7c
KHOA HQC K9 THUAT 145
TIEM NANG PROBIOTIC CUA MQT SO VI KHUAN LACTIC PHAN LAP TlT SUA ME
ThS. Dirong Nhgt Linh' ThS. Nguyin Van Minh'
Trdn Thi Kim Sa' Trdn Cdt Ddng^
TOM TAT
Md' dau: Probiotic Id nhfmg vi khudn sdng cd lgi cho dudng ruot. khi diroc bd .sung vdo ca fhi v&i liiu lirang thich hgp. Nhfmg vi khudn ndy cd anh huang tdt cho siic khoe cua con ngu&i nhu tdng cirang he miin dich, ngdn ngica cdng nhu dieu tri tieu chay do rdi logn vi khudn dir&ng rugt, diiu hod phdn iing viim. Nhiiu vi khudn probiolic dirge su dung di hd trg dieu tri cdc binh dirdTig nidt nhu: lieu chdy, hdi chfrng rugt kich thich. Mac dii chiing probiotic cd thi dugc phdn lgp tir nhiiu ngudn, nhimg neu sir dung cho ngu&i thi dp dung tiiu chi chinh Id cd ngudn gdc tir ngudi (WHO/FAO, 2002),
Sfra m^ Id ngudn dtnh duang quan trgng ddi v&i tri sa sinh. rdt nhiiu nghien cuu chg thdy tdc dung cd lgi ciui sfra ddi v&i sue khde cua tre sa sinh. Tdc dung cd lgi ndy cdn du^c gidi thich Id do cdc vi khudn laclic cd trong sfra me [6].
M^c lieu: Ddnh gid tiim ndng probiotic in vitro cua cdc chimg vi khudn laclic phdn lap tir sua cua nhiing bd me khoe mgnh.
Phuong phdp: Lactobacillus dirge phdn lap bdng mdi tnr&ng MRS cd bd simg CaCO, di phdt hiin acid lactic. Cdc dgc tinh probiotic dugc khdo sal theo Inrang ddn cua WIIO/FAO bao gdm- khd ndng sinh enzym ngogi bdo, chiu acid, mudi mat, vd cdc yiu td an todn. Chting vi khudn duac dmh danh bdng khda phdn logi ciui prokaryote.
Kit qud: Tic 57 chung phdn lap dirge tir sda. chung tdi da sdng Igc vd dinh danh dirr^c 5 chung thugc chi Lactobacillus. Trong dd. 5 chiing cd khd ndng ddi khung v&i vi khudn cd hgi thu&ng gap. cd kha ndng sinh emzyin ngogi bdo. ty le sinh L-lactic acid/D-laclic acid cao. cd khd ndng chiu pH acid dg ddy vd mudi mat, khdng sinh hemolysin, nhgy cam v&i khdng sinh thir nghiem.
Kit lugn: Chung tdi dd chgn 5 chung Lactobacillus ddp irng cdc tieu chi in vitro ddi v&i vi khudn dimg ldm probiolic iheo hu&ng ddn ctia WHO'FAO Kii qua cdng cho thav rdng sfra m^ Id mdt ngudn chiia vi khudn cd tiim ndng ldm probtotic.
Xir khoa: Probiotic. vi khudn lactic, Lactd5acillus. dung ngp acid/mudi mgt. sua me ABSTRACT
Background: Probiotic is viable microbe agent that beneficially affects the host possibly by improving the balance ofthe Indigenous microflora. Many probiolic bacteria liave been used as alternative treatment of some intestinal diseases such as diarrhoea, irritable bowel syndrome. Although probiolic strains can be isolated from many sources:
for human applications the main criteria is being human origin.
Breast milk is an important nutrient source for neonates. Lots of studies showed dial this fluid has beneficial effects on the health of neonates. One reason of being
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beneficial is explaining by the microflora of hitman breast milk including beneficial laclic acid bacteria.
Objectives: This work aimed lo isolating and screening of potential probiolic properties of lactic acid bacteria sfrains from breast milk (milk of healthy mothers)
Methods: iMClobacillus was isolated by MRS medium supplemented with CaCO.
to detect lactic acid. Probiotic characteristics were tested according to the gm'delnjes of WHO/ FAO, which include, producing extracellular enzymes, resistance to low pH and bile salts, and safety aspects Finally, these strains were identified according to Prokaryote - A Handbook on the Biology of Bacteria.
Results: From 57 isolated strains from breast milk we selected 5 strains that showed antimicrobial activity against food poisoning bacteria, ability of producing extracellular enzymes and high ratio of L-lactic/D-lactic acid, resistance to low pH and bile salts, nonhemolytic and sensitive to tested antibiotics. The .selected strains were identified as species belong to Lactobacillus genus.
Conclusion: In this work. 5 Lactobacillus strains have been selected and identified lhat meet Ihe requirements for in vitro probiotic characteristic and safety^ tests according to the guidelines of WHO/ FAO. The results also showed that, breast milk is a source of potential probiotic strains.
Keywords: Probiotics. lactic acid bacteria, Lactobacillus, bile resistance, acid tolerance, breast milk.
Di^T VAN OE
Sira me la ngudn dinh duong ldt nhat cho tre. Trong siia me rat giau cdc ydu id midn djch, cdc yeu td giiip he vi sinh dudng rupl cd lpi phai trien vupt trdi. Dac biet. sCra me gidu prebiolic giup cac vi khudn cd lpi phdt trien. Khong nhung chira hdn hop hodn hao cac chai dgm. cac vitamin va khoang chai cbo nhu cdu phai Iricn ciia Ire. Sua me edn chira mpt lirpng Idn cac vi khuan cd lpi nhu Bifidobacteria va LactobacHlii
|6|. Da cd nhidu nghidn ciru cho ihay rang, tdc dpng cua Lactobacillacae lir sira me cd the gdp phan bao vc ire so sinh. ihiic ddy sy tnrdng thanh cua he thdng mien dich va phdng chong nhidm trung |6.12].
Nhtmg ndm gan day. vice sir dung vi khudn probiotic nhu chdt thay thd khang sinh Irong ihuc pbam dang rat duoc quan lam. Mpi trong nhiimg vi khuan duac
•iu dyng rdng rdi nhal lam probiolic la Lactobacillacae nia chitng [a cd thd thu iihan tir nhidu ngudn khdc nhau nhu phan su em be. luycn siia ngudi mc. hat kclir
va tren cac loai thuc pham ldn men truydn thdng. Sy Ihich nghi ly nhidn ciia nhidu vi kliuan lactic ddi vdi mdi irudng rupl va nhiing chat khang khuan do chiing lao ra nhu acid hO'u co va bacteriocin dd cho vi khuan lactic mdt thuan lpi canh iranh so vdi nhiing vi sinh vdl khac khi dupc dimg lam probiolic.
Tai Vict Nam dang luu hanh mpt sd chd pham probiolic. thanh phan vi khuan irong dd la chu ydu Bacillus va Laclobacillus. nhirng ngudn nguyen lidu hoan todn ngogi nhap. ngogi Irir vai san phdm Irong nudc chira Bacillus cd dac linh chua dupc chirng minh. Dc gdp phan xdy dung mpi bd suu lap nhihig chung vi khuan laclic cd nhu'ng hoal linh idi.
chiing ldi lien hanh phan lap vd sang loc va danh gid liem'nang probiolic cua cdc chung da phan lap. Nghien ciiu gdp phan
\ a a no luc lu lao cac che phdm probiolic cd chira Lactobacillus hodn loan cua Vict Nam. vdi cdc dii lieu khoa hoc duoc chirng minh.
KHOA HOC KY THUAT VAT L i p u VA PHU^OTSG PHAP Vat lif u
Chung vi khudn nghidn ciru dupc thu nhgn tii' 100 mdu sua me tir Benh vien Phu san lir Du va Hiing Vuong.
Chung vi khuan thii nghiem kha ndng doi khdng- Escherichia coli ATCC 25922. Salmonella spp.. Bacillus cereus.
Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa. Vibrio cholerae.
V.parahaemolylicus. Streptococcus feacalis, Streptococcus lieu huyet P nhdm A.
Phuo'ng phdp nghidn ciru Phdn l^p vi khudn tactic Pha loang mdu trong dem PBS (photphatse buffered saline) vdi dp plia loang 10-' ddn 1 0 ' . Hut 100 pL dung dich d mdi dp pha lodng Icn dia thgch MRSA bd sung CaCO, va trdi ddu. U o 35"C trong 24 ddn 48 gid/5% CO,. Quan sat cac khdm khuan trdn mdi trudng. chpn nhung khudn lac cd vdng tan CaCO^ xung quanh. tidn hdnh tao chung thuan qua nhieu lan phan lap tidp. Tidn hanh khao sat dgi thd. vi thd.
catalase. oxidase, dinh tinh acid lactic.
Gidng Ihu dupc nudi trdn mdi trudng MRS dich thd va giiJ gidng iren mdi trudng MRS bd sung 2 0 % glycerol, bao quan a -20"C[11.
Phuvng phdp thu khii niing iinh enzym ngogi biio
Tbii nghidm kiia nang sinh mdt sd loai cnzvmnhu: protease (cascinase. gelalinase).
amylase, lipase.
Chudn bi mdi Irudng TSA cd bd sung chdt cam iing thich hpp. Hoat boa cac cliuiig vi khudn can thir nghidm. Chdm khoang lOpL dich khudn len dTa ISA. ii 37"C.' 48 gid'' S^'o CO,. Doc ket qua bdng cac ihudc ihir Lugol (amylase), thuoc ihu TCA (gelalinase). quan sdt edc vdng due va vdng trong xung quanh khdm (lipase vd caseinase) 13. 7].
Phuimg phdp thir hogt tinh khdng khuan
Chuan bi dich vi khuan chi ihi cd ndng dp 1-2 x 10" td bao/mL. Chung vi khudn can thir nghipm dupc nudi va nhdn gidng Irdn mdi trudng MRS, u 37''C. 5%
CO, sau 48 gid. De tach chidt bacteriocin thd la tidn hdnh ly tam 13000 vdng/phitl.
20 phiit. d 4"C. Djch ndi thu dupc didu chinb vd pH 7 bang NaOII IM. Sau dd dupc tua bang dung djcb ammonium sulphate (40% bao hoa). Khudy dung dich trong 2h/ d 4''C. sau d6 ly tam 20000 vdng/phut, l b . d 4°C. Chat tua dupc huydn phu lai vao 25mL dung djch ddm potassium phosphate (pH"- 7).
Djeh khuan chi ihi dupc trai trdn mdi yudng MHA vd due nhQ'ng gidng cd dudng kinh 6nim. Nhd 50 pL djch ndi da cbuan bj vao mdi gieng va u d 4"C trong 30 phut dd cdc chdt ldng khudch tan dd dang trong thach. Cudi ciJng cac dia dupc u d tirng didu kien phii hop cho timg vi khudn chi thj. Kha ndng khang khudn cua dich bacteriocin thd dupc xac dinh bang each do dudng kinh vdng vd khudn quanh moi gidng [2.5].
Dinh lug'ng D/L- lactic acid Dinh Iupng kha ndng tao D/L -laclic acid cua tung cbiing lactic bang bp Kit phdt hien va dinh lupng D/I.-lac\ic acid ciia hang Megazyme [8].
Khdo sdt khd ndng chiu pH thdp vd muoi mgt
Thu nghiim pH: Cac chiing vi khudn dupc hogt hda. Ly tdm 5000 vdng/
10 phiJl d 4''C. thu cdn vd huyen phii lgi trong PBS (photphatse buffered saline) pH 7,4 tgo thanh huyen djch cd dp due McFarland 0.5 tuong duung 10' C n i / m L . Chuydn huydn dich vi khudn vao binh ndn chira mrdc mudi sinh ly va chinh pH cd gia tri ldn lupt la: 1.5: 2: 2.5.0 a 37"C. 5
% CO,. CJ mdi thdi didm 1 gid. 2 gid. 3 gid Idv ImL dung djch mau thir trung hda vd
TAP CHf KHOA HOC S6 4 (22) 2011 pH = 7. Tidn hdnh pha loang ddn mat dp
thich hpp ddm dupc trong dung dich ddm va Irdi trdn hop thach MRS agar. U aSTC, 5 % CO,, trong 24 - 48 gid. Ddm khdm vd ttnh sd don vi sdng cua vi khuan khao sat.
Mdi ihii nghiem lien hdnh 3 Ian [4. 5].
- Thfr nghiim mudi mat: Cac chimg vi khudn dupc boat hda trong mdi trudng MRS. u qua dem khoang 1 8 - 2 4 gid.
Vi khudn dupc pha loang den dp due McFarland 0.5 luong duong 10" CFU/ml- nhu yen. Ldy ImL djch vi khuan Iy tam, cho tiep xuc vdi dich mudi mat vdi cdc ndng dO ldn lupt 0.3%. 1,5%, 2% trong Igid. 2 gid. 3 gid. Sau do ly tdm nra sach djch mgt. Tidn hdnh pha lodng ddn mal dp thicb hpp ddm dupc irong dung dich dem vd ydi trdn hop thgch MRS agar. U d 37"C.
5 % CO,, trong 24 - 48 gid. Dem khdm va ti'nb sd dan vj sdng cua vi khudn khdo sat.
Mdi ihii nghiem tidn hdnh 3 ldn 14. 9].
Phdn uam sdng sdt dupc tinh: Ni/Nx y I(K), Ni ^ log CFU/mL sau thdi gian nudi cdy, Nx = log CFU/ mL thdi gian 0 gid.
Phuang phdp thu khd ndng huyit gidi Hogt hda cdc cbiing vi khudn can thu nghidm. Diing que cdy vdng ldy vj khuan cdy Idn mdi trudng thgch mdu B A bd sung 5% mdu ciru. song song tidn hanh vdi vi khuan ddi chiing khong dung huydt. U 37^C/5%CO,/24gid[51.
Phuang phdp thie nghi$m tinh nhgy cdm khdng sinh
Xac dinh MIC bdng phuang phap pha loang khdng sinh trong canh Idng theo CLSI. 2006. Cdc khang sinh dupc su dung bao gdm Penicillin. Ampicillin. Imipenem,
Gentamycin. Vancomycin, Erythromycin.
Clindamycin. Pha loang khang sinh vao trong mdi trudng MHB dd dat day ndng dp 128, 64, 32. 16....0.0625pg/mL cho vao cac gidng cua phidn 96 gidng 50 pL.
cung vdi 50 p.L huydn djch vi khuan cd ndng dp 10*^ CFU/mL. 0 37"C/ 16-18 gid va dpe ket qud [5. 10].
Phutmg phdp dinh danh vi khuan Cac chiing chpn Ipc dupc djnh danh bdng phuang phap sinh hda theo khda phdn loai The Prokaryote - A Handbook on the Biology of Bacteria [II].
Phitong phdp xu ly so li$u Cac thunghidm hogt tinh khdng khudn va kha ndng chiu pH thdp vd chju mudi mgt duoc lap lgi 3 lan. Sd lieu dupc xii Iy tbong ke ANOVA bang phan mdm Excel.
KET QUA VA BAN LU^N Ket qua phan l^p Lactobacillus Tir cdc mdu siia m? dupc thu nhdn tir cac Benh vidn phu san TPflCM. da phdn lgp dupc 57 chiing dupc k> hidu theo ngudn gdc mau. Chpn Ipc dupc 13 chung cd dang true khudn. khdng sinh bdo tii, catalase am. oxidase am. sinh acid lactic.
Ket qua sinh enzyme va hoat tinh khang khuan
Tir 13 chiing thu dupc cbung toi thu nghiem khd ndng sinb enzyme (hinh 1) va hogi tinh khang khudn (hinh 2). vdi bai tidu chi ddi khang tdi thieu 4 vi khuan gay bdnh trd Idn hoac doi khang ldi thidu 4 vi khudn gay bdnh trd Idn va sinh ldi thieu 2 trong 4 loai enzyme, chpn dupc 6 chiing thda man didu kidn trdn (SI, S2, S3. S6.S7.S8).
KHOAHPCKYTHUAT
Hinh 1. Thv nghiem fcha ndng sinh enzyme ngogi bdo a: protease; b: amylase
Hinh 2. Thir nghiam kha ning khdng khuSn
• ^ - ^ ^
K^t qua dljnh lu^ng D/L - lactic acid Tir 6 chiing da sang lpc dupc d budc tren, su dyng bp kit djnh lupng, chiing tdi chpn dupc 5 chung cd ty le L/D Idn hon 0,9.
K^t qui khao sAt kha nang chiu pH thip va inu6i mat
5 chiing dupc chpn lpc tiep tuc dupc khao sat kha nang chiu dupc pH tliap, chiing tdi tien hanh thu nghidm cac chung tren d ba miic pH 1,5; 2 va 2,5 va mudi mgt d cac ndng dp 0,3%; 1,5%; 2% trong khoang thdi gian tir 1 gid den 3 gid.
lull* mn
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Bidu dd 1. IV le sdng ciia cac chimg Ihu nghidm theo thdi gian I. 2 va 3 gid d pll 1.5: 2: 2.5 vd inudi mat dcac nong dp 0.3%; 1.5%: 2%.
Dya vao bicu dii 1. cho ihay irong 5 chung thir nghiem chiu dung acid va mudi mal. la ihdy a pH 2.5. cdc chiing cd Iv lc sdng > 60% sau 2 gid. dac bidi chiing S2 cd kha nang ldn lgi va phai uidn dupc Irong dieu kien nay. 6 ndng dp mudi mal 0.3% sau 3 gid hdu hcl cac chung cd ly lc sdng cao iren 90%, mpt sd cbung van cdn cd kha ndng tang trudng.
Ket qua thir nghiem kha nang huyet giai
Then WHO.TAO Ihu ngliiem hcmo- l>sin la mdi budc sang lpc tinh gdy benh cua cdc chung dc dam bao linb an loan cua probiolic. 5 chiing thti nghiem khdng cd enryin hemolysin (khdng cd kha nang lieu huydl) (hinh 3). cac chiing nay ticp tyc duoc thii' nghiem khd nang dc khang khang sinh.
Hinb 3. Kit qua khfing tigu huylt trSn th^cb mdu BA cika cdc chiing thii nghiam
Ket qua thii' nghiem kha nang dd k h a n g khang sinh
Ddy cung la budc danh gia linh an loan ciia cac chimg luydn chpn lam probiolic. cbung ldi khao sal kba ndng nhay cam vdi khang sinh bang phuang phap pha lodng 5 chiing dupc khao sal.
dpe kdl qua .sau 20-24h;37"C vd dupc bic-n giai Iheo MIC Brcakspoinl dp dyng cho Lueioluicillus theo CLSI (2006). Trong 5 cbiing khao sal tat ca cac cbung (SL S2. S6. S7. S8) \ a n cdn nhay cam \'di cac khang sinh thu nghiem.
Kdt qua dinh danh bang sinh hoa Bang \. Ket qua dinh dunh nhotn vi khuiin lactic
Icn men dong hinh group A
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KHOA HOC KY THUAT
Bang 2. Kit qua dinh danb nhom vi khuan lactic Icn men di hinb khong bdt bu$c group B
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Dua trdn bang I vd bang 2. cho thay 5 chiing trdn cho kdt qua sinh bda phil hop vdi 4 loai LactobacilliLs: L gasseri. L.acidophilus. L.salivarius subsp.
salivariiis, L.rhamnosiis.
KET LUi^N
Chiing tdi da chpn 5 chung Lactoba- cillus dap ling cac tieu chi in vitro ddi vdi vi khuan diing Idm probiotic theo hudng ddn ctia WMO/FAO.
TAILISUTHAMKHAO Ml, Nguydn Lan Dung. (1983). Thyc
tap Vi sinh \ a t hpe. NXB Dai hoc &
Trung hpe chuydn nghiep. Ha Npi.
| 2 | . Buniin N.. Cbanihachum S.. Hong- paltarakcrc T. (2008). Screening of lactic acid bacteria from gastroin- testinal tracts of marine fish for Iheir polenlial use as probiotic. Songkla- nakarin 3. Sci. Technol 30: 141-148.
| 3 | . Cabo M.L et al. (1999). A method for bactcriocins quantification. Joumal of applied microbiology 87: 907-914.
141- Cukrowska B.. Mot>'l I.. Kozakova IL. Schv\ar/er M.. Gdrecki R. K..
Klewicka I-".. SUitevtska K.. Libudzisz Z. (2009). Probiotic Laclobacillus Suains: in vitro and in vivo Studies Folia Microbiol. 54(6): 533-537.
| 5 | . Dunne C . Liam CTM. el al. (2001).
In vitro selection crilcria Tor probiolic bacteria of human origin: correlation with in vivo findings. American Soci- ct> TorClinical Nutrition 73; 386-392.
[61. Tederico L.Y.Mo'nica O. et al.
(2007). Beneficial cfTectsof probiotic bacteria isolated from breast milk.
British .lournal of Nutrition, 98, Suppl. I.S96-S100.
[7J. John R.Tagg.. Adnan S.Dajani..
Lewis W.Wannamaker. (1976).
Bacleriocins of Gram-possitive bacteria. American Scocienly for Microbiology 3: 722-726.
f8| Livia Aim. (1982). laTect of Fermentation on L ( ' ) and D(~) Lactic Acid in Milk. Journal of Dairy Science 65: 515-520.
[9]. Mourad K., Nour-Eddine K. (2006).
In vitro preselection crilcria for pro- biotic Lactobacillus plantanrum strains of fermented olives origin.
International Journal of Probiotics and Prebiolics I: 27-32.
flO]. Methods forAnlimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria; Approved Guideline.
(2006). M45-A26. 19:24-25.
I l l ] , Martin D.. Stanley F.. cl al. (2006).
The Prokaryotes - A Handbook on the Biology of Bacteria. Springer 3:
3 2 0 - 3 7 2 .
[12|. Rocio M.. Monica O. et al. (2005).
Probiolic polenlial of 3 Laclobacilli strains isolaied from breast milk.
Journal of human laciaiion official joumal of Intemalionai Laclation Consultant Association. 21 {I): 8-17
