chdng chiu mdi trudng pH thip
3.4.1 Moi trudngpH 3
Cic ddng vi khuin lactic dupe chung vao 2 ml mdi trudng MRS Idng, vd trimg de nudi tang sinh Uong dieu kien ky khi d 37°C Uong 24 gid. Sau dd, tou lay sinh khdi vi khudn bing cich ly tim 5.000 vdng/phut d 4''C trong 10 phdt. Riia sinh khdi tou dupe hai lin vdi dung dich PBS d pH 7,2. Chuyin dich huyin phu vi khuin vao ong nghiem chira mdi ttudng PBS pH 3. Sau mdi todi dilm 0, 1, 2 vd 3 gid, hut 100 pJ dung dich Uong mdi dng chuyin sang mdi trudng MRS long, u d 37*^ ttong 24 gid.
Sau dd, tiln hanh dim mit sd vi khuan d mdi todi dilm khdo sat bing phuang phdp pha loang miu vi phucmg phip dim sdng (Cao Ngpc Dilp vd Nguyin Hiiu Hi^p, 2009). Cdc ddng vi khuin lactic cung dupe khio sit ddng todi Uong mdiJ^OTiggJ^
6,4 dl danh gii sy phat ttiln ttong mdi tt^g^~
toich hop.
3.4.2 Moi trudng pH 2
Cac ddng vi khuin lactic cd khi ning chdng chiu dilu kiln pH 3 ttong 3 gid dupe cbpn dl tilp tuc khio sit ttong dilu kiln mdi trudng pH 2. Thi nghiim dupe tiln hdnh toeo phuaug p b ^ tuong tu nhu ttong dilu kien mdi Uudng pH 3.
3.4.3 Moi trudngpH I
Tuong ty nhu vdy, tilp tyc khio sit ttong dilu kiln pH 1 doi vdi cdc ddng ed khd nang chdng chiu
Phdn A: Khoa hgc Tv nhien. Cong nghi va Mdi trudng: 34 (2014): 8-17
pH 2 sau 3 gid vdi quy trinh thyc hiln nhu khio sat Uong mdi trudng pH 3.
3.5 Khao sat kha naog khang thuic khang sinh 3.5.1 Phucmgphdp Hen hdnh
Thi n ^ l m dupe tiln hdito khio sdt ddi vdi 5 logi khang siito Penicillin V, Stteptomycm, Cephalexin, Ampicillin v i Tetracycline, dya trin phuang phq) khuech tin dia ciia NCCLS (National Committee for Clinical Laboratory Standards) (1997) cd toay ddi vd bd sung phii hop vdi dieu kiln vi yeu cau cua thi nghiim: Tien hanh ttii dung dich chiia vi khuin dd nudi tang sinh ttong mdi trudng MRS Idng trong 24 gid d 37''C tren dia petri. Ddng todi, pha mdi loai khdng siito thdnh day gdm 12 ndng dd giim dan tii 256, 128, 64, 32, 16, 8, 4, 2, 1, 0,5, 6,25, 0,125 mg/I theo phuong phap . pha loang bge hai tir ndng dd gde 256 mg/I da Ipc bing bp lpc vi khuin (Dung et ai, 2008). M§i ndng dp ciia cac loai khang sinh dupe chiia rilng bilt ttong cac lp touy titto vd trung. Diing tou touat vd trimg dat cic dia giiy di tim dung dich khdng sinh lln dia petti vi ii cic dia thi nghiem Uong dilu kiln ky khi d 37°C ttong 48 gid. Sau todi gian u, do dudng kmh vdng lie che sinh trudng vd so sdnh vdi cac gid Uj tieu chuin ve mdc dp nhay cdm vdi khdng sinh.
3.5.2 Phdn tich kit qud
Khd nang khing khing sinh dupe dinh gii toeo 3 mdc dp dya vao dudng kinh vdng uc ehl sinh trudng nhu sau: Khdng manh (K): < 13mm, Khang uung binh (TB): 14-16mm, Nhay cim (N): >
17mm. (Ngudn: White etal., 2003) 4 KET QUA VA THAO LU^N r - 4.1— Gia tri pH cac mlu siia de
Mudi mdu sira de dupe ky hieu mlu tir GMl din GMIO. Kit qui cho thiy gii tri pH mdu dao ddng tii 6,69-6,93. Gid Ui pH toip ttong nhdm d mlu GMl (6,69) khae bilt khdng cd ^ nghia vdi mdu GM2 (6,73) va khdc biet ed y nghia todng ke vdi cic mau cdn lai. Mau GM3 v i GM7 cd gii tri pH khi cao (6,93) vi khac bilt cd y nghTa todng kl vdi cac mdu cdn lai. Tuy nhien, tit c i gii tri pH cac mlu diu nim Uong khoing trung tinh vi phii hpp vdi khi nang tilu hda ciia de con Uong giai d o ^ diu sau khi sinh.
Tgp chi Khoa hgc TntdngDgi hgc Cdn Tho Phdn A. Khoa hgc Tv nhien. Cong nghi vd Mdi trudng: 34 (2014): 8-17
Hinh 1: Sy bien d^ng ciia gia trj pH cic mlu sira d l
Ghi chu: Sd liiu Id trung binh cua 3 ldn lap lai. Giiia cdc mdu, cdc gid tri trung binh cd cung chU cdi ihi hiin sv khdc biil khdng cd y nghia thdng kid do tin cdy 95%
4.2 Kit qua phan lip vi khuin lactic tir siia de vi men tieu hda
Mudi siu ddng vi khuin dupe phdn lip tii 10 mlu siia de vi 2 loai chl pham Bioacimin va Probio. Trong do, co 13 ddng dugc phdn lap tir 10 miu siia de (chilm 81,3%) va 03 ddng phin lap tii 2 chl phim Bioacimin vi Probio (ehilm 18,7%).
Phin ldn cdc ddng vi khuin phdn ldp dupe cd dang khuin lac hinh ttdn; miu tring dye bode tting sua;
dp nli dimg md hay lai; bia nguyen hay chia thdy.
Ngoai ra, hdu hit cdc ddng diu cd khi nang sinh acid manh tao vdng sdng xung quanh kliuin lac
ttIn mdi trudng MRS agar cd bd sung 1,5% CaCOj (Hinh 2). Kit qua khio sat cac d^c tinh smh hpc cho toiy cd 4/16 ddng cd dang binh que (chilm ty le 25,0%) va 12/16 ddng cd dang hinh ciu (chilm ty ll 75,0%) tdn tai d ttang todi don hotle kit ddi.
Tit cd cic ddng vi khuin diu Gram duang vd khdng di ddng. Kit qui toii nghiem sinh hda cho toiy tit cd cdc ddng diu cd thir nghiim oxidase dm tinh v i 7/16 ddng cd tod nghiim catalase im tinh (ehilm tj 11 43,8%). Kit qui myln chpn dupe 7 ddng toudc nhdm vi khuin lactic dya vio hlnb thii khuin lac, hinh dang t l bdo, die dilm Gram va cic tod nghiem sinli hda (Bang 1).
A B Hinh 2; Hinh dang khuin lac cac dong vi Idiuan phSn ISp tir sira dS va che phSm sinh hQc: G6.1 (A) \k
Bio2.1 (B)
Tgp chi Khoa hgc TrudngDgi hoc Cdn Tha Phdn A: Khoa hgc Tv nhiin. Cdng nghi vd Mdi tnrdng: 34 (2014): 8-1 Bang 1: pgc (JQIJ ^JQII ^^^ ^ ^ ddng vi khuan lactic phan lap tur sua dl va ehl pham sinb hpc
Ddng NguoD „ t . L . t i i - ^ 1 Hinh dang Thu nghiem Thir nghifm vi khii^r. ^iT- 1- Hinh thii khuin lac . * t - .L 7 -j VI Knuan phan lap • tebao catalase oxidase Biol.2 Bioacimin
Hinh thii khuan lac Trdn, bia nguyin,
ndi md, ttJng s&a Que ngan Bio2.I Bioacimin Trdn, bia nguyin,
ndi rad, tring siia Que ngan Trdn, chia tou^*,
, tting dye Siia dl (miu
GMl)
Trdn, bia nguyin, ndi md, tring siia Siia d l ( m i u
G M 5 )
Trdn nhd, bia nguyen, ndi md, trang siia Siradl
(mlu GM5)
Trdn, bia nguyen, ndi md, Uang siia Siia de (mlu
GM6)
Trdn, bia nguyen, noi md, Udng si^a Ghi chu: (—): Am tinh
4.3 Ket qua so sanh va dinh gia khi nang ch6ng chiu mdi trufnig pH thap
4.3.J Moi trudngpH6,4
d pH 6,4, mat sd ban ddu vao thdi dilm 0 gid cua cdc ddng vl khuan p h ^ lap cd sy kliac nhau vd dao dOng trong khoang tu 7,69-8,91 log(CFU/ml).
Bing 2: M^t s6 (logCFU/ml) ciia cac ddng vi khuin lactic trong moi trudng pH 6,4
Den tocri diem 3 gid, mat sd dao ddng Uong khoing 7,74-8,89 log(CFU/ml), till hiln sy thay ddi rit it hay gdn nhu khdng thay ddi so vdi thdi dilm ban ddu. Kit qui khdo sdt chiing td pH 6,4 la dilu kiln pH thich hpp cho sy ton tai vd dn dinh mit sd ciia cie dong vi kliuan phan lap.
Ddng vi khuan
Thdi gian (gid)
CV(%) Biol.2
Bio2.1 Probi Gl.l G5.1 G5.6 G6.1
8,87 8,91 7,69 8,72 8,79 8,74 8,78
8,87 8,90 7,82 8,74 8,80 8,74 8,78
7,82 8,73 8,79 8,74 8,79
8,87 8,89 7,74 8,72 8,80 8,74 8,78
0,24 0,25 2,81 0,22 0,37 0,30 0,-28^
Ghi chu: ns: khdc biit khong eo y nghta
Sd liiu Id kit qua trung binh eia 3 ldn lap lgi. Trong cUng mgt hdng, ede gid tri trung binh ihi hiin sv khdc biil khdng cd y nghia thdng kid do lin cay 95%
4.3.2 Moi trudng pH 3
Kit qud khdo sdt d dilu kiln pH 3 cho toiy mat so ban diu tai thdi dilm 0 gid cda cie ddng vi khuan phdn ldp cd su khic nhau vd dao ddng Uong khodng tir 7,93-9.10 log(CFU/ml). Dong G5.1 khdng cd khi ning ch6ng chiu sau I gid vdi mit sd bdng 0 log(CFU/ml). Sdu ddng vi khuin cdn lgi (chilm t j ll 85,7%) diu cd mdt so giim sau 3 gid toi nghiim va dao dpng ttong khoing tir 7,82-9,05 log(CFU/ml). Sy khdc bilt ed ^ nghia ye mit todng ke cho thiy sy toay ddi dang kl mat sd cdc ddng vi kbuin so vdi todi dilm ban diu (Bing 3). Tuy
nhiin, xet trong tieu ehi danh gii khi nang cbdng chiu mdi trudng pH thdp, cdc kit qui khdo sit trin di chiing minh phdn ldn cic ddng vi khuin lactic phin lap deu cd kba nang chdng chiu dupe dilu kiln pH 3 Uong 3 gid. Mdi trudng pH dich da diy cua ngudi cd toe dat pH 3 hoic cao hon khi chua toye phdm va eic sin phdm tir sua (Matijasic v i Rogelj, 2000). Kit qua khio sat da chiing minh hiu hit cic ddng vi khudn lactic phdn lip tir sUa de va ehl phim men tilu hda deu cd khd ning chiu dyng dupe pH 3 ttong 3 gid. Ndi cich khic, chung cd kha nang chdng chiu vdi dieu kien acid ciia d? diy de di den rudt non.
Tgp chiKhoa hgc TrudngDgi hgc Cdn Tho Phdn A: Khoa hgc Tu nhiin, Cdng nghi vd Mdi Irudng: 34 (2014): 8-17 Bang 3: Mat so (logCFU/ml) cua cac ddng vi khuin lactic trong moi tmdng pH 3
DAng Biol.2 Bio2.l Gl.l G5.1 G5.6 06.1
0 8.95' 8,81- 7,93' 9,02"
8^5"
9,04"
9,10"
ThM aian (gi6>
1 2 8,88* 8,81=
8,80- 8,78"
7,89- 7,86- 9,01' 9,00"
0,00' 0,00' 9,04' 9,03"
9,09" 9,08"
3 8,82'»
8,76"
7,82' 8,98- 0,00' 9,03' 9,05'
CV(%) 0,30 0,45 1,26 0,25 0,63 0,22 0,15 Ghi chu: Sd lieu Id kit qud trung binh cua 3 ldn lap lgi. Trong cung mgi hdng, cdc gid Iri trung binh cd cimg chir cdi thi hiin su khdc biet khdng cd y nghia Ihdng kid do lin cdy 95%
gid, tit c i eic ddng vi khudn khdng cdn kha ning 4.3.3 Moi Iruang pH 2
Sdu d6ng vi khudn cd kha nang chong chju mdi trudng pH 3 ttong 3 gid dupe tuyln chpn ^z. khdo sdt Uong dilu kiln pH 2. Khic vdi kit qui khao sat ttong dilu kien pH 6,4, Uong mdi trudng pH 2 tit ci cie ddng vi Idiuin diu cd mat sd gidm ro ret sau 3 gid thf nghiem. Sau 1 gid khdo sit, tdt ci cdc ddng vi khu^ diu thi hi?n khi nang chdng chiu pH 2 vdi mat sd thay dii khong ddng kl so vdi thdi dilm 0 gid. CJ thdi diem 2 gid, van edn 2 ddng vi khuan (ehilm ^ le 33,3%) cd khi ning chdng chju vdi dilu ki^n pH 2. Cdc ddng vi khuan cd khi nang chdng chiu bao gdm Biol.2 vdGd.l vdi gii tti mat sd 9,01 log(CFU/ml). Tuy nhien, den thdi diem 3
Bing 4: Mdt so (logCFU/ral) ciia cic ddng vi khuin lactic trong moi trudng pH 2
chong chiu vdi gii tti mat sd bang 0 (Bdng 4). Sy khdc bilt vl khi ning cbdng chiu dilu kiln pH 2 cLia cie ddng vi khuan cd toe giii toich do diy Id dilu kiln pH rit thip ddi vdi sy tdn tai cua nhilu nhdm vi sinh v|t, ttong dd cd nhdm yi khuin lactic.
Dilu kiln pH niy cd anh hudng rdt Idn den ho?t dpng ciia enzyme va sy Uao ddi chdt ciia vi sinh vgt. Do do, phdn ldn cie vi sinli vgt khdng co khd ning tdn tai vd phit triln Uong dilu kiln pH 2 den tiii^ ban (Cakir, 2003). Kit qua khdo sdt chua tuyln chpn dupe ddng chdng chju dupe mdi trudng pH 2 ttong 3 gid nen khdng tiln hdnh thi nghiim d dilu kien pH 1.
Ddne vi l{huiin Bio 1.2 Bio2.1 Probi Gl.l G5.6 06.1
0 9,07' 9,06- 8,05' 8,97"
9,07"
9,06"
Thcri gian 1 9,06' 8,90' 7,50' 8,87' 9,02' 9,04f , ,
(R'il-) 2 9,01' 0,00' o.oo- 0,00- 0,00"
-_9,01*
3 0,00' 0,00' 0,00' 0,00' 0,00' 0,00'
CV(%) 0,26 0,22 2,31 0,39 0,22 0,36 Chi chu: Sd liiu Id kit qud trung bmh eiia 3 ldn IdpTgi Trongcung mgthdng, cdc gid tri trung binh co cing chU cdi thi hiin sv khdc biit khdng ed y nghia ihong kiddg Tin cgy 95%
4.4 Kit qua khio sit kha nang khing thuoc khdng sinh
4.4.1 Khdng sinh Penicillin V
Ndng dd lie chl tdi thilu MIC (Minunum Inhibitory Concentration) la ndi^ dp todp nhit cua mpt logi Idling sinh cd khi nang lic chl sy phdt Uiln cda vi khuin sau klioing 24 gid nudi ciy (Dung el ai, 2008). Do dd, ti^ng toi nghi?m nay hipu qud khdng khing sinh ciia cac ddng vi khuin phdn l§p dupe danh gii dya vio gid Uj MIC khio sdt trin vi sinh v^t gay b^nh. Penicillin V Id lo£u khing sinh toupc nhdm lie chl tdng hpp vich tl bio vi khuan vd cd gii Uj MIC toudng ndra trong khoing 0.01-0,1 mg/l.
Kit qua thi nghi?m cho toiy tit cd cdc dong vi khudn lactic phin lap diu cd khd nang khdng Penicillin V ndng dp tii 0,125-8 mg/l. CJ ndng dd t&
16-128 mg/l, cd din 5/6 ddng vi khuin (chilm ^ I§
83,3%) bilu hiln khang vdi Penicillin V; ttong khi dd, ddng Probi bilu hien nhay cdm chi d ndng dO 16 mg/l. Den ndng dp 256 mg/l, ddng Bio2.1 vdn cdn bilu hiln khing vdi Penicillin V (chilm t5' le 16,6%). Kit qud toi nghiem da chiing mmh ede ddng vi khuin phin lap ed khd ning khang Penicillin V d ndng dp rit cao so vdi eic nhdm vi khudn gdy hlnb trin ngudi v i d^ng v^t khi so sdnh vdi gia tti MIC.
Tgp chi Khoa hgc Trudng Dgi hgc Cdn Tho Phdn A: Khoa hoc Tv nhiin, Cong nghi vd Moi trudng: 34 (2014): 8-17
Bang 5: Kha nang khang khang sinh Penicillin V caa mdt so ddng vi khuin lactic phan lip tir sua dl v i chl pham sinh hpc
STT 1 2 3 4 5 6
Dong vi IdiuSn
Bio 1.2 Bio2.1 Probi Gl.l G5.6 G6.1
0,125 K K K K K K
0,25 K K K K K K
0,S K K K K K K
1 K K K K K K
Nong do 2 K K K K K K
Idiang sinh (i 4 K K K K K K
8 K K K K K K
mg/l) r6 K K N K K K
17, K K N K K K
64 K K N K K K
128 K K N K K K
256 TB K N TB TB TB Ghi chit: K: Khdng mgnh; TB: Khdng trung binh. N: Nhgy cdm
4.4.2 Khdng sinh Streptomycin vd Cephalexin Stteptomycin la khang sinh thudc nhdm ire chl tong hop protem ciia vi khuin. Theo Korhonen (2010), gia tti MIC cua Stteptomycin ddi vdi phin ldn vi khuin lactic ndm ttong khoang 2-256 pg/ral.
Ngodi ra, toeo nghiin eiiu ciia Dung et ai (2008), cd din 98,7% (trong tdng sd 50 ddng vi khuin gdy benh gan todn mu trin c i tta) bi nhay cim vdi khang sinh Cephalexin ndng dp 30 pg/ml. Hieu qui cao ciia khang sinh Cephalexin nhd vio kbi nang Bang 6: Kha nang khing khang sinh Streptomycin
phin lip tu sfia de va chl phim sinh bpc
lie che tSng hpp vach te bio vi khuan va ben viing vdi enzyme penicilinase. Kit qua thi nghiem nay cho thiy tit ca cac ddng vi khudn lactic phin lap deu ed khi nang khing SUeptomyein va Cephalexin d ndng dd td 0,125-256 mg/l (Bdng 6).
Dieu nay chung minh khang sinh Stteptomycin vi Cephalexin d gidi ban ndng dp khio sat lln den 256 mg/l vdn chua anh hudng din sy tdn tai vd phit triln eiia eic ddi^ vi kbuan phan lap.
va Cephalexin ciia mot so ddng vi khuan lactic
STT 1 2 3 4 5 6
Dong vi IthuSn
Biol.2 Bio2.1 Probi Gl.l G5.6 G6.1
0,125 K K K K K K
0,25 K K K K K K
0,5 K K K K K K
1 K K K K K K
Nong do 2 K K K K K K
l(hang 4 K K K K K K
sinb 8 K K K K K K
(mg/l) 16 K K K K K K
32 K K K K K K
64 K K K K K K
128 K K K K K K
256 K K K K K K Ghi chu. K: Khdng mgnh; TB: Khdng trung binh; N: Nhgy cdm
Hinh 3: Kha nang khang khang sinh Penicillin V (P), Streptomycin (S) v i Cephalexin (C) d ndng do 256 mg/l eua ddng vi khuin Bio2.1 (A) va G6.1 (B)
Tgp chi Khoa hgc Trudng Dgi hgc Can Tho Phdn A • Khoa hgc Tv nhien, Cong nghi va Moi Irudng: 34 (2014). 8-
.A
4.4 3 Khdng sinh Ampicillin
Ampicillin Id loai khdng sinh phd rpng tiiupc nhdm beta-lactam cd tic ddng chu ylu vio qud Uuto nhin ddi cua vi khuin, lie chl sy tdng hpp mucopeptide eiia ming tl bio vi khuin. Theo Dung et al. (2008), ed din 86% (ttong tdng sd 50 ddng vi khuan gay benh gan tiidn mii Uen ca Ua) nhay cdm Bang 7: Kha ning khang khang sinh Ampicillin ci
chl phim sinh hpc
vdi Ampicillin ndng dp 10 ^ra\. Kit qui khdo sat toe hien, tdt ci cdc ddng vi khudn phdn ldp khdng Ampicillin d ndng dp tii 0,125-64 mg/l. Tuy nhien, d ndng dp 128 mg/l, vdn cdn 4/6 ddng vi khuin (chilm tj le 66,6%) bilu hiln khdng (bao gim cdc ddng Bio2.1, G l . l , G5.6 va G6.1). Den ndng dp 256 mg/l, khdng cd ddng vi khuin ndo bilu hiln khing vdi khing suih Ampicillm.
I mpt so ddng vi khuan lactic pbin lip tir sua de va
STT 1 2 3 4 5 6
Dong vi Ithnan
Biol.2 Bio2.1 Probi Gl.l G5.6 06.1
0,125 K K K K K K
0,25 K K K K K K
0,5 K K K K K K
1 K K K K K K
Ndngi 2 K K K K K K
So khSng 4 K K K K K K
sinb (1 8 K K K K K K
ng/l) 16 K K K K.
It K
32 K K K K K K
64 K K K K K K
128 TB K TB K K K
256 TB TB N N N N Ghi chu: K: Khdng mgnh: TB: Khdng trung binh; N Nhay cdm
4.4.4 Khdng sinh Tetracycline Kit qui khio sdt cho tody, tit ci cic ddng vi khuin phan Igp cd khd nan^ khdng Tetracycline d ndng dd tii 0,125-16 mg/l. 6 ndng dp 32 mg/l, cd 5/6 ddng vi khuin (chilm ty le 83,3%) bilu hi?n Idling. Vdi ning dd 64 mg/l, chi cdn 3/6 ddng vi khuin (chilm ty ll 50,0%) bilu hiln khdng, cdc ddng cdn lgi tbi hiln miic do tmng gian vdi TeUacycline d nong dp niy. Din nflng dd 128 mg/l vi 256 mg/l, ehi cd ddng vi khudn I'robi (chiem ty ll 16,6%) bieu hi^n khing vdi TeUacycline.
Tetracycline lic chl sy tdng hpp protein cua te bdo vi khuan bing cdch gin vao phdn 30S ciia ribosome, do dd lie che gan aminoacyl-tRNA mdi vio vj tti tilp nhan. Theo Dung et ai (2008) chi cd khoing 30% (Uong ting si 50 ddng vi khuan gay
Bing 8: Khi ning khing khing sinh Tetracycline ciia m$t so ddng vi khuan lactic phan l^p tir su^ dl va chl pham sinh hpc
benh gan togn mii tten cd tta) nhgy cdm voi TeUacycline ndng dp 30 pg/ml.
Cie nghien eiiu hien nay da ung hd vi?c sii dyng probiotic dl phdng ngira binh tilu chdy co liln quan din khdng sinh. Hiiu qud khing thuic khdng smh d ndng dp cao chi ra rdng, nlu sii dung cie probiotic phan lap tir siia dpng vdt cho b?nh benh nhdn dilu tti bang khing sinh se rit hftu ich trong vile phuc hdi binh nhanh ban bdi sy thiet ldp hi vi sinh v^t cd ich Uong dudng rudt mdt cdch nhanh chdng. Kha ning khdng cua vl khudn probiotic vdi mot sd khdng sinh cd the dupe sii dung cho c i hai muc dich phdng ngira vi dieu trj cac binh nhilm triing dudng rudt (EI-Naggar, 2004).
STT 1 2 3 4 5 6
D6ng vi llhuan
Biol.2 Bio2.I Probi Gl.l G5.6 06.1
0,125 K K K K K K
0,25 K K K K K K
0,5 K K K K K K
1 K K K K K K
Nong do 2 K K K K K K
Idiang sinh (ing/1) 4
K K K K K K
8 K K K K K K
16 K K K K K K
32 K TB K K K K
64 TB TB IC K TB
K 128
N N K TB TB TB
256 N N K N N N Ghi chu: K. Khdng mgnh: TB Khdng trung binh; N: Nhay cam
Tgp chi Khoa hgc TrudngDgi hgc Cdn Tho Phan A: Khoa hgc Tv nhiin, Cdng nghi va Mdi Irudng: 34 (2014): 8-17
Hinh 4: Kha nang khiug khing sinh Ampicillin (A) vi Tetracycline (T) d nong dp 64 mg/l eua ddng vi khuan Probi (A) va G6.1 (B)
5 KETLUIN TAI LIEU THAM KHAO Mudi sau ddng vi khuin dupe phdn l|p tten
mdi trudng MRS ^ a r tir ngudn sOa de va ehl phdm sinh hpc. Phin ldn cie ddng vi khuin cd dEing khuin lgc hinh udn, mau uing due din tring siia, dd ndi dgng md hogc ldi, bia nguyen hay chia tody; hinh d ^ g chii ylu cd dang que bode cdu tdn tgi d trang toii dan hay kit ddi. Tit e i cdc dong vi kliuin phdn l^p diu Gram duang, khong di ddng vd cd thii nghiim oxidase im tinh; ben canh dd, cd 7/16 ddng cho kit qua toii nghiim catalase am tinh.
Tir cic ket qua khdo sdt tuyln chon duac 7 ddng thu^c nhdm vi khudn lactic. Trong 7 ddng dupe khdo sdt, cd den 6 ddng cd khd nang chdng chiu mdi trudng pH 3 ttong 3 gid. Dgc bilt, ddng Biol.2 vi G6.1 cd khd nang tdn tgi ttong dilu ki^n mdi trudng pH 2 ttong 2 gid. Ddng Bio2.1 ed khi ning khing 4 logi khang sinh Stteptomycin, Cephalexin v i Penicillin V. d. ndng- d6r256 mg/Lva Ampicillin d ndng dd 128 ' ^ / i r l ^ ^ ^ ^ l f G 5 . 6 va G6.1 ed kha ndng khdng 3 loai khdng sinh Stteptomycin, Cephalexin d ndng dp 256 mg/l va Ampicillin d ndng dp 128 mg/l. Ngoai ra, ddng Probi ed khi ning khing 3 logi khang sinh Stteptomycin.
Cephalexin va TeUacycline d ndng dp 256 mg/l.
L<n CAM TA
Nhom tic gia xin chin toanh cam cm Ban lanh dgo Vien NC & PT Cdng nghe Sinh hpc, cdc anh chi vd cic ban d Phdng toi nghiim Vi sinh v^t, Cdng nghe Sinh hpc Thyc phim vd Sinh hpc phdn tii Thue v^t da tan tinh ho Up rat nhieu tir qua Uinh thyc liien din khi hoin toanh nghiin ciiu niy.
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