DANH SCH BNG Hnh Tn hnh Trang

Bng 1.1 Cc ging khc nhau ca vi khun lactic .........................................4 Bng 1.2 c im phn bit mt s loi ca chi Lactobacillus ......................6 Bng 1.3 Cc c im phn bit mt s loi Pediococcus ...........................10 Bng 3.1 Cc chng vi khun dng trong th nghim ...................................23 Bng 3.2 Thnh phn mi trng MRS rn/Lt .............................................27

DANH SCH HNH Hnh Trang Tn hnh

Hnh 1.1 Hnh thi t bo ca Lb. axitophilus v Lb. plantarum ....................5 Hnh 1.2 Hnh thi t bo ca Leuconostoc mesenteroides ..............................8 Hnh 1.3 Hnh thi t bo ca Pediococcus halophilus v Aerococcus urinaeequi....................................................................................................... 9 Hnh 1.4 Hnh thi t bo ca Bifidobacterium ..............................................11 Hnh 3.1. Hnh thi t bo v nhum gram ca cc chng vi khun lactic s dng trong th nghim ....................................................................................24 Hnh 4.1. Thi gian tng OD 0,3 ca cc chng vi khun lactic trong mi trng MRS c 0,3% mui mt .....................................................................33 Hnh 4.2. Kh nng sng st ca cc chng vi khun lactic pH 2 ..............36 Hnh 4.3 Hnh nh minh ha kh nng chu axit ca chng DC1 trn a peptri ........................................................................................................................38 Hnh 4.4 Hnh nh minh ha kh nng chu axit ca chng DC2 trn a peptri...............................................................................................................39 Hnh 4.5 Hnh nh minh ha kh nng chu axit ca chng MC5 trn a peptri...............................................................................................................40 Hnh 4.6 Hnh nh minh ha kh nng chu axit ca chng MC9 trn a peptri...............................................................................................................41 Hnh 4.7 Hnh nh minh ha kh nng chu axit ca chng MC10 trn a peptri ..............................................................................................................42 Hnh 4.8. ng cong tng trng ca chng MC9 .....................................43 Hnh 4.9. Trnh t mt on gen rDNA 16S ca chng MC9 v Lactobacillus fermentum SFCB2-3 .......................................................................................45 Hnh 4.10. Cy phn loi da trn trnh t rRNA 16S ..................................46

CC T VIT TT Lb Lc MRS CFU Lactobacillus Lactococcus Man, Rogosa, Sharpe Colony Forming Unit

WHO World Health Organization FAO OD Food and Agriculture Organization Optical Density

MC LC PHN 1 : M U ................................................................................................................... 1 PHN 2 : TNG QUAN TI LIU ..................................................................................................................... 3 2.1 Tng quan v vi khun lactic ................................................................................................. 3 2.1.1 c im sinh l, sinh ha ca vi khun ..................................................................................................................... 3 2.1.2 Cc nhm vi khun lactic ................................................................................................. 3 2.1.3 ng dng ca vi khun lactic ...............................................................12 2.2 H vi sinh vt trong ng tiu ha ca ngi ........................................12 2.2.1 c im chung ca h vi sinh vt ng tiu ha ngi ....................12 2.2.2 Chc nng cc nhm vi sinh vt ch yu trong ng tiu ha ngi 13 2.3 Tng quan v probiotics ..........................................................................14 2.3.1 Khi nim probiotics .............................................................................14 2.3.2 Nhng tnh cht c li ca probiotics ...................................................16 2.3.3 Cc tiu chun sng lc mt chng vi khun lm probiotics ................19 2.4. Kh nng tn ti v sinh trng ca vi khun lactic trong ng tiu ha ngi ..............................................................................................................20 2.5 Tnh hnh nghin cu kh nng chu axit v mui mt ca vi khun lactic trn th gii ....................................................................................................21 2.6 Tnh hnh nghin cu kh nng chu axit v mui mt ca vi khun lactic Vit Nam .....................................................................................................21 PHN 3 : I TNG V PHNG PHP NGHIN CU ............23

3.1 i tng nghin cu ..............................................................................23 3.2 Thit b, ha cht s dng ........................................................................26 3.2.1 Thit b ..................................................................................................26 3.2.2 Ha cht ................................................................................................26 3.3 Phng php nghin cu .........................................................................26 3.31. Phng php hot ha ging ...............................................................26 3.3.2. Phng php nui cy tng sinh .........................................................27 3.3.3. Phng php bo qun ging .............................................................27 3.3.4. Phng php xc nh mt t bo trong mi trng lng .............28 3.3.5. Phng php kho st kh nng chu mui mt ..................................29 3.3.6. Phng php kho st kh nng chu axit ...........................................30 3.3.7. Phng php xy dng ng cong sinh trng .................................30 3.3.8 Phng php x l s liu .....................................................................31 PHN 4 : KT QU V BIN LUN ....................................................32 4.1. Kt qu kho st kh nng chu mui mt ..............................................32 4.2. Kt qu kho st kh nng chu axit .......................................................34 4.3. Xc nh kh nng sinh trng v tch ly sinh khi ca MC9 .............43 4.4. Kt qu nh danh ...................................................................................44 PHN 5 : KT LUN V KIN NGH ..................................................48 TI LIU THAM KHO ...........................................................................49

PHN 1 M U 1. X hi cng pht trin th vic nng cao sc khe, phng chng bnh tt li cng c ch trng. Lo s trc cc tc ng c hi ca vic iu tr bnh bng khng sinh, con ngi ngy cng a thch nhng phng php gi gn sc khe, phng trnh v iu tr bnh tt c ngun gc t nhin, to ra s cn bng, n nh trong c th. Mt trong s nhng phng php ph bin rng ri nht hin nay l s dng cc ch phm probiotic. Probiotic l ch phm gm cc vi sinh vt sng, ch yu l vi khun lactic. y l nhng vi khun sng t nhin trong ng tiu ha ca con ngi v cc loi ng vt khc. Chng c nhiu tc ng c li cho sc khe nh gip n nh h vi sinh vt ng rut, c ch vi khun gy bnh trong ng rut, h tr tiu ha, gim nng cholesterol trong mu v gim nguy c b ung th. thng xuyn duy tr nhng tc ng ny, c th b sung thm t ngun thc phm ln men c cha chng nh sa chua, tm chua, nem chua hoc t cc ch phm chuyn dng. Hiu qu ca chng s cng c tng cng khi cc chng vi khun lactic trong qua nhng nghin cu sng lc m bo chng c tim nng probiotic mnh. Mt trong nhng tnh cht quyt nh tim nng probiotic ca cc chng vi khun ny l kh nng tn ti, thch nghi c vi mi trng trong ng tiu ha ng vt. Chng phi chu c mi trng axit d dy v

thch nghi c vi nng mui mt rut v cc tc ng ca cc enzyme trong ng tiu ha. Nhng nm gn y, cc loi thc phm c cc vi sinh vt probiotic rt c a chung Vit Nam. Tuy nhin, cha c nhiu cc nghin cu phn lp v kho st tim nng probiotic ca h vi sinh vt ln men lactic nc ta. Do , chng ti tin hnh ti: Tuyn chn chng c kh nng chu axit v mui mt cao t cc chng vi khun lactic trong mt s sn phm ln men lactic trn a bn thnh ph Hu, nhm xc nh c mt s chng c kh nng tn ti v thch nghi trong ng tiu ha ng vt, lm tin cho cc nghin cu tuyn chn cc dng vi khun lactic c tim nng probiotic ng dng vo sn xut.

PHN 2
TNG QUAN TI LIU 2. 2.1. Tng quan v vi khun lactic 2.1.1. c im sinh l, sinh ha ca vi khun lactic Vi khun l nhng nhng vi sinh vt n bo, c cu to n gin. Chng c th h hp hiu kh hoc ym kh, trong cu to t bo ca chng khng c dip lc t [5]. Ngoi nhng c im chung trn th i vi mi nhm vi khun khc nhau lun c nhng c im ring c trng cho nhm . Vi khun lactic cng vy chng cng c nhng c im sinh l sinh ha khc so vi cc nhm vi khun khc v trong mi loi s khc nhau cng thy r hn. Mc d nhm vi khun ny khng ng nht v mt hnh thi nhng nhn chung tt c nhng vi khun lactic u c nhng c im sau. 2.1.1.1. c im sinh l - l nhng vi khun gram dng, ni chung l bt ng, khng sinh bo t. - Chng l nhng vi khun k kh ty nghi, vi hiu kh, l loi c th c nht c kh nng ln men hiu kh cng nh k kh, chng c kh nng sinh trng c khi c mt oxi. - Chng khng cha cc cytochrom, catalase v nitratoredutase m tnh 2.1.1.2. c im sinh ha - Dinh dng ca nhm vi khun lactic kh phc tp do kh nng tng hp cc cht cn cho s sng ca chng rt yu. Khng mt i din no

thuc nhm ny c th pht trin trn mi trng mui khong thun khit cha glucose v NH4+. a s chng cn hng lot vitamin (lactoflavin, tiamin, axit pantotenic, axit nicotinic, axit folic, biotin), v cc axit amin, cc baz purin v pirimidin [5]. - Chng c kh nng ln men to axit lactic bng cch phn gii ngun hydrat cacbon c trong mi trng. 2.1.2. Cc nhm vi khun lactic Nm 1857 Pasteur chng minh c rng vic lm sa chua l kt qu hot ng ca mt nhm vi sinh vt c bit l vi khun lactic. Nm 1878 Lister phn lp thnh cng vi khun lactic u tin v t tn l Bacterium lactics.V sau cc nh khoa hc lin tip phn lp c nhiu loi vi khun lactic khc nhau [1]. V cc loi vi khun ny c xp chung vo h Lactobacteriaceae. Bng 1.1 Cc ging khc nhau ca vi khun lactic [2] T bo Kiu ln % Ti liu dn Ging G+C Hnh Sp xp men Streptococcus Cu Chui ng hnh 34- 46 Schleifer 1986 Leuconostoc Cu Chui D hnh 36- 43 Farrow v cng s 1989 Pedicoccus Cu T cu Schleifer 1986 Lactobacillus Que Chui ng hnh 34 -42 Kandler v welss, 32 -53 1986 Bifidobacterium Khc Nhiu ng hnh 55 -67 nhau dng v d hnh Scardovi, 1986 ty loi Lactic v acetic

Nhm vi khun lactic rt a dng bao gm rt nhiu chi khc nhau. Vic phn loi cc chi ny da trn s khc nhau v hnh thi ca vi khun (T bo ca chng c th hnh que hoc hnh cu) v kh nng sinh trng v pht trin trn cc iu kin pH khc nhau (4,4; 9,6); cc nhit khc nhau

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(100 C; 45 0C) cng nh kh nng ln men ng hnh hay d hnh ca chng. Ngi ta tin hnh phn loi v chia nhm vi khun lactic thnh 5 chi ch yu sau (Th hin trn bng 1.1). S tp hp ca 5 chi ny vo cng mt nhm vi khun lactic c khng nh qua phng php phn loi phn t [2]. Ngoi ra c mt s loi vi khun sinh ni bo t c kh nng ln men to axit lactic thuc chi Bacillus, Tuy nhin, cc loi ny khng c xem l vi khun lactic do cc c im sinh l v ha sinh ca chng. 2.1.2.1. Lactobacillus Ging Lactobacillus l chi ln nht trong nhm vi khun lactic, gm khong 80 loi [27]. Vi mc khc nhau rt nhiu v hnh thi, c im sinh ha v sinh l. S khng ng nht th hin ph t l mol G+C rt rng, t 32-55% [27]. Chng l nhng trc khun khng sinh bo t thuc lp vi khun Gram dng, catalase m tnh, sinh trng trong iu kin k kh khng bt buc hoc vi hiu kh. Nhng trc khun ny thng ng ring l hoc thnh chui, thy phn ng saccharosa mnh to axit lactic, khng kh nitrat, chng phn gii gelatin, indole v H2S. Chng pht trin mnh trong mi trng c tnh axit, ty loi m pH dao ng t 4,5 n 6,4. y l nhng vi khun khuyt dng nhiu loi vitamin, axitamin. Ngi ta c th tm thy Lactobacillus trong cc sn phm sa, tht, hoc trong nc sch, bn, b mt thc vt, tri cy. Cc loi thuc chi ny cng l mt phn quan trng trong h thng vi sinh ng rut ngi v ng vt bc cao, c trong khoang ming, ng rut v m o. Nhiu kt qu nghin cu phn loi sinh ha v lai DNA cho thy Lactobacillus l mt nhm rt c bit.

INCLUDEPICTURE "http://bp0.blogger.com/_FlhB437Wa_U/SClyFlwHv7I/AAAAAAAAArc/TeX5R_U81vA/s400/a

a
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Hnh 1.1 Hnh thi t bo ca (a) Lb. axitophilus v (b) L

Bng 1.2 c im phn bit mt s loi ca chi Lactobacillus [2] (Theo Bottazzi, 1988)
Nhm Loi GC% Loi peptido Dng Ni sng chnh ADN Glycane Lb.delbrueckii subsp.delbruceckii Lb.delbrueckii subsp.bulgaricus I Lb delbrueckii subsp lactics Lb.acidophilus Lb.gasseri Lb.helveticus 49-51 Lys Asp 49-51 Lys Asp 49-51 Lys Asp 34-37 Lys Asp 33-37 Lys Asp 38-40 Lys Asp axit lactic D D D DL DL DL Mt thc vt Sa chua Fomat Ming, m o Ming, m o Fomat

Lb.casei subsp Casei Lb. casei subsp pseudoplantarum II Lb casei subsp Tolereas Lb.casei subsp rhamnosus Lb. sake Lb.bavaricus Lb. Plantarum

45-47 Lys Asp 45-47 Lys Asp 45-47 Lys Asp 45-47 Lys Asp 42-44 Lys Asp 42-44 Lys Asp 44-46 m-ADP

L DL L L DL L DL

D dy v nhai li Fomat, rm r Mm, m o Rut non ngi B mt thc vt B mt thc vt Thc vt,fomat

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Lb.bifementans III Lb. brevis Lb buchneri Lb. kefir Lb.reuteri Lb. fermentum Lb.confusus Lb. Viridescens Lb. Sanfrancisco

44-46 Lys Asp 45-47 Lys Asp 44-46 Lys Asp 40-42 Lys Asp 40-42 Lys Asp 45-47 Lys-Ala 36-38 Lys-Ala

DL DL DL DL DL DL DL

Fomat Thc vt,fomat Thc vt,fomat Kefir Rut non Thc vt,fomat Thc vt Ccc sn phm tht Cc loi bnh

52-54 Orn-D-Asp DL 45-47 Lys-Ala-Ser DL

Ging Lactobacillus c chia lm 3 nhm (Bng 1.2) [6]: Nhm 1: c gi l Thermobacterium, ln men ng hnh v a nhit. Khng ln men glucose v ng pentosa. Nhng vi khun ny c fructose 1,6 diphosphate aldolasa, chng pht trin 450C. Nhm 2: c gi l Streptobacterium, y l nhng trc khun ln men ng hnh v a m, nhiu loi ca nhm ny c kh nng ln men d hnh. Cc hexosa c ln men theo con ng EMP to thnh axit lactic, nhng cc pentosa c th ln men d hnh. Nhm 3: gm nhng trc khun c tn gi l Betabacterium, chng l nhng vi khun ln men d hnh bt buc. Chng sinh axit lactic, axit acetic, CO2 v ethanol. 2.1.2.2. Streptococuss y l nhng vi khun gram dng bao gm cc cu khun, a s ln men ng hnh v bt ng. Sau khi phn chia chng thng to thnh chui, sng hoi sinh trong t, b mt cy, da ngi, rut Chi ny cng l mt chi rt ln trong vi khun lactic, bao gm nhiu nhm khc nhau. Ngoi Lactococcus th trc y ngi ta xp thm Enterococcus vo chi Streptococcus. Mc d ngy nay c s phn tch v to thnh nhng chi mi, chi Streptococcus vn l mt chi rt ln v kh phn loi mt cch hon ho. Enterococcus khng c vai tr quan trng trong cng ngh

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thc phm. Mt s loi trong chi ny, v d nh E. faecalis c th l nhng vi khun gy bnh c hi, v th khng c a chung trong ch bin thc phm. Ngi ta quan tm nhiu n kh nng khng khng sinh v kh nng chuyn mt s tnh trng thng qua cc yu t di truyn c kh nng dch chuyn. Tuy nhin, vn c nhiu nghin cu s dng E. faecium v E. faecalis lm probiotic do nhiu loi trong chi Enterococcus c tr t nhin trong ng rut ca ng vt bc cao v ngi [10] [36] . 2.1.2.3. Leuconostoc Chi Leuconostoc l nhng vi khun hnh cu, gram dng, phn ln chng ln men d hnh theo con ng Hecxoso-monophosphat to D-lactic t glucose. Mt s loi Leuconostoc cng vi Lactobacillus gy nn qu trnh ln men malolactic, y l qu trnh ln men th hai sau ln men ru vang, n lm chua ru vang, qu trnh xy ra mnh trong vang cn nhiu axit tactric v axit malic. V vy rt d nhm ln gia Leuconostoc vi Lactobacillus ln men d hnh c hnh thi dng cu-que. Do c s tch ra mt s loi t c chi Lactobacillus v Leuconostoc hnh thnh nn chi mi: Weissella, gm cc vi khun ging nh Leuconostoc, chnh l cc loi trc y c xem nh Ln.paramesenteroides; Lb.confusus; Lb.viridesens [27]. Chi ny v th c c dng hnh cu v que. Ngi ta nhn thy loi Ln. oenos, cn c gi l Leuconostoc ru, c kh nng chu nng ru v axit cao, v th c tch ra to thnh mt chi mi, Oenococcus. Trong khi vic phn bit gia Oenococcus v Leuconostoc kh n gin, th vic phn bit gia Weissella v Leuconostoc vn l mt vn nan gii.

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Hnh 1.2 Hnh thi t bo ca Leuconostoc mesenteroides

2.1.2.4. Pediococcus Pediococcus l mt chi trong nhm vi khun lactic gm nhng t cu hoc song cu ln men lactic ng hnh, to axit lactic dng DL hoc (L+),c kh nng chu axit v thy phn protein yu. Hu ht chng khng c kh nng s dng lactosa v c c trng bi GC% ADN t 34-42 %. Ngi ta phn bit Pediococcus bi kh nng chu nhit, chu pH, chu NaCl v kh nng ln men ng (Bng 1.3). Trong cng ngh thc phm vai tr ca chng va c ngha tch cc va tiu cc. Chng hn nh v P. pentosaceus c s dng lm ging nui cy khi u trong sn xut xc xch v thc n gia sc [27]. Chng cng c th c vai tr quan trng gp phn vo s chn ca phomat. Ngc li th P. damnosus li l mt tc nhn chnh lm hng bia, v s pht trin ca chng c th dn n s to thnh diacetyl/acetoin, lm cho bia c v ging b. Ngoi Pediococcus th Aerococcus, Tetragenococcus cng to nn cc vi khun lactic dng tetrad (4 cu khun lin kt thnh b 4). Trong chi Aerococcus nhn chung ch c vai tr th yu trong cng ngh thc phm. Cn chi Tetragenococcus ban u c coi l Pediococcus halophilus.

a
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Hnh 1.3 Hnh thi t bo ca Pediococcus halophilus(a) vAerococcus urinaeequi(b)

Bng 1.3 Cc c im phn bit mt s loi Pediococcus [2] Pc.damnvulus Pc.dextrinicus Pc.pentosaceus Pc.axitolactici Pc.halophilus Pc.urinaequi Pc.damnosus Pc.inopinatus

c im

- Sinh trng pH 4,5 - mn cao nht c th chu (%) - Ln men : Lactoza Maltoza Melezitoza Riboza Saccharoza Trehaloza Dextrine Amidon - Thy phn agrinin

+ <4

+ 6.5

ND 4-6.5

+ 6.5

ND 6.5

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6.5

+/+/+/+ -

+ +/-

+/+ + +/-

+/+ -/+ +/+ + -

+/+ + + +

+/+ +/+

+ + + + + -

+/+ ND + + + -

+: dng tnh; -: m tnh; +/-: khc nhau ty chng; ND: khng xc nh.

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Ngoi c im l chu c nng mui rt cao (18%) th chng cn mt c im na phn bit vi cc vi khun lactic khc l chng cn nng mui khong 5% pht trin. Chi ny c vai tr quan trng trong cc loi thc phm c hm lng mui cao, nh nc chm u nnh. 2.1.2.5. Bifidobacterium Chi Bifidobacterium l nhng trc khun k kh khng sinh bo t, Gram dng, bt ng. Khi mi phn lp chng l nhng trc khun c th phn nhnh dng ch Y, V tp hp thnh khi. Sau nhiu ln cy chuyn chng tr thnh nhng trc khun thng hoc hi un cong vi nhng ht d tr nhum mu xanh metylen. V vy khi pht hin vo nm 1899, vo u th k XX chi ny chi c xem nh thuc vo chi Lactobacillus. Theo kha phn loi Bergey nm 1920, chng c tn l Lactobacillus bifidus. Nm 1967, Devries v Stouthamer chng minh s c mt ca enzyme fructose-6phosphate phosphoketolase. nhm vi khun ny v s vng mt ca hai enzyme l aldolase v glucose-6-phosphatase dehydrogenase hai enzyme c mt trong Lactobacilli. T ngi ta kt lun rng vic phn loi nhm bifido trong chi Lactobacillus l khng hp l. Ngy nay, nhm ny c xp thnh mt chi ring bit, chi Bifidobacterium, thuc h Actinomycetaceae, c 25 loi [2]. Chng ln men lactic d hnh sn phm chnh ca chng l axit acetic v axit lactic, cng vi mt lng nh axit formic, ethanol, axit succinic v mt iu c bit khc na l trong qu trnh ln men khng sn sinh ra kh CO2. L nhng c th catalasa m,nitrat dng, indol v gelatine m. Chi ny ch yu sng trn c th ng vt bc cao, c th gp chng trong khoang ming v ng tiu ha ca ng vt v c cn trng, thm ch c bn, chng l vi sinh vt tin phong trong h tiu ha ca ngi. Chng c ng dng quan trng trong vic sn xut cc ch phm vi sinh lm probiotic.

Hnh 1.4 Hnh thi t bo ca Bifidobacterium

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2.1.3. ng dng ca vi khun lactic Vi khun lactic c ng dng nhiu trong cng nghip bo qun, ch bin thc phm v thc n gia sc [5]. T lu nhn dn ta bit bo qun v ch bin cc sn phm rau qu bng cch mui chua ko di thi gian s dng. Chnh cc vi khun lactic l tc nhn ch yu tham gia qua trnh ln men chua. Chng c kh nng ln men ng to axit lactic trong mi trng k kh lm cho pH gim xung di 5, lm c ch cc loi vi khun gy thi pht trin. Ngy nay, vi khun lactic c s dng trong sn xut sa chua, phomat, ln men cc sn phm rau qu, tht, hi sn. Ngoi ra, nhm vi khun ny cn c nhiu c tnh c tim nng probiotic nn c nhiu nh nghin cu v ang quan tm. Vic s dng cc chng vi khun lactic trong phng nga v iu tr bnh cng nh phc hi v duy tr sc khe ngy cng ph bin. Ngy nay, chng cn c s dng trong iu tr lm tng h min dch, gim cholesterol, ngn nga ung th [20] [21]. Cc chng vi sinh vt c s dng lm probiotic thng mi hin nay u thuc nhm vi khun lactic, hai chi l Lactobacillus v Bifidobacterium [16]. Trong Lactobacillus l nhm vi khun lactic c s dng nhiu nht trong lnh vc probiotic [20] [19]. Nhng loi ny u c xem l cc vi khun khng gy bnh ngi. Hin nay ang c mt s nghin cu s dng c Enterococcus, Streptococcus lm probiotic [14]

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[36]. Vit Nam, vic nghin cu v s dng cc sn phm probiotic c ngun gc t vi khun lactic cn rt hn ch. 2.2. H vi sinh vt trong ng tiu ha ca ngi 2.2.1. c im chung ca h vi sinh vt ng tiu ha ngi H tiu ha ca chng ta ch chim mt khong khng gian kh khim tn trong bng nhng din tch b mt tiu ha ca rut ln rt ln. Ngi ta c on trong rut chng ta c hng h sa s cc loi vi sinh vt khc nhau, gp hng chc ln tng s t bo trong c th mt ngi trng thnh. [19]. Khi mi sinh ra, ng tiu ha ca chng ta khng c bt k mt vi sinh vt no k c vi khun, do nhng tip xc u tin vi mi trng, t sa m... vi khun bt u xm nhp. Nhng c th vi khun ny sau khi thot qua c d dy n rut s tng sinh nhanh chng, tn ti v pht trin, dn dn to nn qun th vi sinh vt vi s cn bng t nhin. Khi trng thnh, vi sinh vt ng rut n nh v cn bng hn. Tuy nhin cng c th b mt cn bng do nh hng ca mi trng, v sinh an ton thc phm, bia ru, thuc khng sinh, thuc ha tr liu, hoc ngay c cng thng cng vic... V vy ri lon ng rut vn thng xuyn xy ra. Vi ngi gi, vi sinh vt c li c xu hng gim nhng vi sinh vt gy bnh li tng, do ngi gi thng xuyn b ri lon tiu ha nh tiu chy, to bn, vim i trng, snh bng, i phn sng..., gim kh nng hp th dinh dng, gim khng, bnh tt, k c ung th rut kt. V mt khoa hc, chng c bit l mt h vi sinh vt sng. Chng sinh si ny n trong ng rut non, i trng ca ngi v phn ln chng tn ti m khng cn oxy [19]. Hng trm ngn t vi sinh vt sng ny l s cn bng gia vi khun c li v vi khun c hi. Bt u t d dy, vi khun gn nh bng khng, ch c vi khun gy lot d dy Helicobacter pylori l c th hin din, s lng tng dn rut non v rut gi. Trong s c mt ca vi khun c li ng vai tr v cng quan trng cho chc nng tiu ha v gip cn bng h vi khun rut. 2.2.2. Chc nng cc nhm vi sinh vt ch yu trong ng tiu ha ngi

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Cc nhm vi sinh vt ch yu trong ng ng rut ca chng ta l nhng vi sinh vt sng c hi, c li v cc loi trung tnh khc nhau. Cc vi sinh vt c li ch yu trong ng rut ca chng ta l cc loi cng sinh Bifidobacterium, Lactobacillus, Streptococcus, Enterococcus, Bacillus v nm men [11] [19]. Cc tc ng ca nhm vi khun c li : Chng em li li ch bng cch ti lp s cn bng t nhin trong h tiu ha, gip ng rut khe mnh bng cch bm cht, chim lnh thnh rut v cnh tranh cht dinh dng vi vi khun c hi; ngn nga vi khun c hi tn cng v pht trin, tng cng chc nng chng ca nim mc rut, gip khng ch vi khun gy hi. Mt s loi vi khun sinh ra cc cht khng khun nh bacteriocins gip ngn nga v tiu dit mm bnh. Ngoi ra vi khun cng sinh cn gip tng c phn ng min dch c hiu v khng c hiu bng cch kch thch cc t bo phn ng min dch (macrophages, lymphocytes), tng cng s sn xut ra cc thnh phn min nhim (cytolines, immunoglobulins, interferon). Mt s vi sinh vt c li khc trong rut non li hn ch hp thu cholesterol, gip gim cholesterol trong mu; sn sinh ra cc enzym khc phc qu trnh tiu hy carbohydrat, lm thun tin hn vic hp th nng lng t cc cht dinh dng. Vi sinh vt c li gip tng kh nng dung np lactose nh kch thch sn sinh lactaze. Bn cnh li ch ca vi khun c li th lun c s tn ti ca cc vi khun c hi nh Escherichia coli, Shigella, Staphylococcus aureus, Clostridium, Veillonella [19]. Cc vi khun c hi ny khi xm nhim vo ng rut s gy ra cc tc ng xu n sc khe mgi tiu dng nh ng c thc phm ( Salmonella), vim nhim ng rut (Campylobacter), ri lon tiu ha (Enterotoxigenic E. coli, Enterotoxigenic E. coli, Enteroinvasive E. coli, Enterohaemorrhagic E. coli) Tuy nhin khi no vi khun c li v vi khun trung tnh ny cnh tranh nhau ginh ly khng gian trong ng rut th vi khun c hi khng c

20

c hi nhn ln. Thng th h vi khun trong ng rut ca chng ta rt bn vng. Ngay c khi h khun rut b ri lon rt nng do dng khng sinh hoc tiu chy th chng vn c th phc hi mt cch nhanh chng khi nguyn nhn gy nn ri lon ny c loi b. Tuy vy i khi vn c trng hp nhng vi khun c hi chim u th trong ng rut. 2.3. Tng quan v probiotic 2.3.1. Khi nim probiotic Vic s dng vi sinh vt sng nhm tng cng sc khe con ngi khng phi l mi. Hng nghn nm v trc, trc khi tm ra thuc khng sinh, con ngi bit tiu th cc thc phm cha vi sinh vt sng c li chng hn nh cc sn phm sa ln men. Cc nh khoa hc u tin, nh Hippocrates v nhng ngi khc cng ch nh sa ln men vi tnh cht dinh dng v dc tnh ca n, cha tr ri lon rut v d dy (Oberman, 1985) [10]. Cn i vi nh sinh l hc ngi Nga, Eli Metchnikoff (1907) th "S ph thuc ca h vi sinh vt trong rut i vi thc phm lm cho n c kh nng chp nhn bin php thay i h vi sinh vt trong ngi ca chng ta, tc l thay th vi sinh vt c hi bi vi sinh vt hu ch " [21]. Mt vi nm trc bi tho lun cu Metchnikoff, Pastuer v Joubert (1877) [10], trong khi quan st s i khng gia cc chng vi khun, nhn thy s tiu th vi khun khng gy bnh c th kim sot cc vi khun gy bnh. Ngoi ra, cng thi gian ny, Henry Tissier, mt bc s khoa nhi ngi Php phn lp Bifidobacterium, mt thnh vin ca nhm vi khun lactic, t phn ca tr c nui bng sa m (Tissier, 1906) [18] v nhn thy chng l mt thnh phn ni bt ca h vi sinh vt rut (Ishibashi v Shimamura, 1993). Nh vy nn tng cho khi nim hin i v probiotic c thnh lp t rt lu v vic nghin cu v vic s dng vi khun lactic trong ch n ung c tip tc sut cc th k va qua. Vy th probiotic l g? T probiotic c ngun gc t Hy Lp c ngha l cho cuc sng. Tuy nhin, nh ngha v probiotic pht trin rt nhiu theo thi gian. Lily

21

v Stillwell (1965) m t trc tin probiotic nh hn hp c to thnh bi mt ng vt nguyn sinh c tc dng thc y s pht trin ca i tng khc [21]. Sau , Parker (1974) p dng khi nim ny i vi phn thc n gia sc c mt nh hng tt i vi c th vt ch bng vic gp phn vo cn bng h vi sinh vt trong rut ca n. nh ngha chung ny sau chnh xc hn bi Fuller (1989), ng nh ngha probiotic nh mt cht b tr thc n cha vi sinh vt sng m c nh hng c li n vt ch bng vic ci thin cn bng h vi sinh vt rut ca n [21]. Khi nim ny sau c pht trin xa hn : vi sinh vt sng (vi khun lactic v vi khun khc, hoc nm men trng thi kh hay b sung trong thc phm ln men) m th hin mt nh hng c li i vi sc khe ca vt ch sau khi c tiu ha nh ci thin tnh cht h vi sinh vt vn c ca vt ch (Havenaar v Huis in't Veld, 1992) [21] [10]. Vi Tannock et al., 2000 th probiotic l vi sinh vt sng c li cho vng rut, gp phn vo vic cn bng h vi sinh vt ng rut v c tc ng tt n sc khe ca ngi tiu dng [38]. Tm li, l nhng vi khun tt sng trong ng tiu ha v ngay c trong m h. Th d in hnh v probiotic l vi khun Bifidobacterium v vi khun Lactobacillus trch t h vi sinh ng rut [19]. 2.3.2. Nhng tnh cht c li ca probiotic Probiotic c coi nh l mt thc phm chc nng rt c a chung bi cc tnh nng ca n mang li. Nhng tc ng ch yu ca cc chng vi khun probiotic ti sc khe con ngi gm nhng mt chnh nh sau: Ngn chn s xm nhp ca cc vi khun gy bnh bng cch cn chng bm vo thnh rut Probiotic khi c b sung vo c th, cc vi sinh vt ny sau khi vt qua cc ro cn v l hc v ha hc trong h d dy- rut non th chng s tin hnh bm dnh vo thnh rut thc hin cc chc nng c li cho c th vt ch. Chng cnh tranh ni vi vi khun gy bnh, ngn cn khng cho cc vi khun ny bm vo thnh rut, t lm gim s lng vi khun ngn chn cc mm bnh [21]. Gip cho vic tiu ha thc n c hu hiu hn

22

i vi nhng ngi thng b chng bt dung np ng lactose trong cc sn phm sa do trong rut khng c enzyme tiu ha loi ng ny th probiotic s gip h tiu ha c d dng hn [21]. Gim nguy c b tiu chy do ung nhiu thuc khng sinh Vic s dng probiotic phng nga tiu chy gy ra bi thuc khng sinh rt c quan tm. S pht trin mt cch rng ri v c hiu cao ca thuc khng sinh khin cho ngnh y hc qu tin tng vo phng php tr liu khng sinh. Tuy nhin, tc ng loi b ca thuc khng sinh khng phn bit c mm bnh tht s v h vi sinh vt c li trong rut. V vy, phng php tr liu khng sinh cng dn n thay i h cn bng ca rut v gy ra mt vi nh hng xu m c th tn ti lu di ngay c sau khi ngng s dng cch iu tr ny. Bn cnh , s xut hin nhanh ca nhiu chng vi khun khng li khng sinh chng hn nh Enterocci khng vancomycin v Staphylococcus aureus khng methicillin l mt vn ng quan tm. Hn na, mt vi bnh ly nhim li gia tng v tr nn trm trng hn trong iu tr thuc khg sinh. V d, bnh tiu chy c th do Clostridium difficile, mt mm bnh c hi, hot ng v tng nhanh khi c s mt cn bng ca h vi sinh vt trong rut thng thng din ra trong sut qu trnh iu tr khng sinh [21]. Mc d bnh ny ni chung c iu tr thnh cng vi mt loi thuc khng sinh th hai, tuy nhin mt vi bnh ly nhim vn ti din mc cho cc phng php tr liu khng sinh khc (Sanders, 1999). V sau vic s dng probiotic ci thin ng k h vi sinh vt ni sinh (Markowitz v Bengmark, 2002) bng cch thay th cc vi khun c ch b mt. So vi cc nhc im nh t, tnh ha hc v xm hi rut ca thuc khng sinh, u im ca probiotic l an ton, ngun gc t nhin, v phn ln khng c bt c nh hng c hi no. Ngoi tc dng gim nguy c b tiu chy do ung nhiu thuc khng sinh th vic s dng probiotic cn c tc dng ngn nga nguy c tiu chy gy ra bi cc loi virut gy bnh [21]. Tng cng h min dch

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Probiotic nh mt v thuc cha cc phn t chng vim cho ng rut. ng thi gp phn y mnh s bo hiu cho t bo vt ch gim cc phn ng vim bng cch to cc phn ng min dch cho c th. Nga ung th rut Mt vi nghin cu cho thy rng cc vi sinh vt probiotic c th ngn chn hoc lm chm qu trnh ung th rut. Theo nh cc ghi nhn trc th cc thnh vin ca h vi sinh vt ng rut c kh nng sn sinh ra cht gy ung th nitrosamines. Do d vic s dng Lactobacilii v Bifidobacterium v mt l thuyt c th gim bt c mc sn sinh ra cc cht gy ung th ny (Hosada et al, 1996). Tuy nhin cng qu sm a ra kt lun cui cng v hiu qu ca probiotic trong vic nga ung th rut[ 21] [34] Gim cholesterol trong mu. Hm lng cholesterol trong huyt thanh nh hng rt ln n cc bnh v tim mch. Nu hm lng ny cao hn 1mmol so vi mc bnh thng th ngi bnh ang mc nguy him ca cc bnh v tim mch (>35%) v c th gy cht ngi nu cao hn 45 %. V vy ch cn gim 1 % hm lng cholesterol trong mu l c th gim c mc nguy him ca cc bnh v tim mch t 2 n 3 % (Masnon etal, 1992) [27]. V vic s dng cc sn phm probiotic c kh nng lm gim hm lng cholesterol trong mu [34] [10]. Mt trong nhng bng chng u tin l vic s dng probiotic lactobaccilii trong cc sn phm c nh hng tt n tim mch, bao gm c kh nng ngn nga v iu tr cc hi chng thiu mu cc b ca tim (Oxman et al, 2001) v gim ng k hm lng cholesterol trong mu (De Roos v Katan) , 2000 [18]. Gim thiu hin tng d ng. Lb. rhamnousus GG c th nghim trn hai i tng c mc d ng cao l ph n 6 tun trc khi sinh v tr s sinh trong vng 6 thng. Kt qu cho thy c s gim c ngha v mc d ng (Kalliomaki et al , 2001). Nghin cu ny cho thy tim nng ca probiotic trong vic to ra cc phn ng gip c th chng li cc cht l xm nhp vo c th v ngn nga hin tng d ng xy ra. Trong cc nghin cu y hc khc v bnh d ng ca tr em vi sa b, ngi ta thy rng chng vim da di ng

24

ny c th gim bt khi s dng thc phm probiotic ca chng Lb. rhamnousus v B. lactics BB-12 (Majamaa v Isolauri, 1996; 1997; Isolauri et al, 2000) [18]. Ngoi cc ng dng chnh trn th probiotic theo nh cc nghin cu khc cn c kh nng thuyn gim triu chng ng rut ca hi chng rut b kch thch, nhim nm m o, nhim Helicobacter pylori d dy, bnh vim rut, v nhim trng ng tit niu [21] [34].

2.3.3. Cc tiu chun sng lc mt chng vi khun lm probiotic Khng phi bt c loi vi khun, nm men hay vi sinh vt n bo no cng c kh nng s dng lm probiotic. Chnh s lng v s a dng ca cc loi vi sinh vt lm cho vic sng lc tr nn kh khn. V vy qu trnh sng lc c hiu qu hn trong ng dng th i hi phi tin hnh c th nghim in vitro v in vivo [19]. Cc nghin cu in vitro c s dng nh gi cc c tnh ca vi sinh vt, lm c s cho vic sng lc cc chng c tim nng lm probiotic. Cn th nghim in vivo i hi nhiu thi gian v s lng ln ng vt cho vic th nghim, do , phng php ny ch c th s dng sau khi chn lc c mt s chng c tim nng lm probiotic (Martins v cng s , 2008) [31]. Nhng vi sinh vt c s dng lm probiotic phi chu s tc ng ng thi hay lin tip cc iu kin bt li nh sc nhit nh (nhit c th), tnh axit ca dch d dy, cc enzyme trong dch ty, lysozyme v mui mt (Klaenhammer v Kullen, 1999)[26] v kh nng c ch s pht trin ca cc vi sinh vt gy bnh, kh nng cnh tranh s bm dnh vi vi khun gy bnh v gim s bm dnh ca vi khun gy bnh vo ng rut.Trng hp probiotic khng c ngun gc t ng tiu ha ca ng vt th vn ny rt cn c quan tm. WHO v FAO trong tuyn b chung v tiu chun probiotic gii thiu mt s tiu chun ch yu nh sau [22]: - Kh nng chu pH thp ca d dy.

25

- Kh nng chu mui mt. - Kh nng phn gii mui mt. - Kh nng c ch vi khun gy bnh. - Kh nng bm dnh ng rut. - Kh nng bm dnh biu m m o (chuyn dng cho cho sn phm probiotic gip cn bng h vi sinh niu sinh dc. y cng l cc kim nghim in vitro ang c dng rng ri trong qu trnh sng lc cc chng vi khun lm probiotic trn th gii. Sau khi hon thnh cc th nghim in vitro chng ta s c c cc nhn xt s kho v c tnh ca cc chng ang test, lm c s cho qu trnh sng lc cc cc chng c tim nng probiotic [22] [28]. T tin hnh cc th nghim in vivo tip theo trn ng vt v i tng cui cng l con ngi [22]. 2.4. Kh nng tn ti v sinh trng ca vi khun lactic trong ng tiu ha ngi H tiu ha ca con ngi l mt h thng cc c quan, l mt ng di t ming n hu mn, gm thc qun, d dy, rut non, rut gi. Khi ta nut, n c thc qun dn xung d dy. D dy co bp, trn ln n vi dch v, gm axit v men tiu ha. Sau n c y xung rut non, di tc dng ca nhiu men tiu ha khc, thc n c chuyn thnh cc cht d hp thu hn v c rut non hp thu vo mu, cn cc cht b c y xung rut gi. Rut gi ht bt nc, lm c cht b, ng khun v co bp thi cht b ra ngoi. Tuy nhin i km vi thc n vo ming l rt nhiu loi vi khun k c vi khun c li v c hi t mi trng. Cc nghin cu cng cho thy rng vi khun lactic l nhng vi sinh vt c s lng v kh nng tn ti trong rut ngi kh cao. in hnh l cc vi khun lactic 2 chi Bifidobacterium v Lactobacillus [19] [21]. Rt nhiu nghin cu cho thy vi khun lactic l nhm vi khun c li c b sung vo c th dui dng probiotic [30] [13]. Tr ngi u tin ca cc chng probiotic ny l dch axit trong d dy, n c th hy dit mt s lng ln cc vi sinh vt i vo trong c th. c th tn ti c i hi cc chng probiotic phi cn sng sau 2gi - 4gi trong d dy di iu kin

26

pH thp. Trong trng hp nhng vi khun ny vt qua c ro cn ha hc l lp dch c tnh axit tng i mnh ca d dy th mi tng tc gia chng vi cc vi khun trong rut cng s lm hn ch s ly lan ca vi khun xm nhp c hi. Sau l kh nng chu mui mt trong rut non cng nh cc enzyme trong rut non. Nhng dng c xem l khng mui mt tt l nhng dng c kh nng tng trng tt trong mi trng c cc nng mui mt trong khong 0,15 0,3% [33]. 2.5. Tnh hnh nghin cu kh nng chu axit v mui mt ca vi khun lactic trn th gii Hin nay, c rt nhiu nghin phn lp v tuyn chn cc dng vi khun lactic lm probiotic [10] [36]. V mt trong nhng tiu chun quan trng nht c ch nghin cu khi chn lc chng probiotic l kh nng sng st v thch nghi trong ng tiu ha, vi 2 c im ni bt l chu axit v chu mui mt [ 35] M. T. Liong v N. P. Shah (2005) [33] tin hnh kho st kh nng chu axit v mui mt ca 11 chng Lactobacillus v cho kt qu kh quan v kh nng chu axit v mui mt ca cc chng ny, thi gian trung bnh tng OD ln 0,3 n v trong khong t 2,3 n 4,6 gi (tnh trong pha logarit). Kh nng sng st pH 2 gim rt mnh v thay i ln ty theo chng. Sau 120 pht pH 2, hai tc gi ny nhn thy lng t bo sng gim t khong 10 (log CFU/ml) xung cn t 4,2 n 7,5 (log CFU/ml) ty theo chng . Suskovic v cng s (2000) [22] tin hnh nghin cu nh hng ca cc loi mui mt ln kh nng tng trng ca chng probiotic M92, kt qu cho thy chng b gim khong t 40-60 % kh nng tng trng trong mi trng mui mt. Khalil v cng s (2007) [24] cng kho st tim nng probiotic ca mt s chng vi khun lactic phn lp Ai Cp, kt qu cho thy kh nng chu axit v mui mt tt nht ri vo cc chng thuc cc loi Bifidobacterium longum, Lb. fermentum, Lb. plantanum.

27

2.6. Tnh hnh nghin cu kh nng chu axit v mui mt ca vi khun lactic vit Nam. Vit Nam, s lng cc nghin cu v kh nng chu mui mt cn t v phm vi nghin cu cn hn ch. Cc nghin cu in vitro v cc chng c tim nng probiotic nh ca H Trung Thng, H L Qunh Chu (2009) [7]. Nghin cu kh nng sng trong mi trng ng tiu ha ca ng vt mt s chng vi sinh vt nhm tng bc chn lc to nguyn liu sn xut probotic. Nhm tc gi phn lp c mt s chng nm men c kh nng thch nghi cao vi cc iu kin ng rut v c th lm probiotic. Tuy nhin, hin nay cc sn phm probiotic c cha vi khun lactic vn c a chung hn do c nhiu u im vt tri. Cc tc gi Nguyn V Tng Vy, Nguyn Vn Thanh, Trn Thu Hoa (B mn Vi sinh K sinh Khoa Dc, H Y Dc TP.HCM) (2007) [9] thc hin kho st vi sinh vt probiotic gm vi khun Lactic (Lactobacillus aci-dophilus, Lactobacillus casei, Streptococus fae-calis), vi khun to bo t Bacillus subtilis v nm men Saccharomyces boulardii nhm kho st kh nng chu ng axit, mui mt v khng sinh ca mt s vi sinh vt l nguyn liu sn xut probiotic dng ng ung. Tuy nhin, cc tc gi vn cha phn lp c cc chng sng st c pH 2 sau 2 gi. Kt qu cho thy sau 2 gi tt c cc chng u mt kh nng sng pH 2. Nh vy Vit Nam mc d cha c nhiu nghin cu nhng thng qua cc nghin cu trn cho thy probiotic tr thnh mt i tng c ch nhiu trong cng ng khoa hc v sn xut. Vit Nam c mt tim nng rt ln v a dng vi sinh vt cha c khai thc. Trong , Hu vn ni ting vi cc sn phm ln men chua lactic truyn thng, v c th tim n nhng chng vi khun lactic c tim nng probiotic mnh. Do , chng ti chn ti Tuyn chn chng c kh nng chu axit v mui mt cao t cc chng vi khun lactic trong mt s sn phm ln men lactic trn a bn thnh ph Hu nhm s b kho st mt s c im ban u chn lc cc dng vi khun lactic c tim nng probiotic.

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PHN 3 I TNG V PHNG PHP NGHIN CU 3. 3.1. i tng nghin cu i tng nghin cu l 12 chng vi khun lactic phn lp t cc sn phm ln men lactic truyn thng (tm chua, nem chua, da ci chua, mng chua, nc chua trong sn xut u ph) trn a bn thnh ph Hu, c phn lp v nh danh mc chi ti phng th nghim Cng ngh vi sinh, Vin Ti nguyn mi trng v Cng ngh sinh hc Hu. (Bng 3.1, Hnh 3.1) Bng 3.1. Cc chng vi khun dng trong th nghim STT 1 2 3 4 5 6 7 8 9 10 11 12 Dch Whey u ph Mng chua Nem chua Da ci chua Tm chua Ngun phn lp Tn chng Catalase/Gram Kt qu nh danh T9 T10 N1 N5 N10 DC1 DC2 MC5 MC9 MC10 NC10 NC13 -/+ -/+ -/+ -/+ -/+ -/+ - /+ - /+ - /+ - /+ - /+ - /+
+ : Gram dng tnh

Lactobacillus Lactobacillus Lactobacillus Lactobacillus Leuconostoc Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus

- : Catalase m tnh

29

DC1

DC2

N1

N5

N10

NC10

30

NC13

MC5

MC9

MC10

T9 T10 Hnh 3.1 Hnh thi t bo v nhum gram ca cc chng vi khun lactic s dng trong th nghim

31

3.2. Thit b ,ha cht s dng 3.2.1. Thit b T m Incubactor MiR.262 - Nht T lnh Sanyo - Nht. My o pH Hanna - . My o quang ph quang ph k UV/VIS - Nht. T cy Labgard - Nht V mt s thit b thng dng ca phng th nghim. 3.2.2. Ha cht NaH2PO4.H2O Na2HPO4.7H2O Pepton NaCl HCl No.3, Merck ( Sodium chocolate v sodium deoxycholate) Cc ha cht c cung cp bi Meck 3.3. Phng php nghin cu 3.3.1. Phng php hot ha ging Nguyn tc : Ging ban u thng c bo qun trong iu kin lnh -30 oC. Khi s dng nui cy v tin hnh cc th nghim tip theo cn phi hot ha ging c c kh nng sinh trng v pht trin bnh thng. Tin hnh: Ly 100l dch r ng ca ging vi khun cho vo eppendoft c cha 900l dch pepton 0,1%. em trong 1gi t m 370 C. Sau s dng cy chuyn qua cc mi trng phc v nghin cu. Thnh phn dch pepton 0,1%; pH 7,2 Pepton 1g NaCl 0,5g Nc ct 1lt 3.3.2. Phng php nui cy tng sinh

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Nguyn tc: Trong mi trng lng vi khun c kh nng tip xc cc cht dinh dng d dng hn, kh nng pht trin n nh v t thu c sinh khi cho cc nghin cu tip theo. Tin hnh: Dng que cy trn ly 1 khun lc n nh u vo eppendoft c cha 1 mL MRS lng, nui t m 37 0C trong 24 gi. Bng 3.2. : Thnh phn mi trng MRS rn/Lt [7, 37] ( Mi trng MRS lng khng b sung agar) Thnh phn S lng (g) Pepton 10 Cao tht 8.0 Cao nm 4.0 Glucose 20.0 Sorbitan monooleate (Tween 80) 1.0 K2HPO4 2.0 MgSO4.7H2O 0.2 MnSO4 .4H2O 0.05 (NH4 )2 C6H6O7 2.0 CH3COONa.3H2O 5.0 Agar 15.0 Nc ct 1l S dng HCl 1N v NaOH 1N iu chnh pH mi trng v 6,2. Mi trng c thanh trng 121oC trong 15 pht. 3.3.3. Phng php bo qun ging Ging c bo qun nhm mc ch s dng thng xuyn trong nghin cu th nghim v bo qun lu di [4] [17]. bo qun ging cc chng vi khun lactic, chng ti tin hnh nh sau: * Bo qun thng xuyn: ging c cy chuyn trong eppendorf c cha 1ml mi trng MRS lng v 37oC trong 24 gi, sau c gi lnh 4oC. Ging bo qun c th c s dng cy tng sinh thu sinh khi

33

nghin cu m khng cn phi hot ha, tuy nhin ging ny ch s dng trong khong 10 ngy v phi thng xuyn cy chuyn. * bo qun ging lu di chng cng c nui tng sinh ging nh bo qun thng xuyn, sau li tm thu sinh khi tc 5000 vng/pht, loi b dch ni, b sung 1ml mi trng MRS c glycerol (30%) vo eppendoff v gi -30oC. 3.3.4. Phng php xc nh mt vi khun trong mi trng lng 3.3.4.1. Phng php m khun lc trc tip (phng php Koch) Nguyn tc : Mu c cha sinh khi vi sinh vt khi c pha long nng thch hp v trang ln mi trng rn th mi t bo vi khun s pht trin thnh mt khun lc n. Do , m s khun lc n s xc nh c s t bo ban u trong dch pha long v qua tnh c s t bo ban u trong dch gc. S t bo c xc nh bng n v log CFU/ml. Tin hnh : Mu c pha long lin tip theo t l pha long 10 ln thnh cc mu t 100 n 10-5 bng pepton 0,1% v ly 50l trang trn a petri ng knh 10cm c cha mi trng MRS. Mu c 48h 37oC v m s khun lc n. Kt qu c tnh nh sau : Log CFU/ml = log (Ai . Di / V) (3.1) Trong : Ai l s khun lc trung bnh trn mt a Di l pha long V l dung tch huyn ph cho vo mi a 3.3.4.2. Phng php o mt quang [1] [8] Nguyn tc : Khi nh sng i qua mi trng lng c nhiu ht rn l lng s b tn x tr li v dn n cng tia sng l ra thp hn so vi cng tia sng i vo, vic so snh chnh lch cng tia sng s phn nh mt cch tng i lng cht rn l lng trong mi trng. Ngoi ra, chnh bn thn cht lng cng hp th mt phn nh sng, do , lng nh sng i qua cng b nh hng bi bn cht ca mi trng lng. nh gi mt t bo trong dch nui cy, ta c th xem t bo nh cc ht rn l lng, v tin hnh o mc hp th ca dch c t bo nui cy, i

34

chiu vi mi trng lng khng c sinh khi s cho ra lng tng i ca sinh khi vi khun. Lng nh sng b hp th t l tuyn tnh vi mt t bo theo nh lut Lumber-Beer, iu kin nghim ng ca nh lut l mc OD thp (di 1), do , cn tin hnh pha long mu o lun c OD thp hn 1. OD tht s ca dch c tnh li bng cch nhn vi pha long. Tin hnh : Chng vi khun c nui trn mi trng MRS lng trong eppendorf 1ml. Sinh khi c xc nh bng cch o OD bc sng 600nm vi mu trng (blank) l mi trng MRS khng nui cy. Mu c pha long (nu cn) bng mi trng MRS lng. 3.3.5. Phng php kho st kh nng chu mui mt [33] [11] Nguyn tc: Kho st kh nng chu mui mt ca cc chung vi khun lactic trong mi trng MRS lng c b sung 0,3% mui mt (No.3, Merck) da trn phng php ca Gilliand v Walker (1990) [15]. Cc bc tin hnh: Qu trnh kho st c tin hnh theo cc bc sau : 1. Hot ha ging gc chng vi khun cn kho st (phn 3.3.1) 2. Tin hnh nui cy tng sinh (phn 3.3.2) 3. Tin hnh phn phi ging sau khi tng sinh vo cc eppendoff (t l th tch 1/10) trong mi trng MRS lng c b sung mui mt 0,3 %. 4. cc eppendoff c cha dch vi khun trn 370C v tin hnh ly mu 0 gi, 4 gi (mi gi 3 eppendoff ) 5. o OD sau mi mc gi ti bc sng 600nm v tnh kt qu theo cng thc 3.2 Tnh kt qu : Mc chu mui mt c x nh bng thi gian trung bnh tng OD ln 0,3 n v
t= 4 0,3 (gi) OD 4 OD 0

(3.2)

Trong :

t : Thi gian tng OD ln 0,3 n v. OD4: OD mc thi gian 4 gi. ODo: OD mc thi gian 0 gi.

35

3.3.6. Phng php kho st kh nng chu axit [33], [11] Nguyn tc: Kh nng chu axit ca cc chng kho st c nh gi qua lng vi khun sng st sau khi pH 2, s t bo vi khun sng st c xc nh theo phng php Kock (Do lng t bo sng st t nm di ngng o OD). Tin hnh: Qu trnh kho st c tin hnh theo cc bc sau 1. Hot ha ging gc chng vi khun cn kho st (phn 3.3.1) 2. Tin hnh nui cy tng sinh (phn 3.3.2) 3. Ly tm thu sinh khi 500 vng/pht trong 10 pht, nhit phng 4. Ra sinh khi bng m phosphate 0,1M pH 7 2 ln (1 ln 1ml m/eppendoff) 5. Ti huyn ph trong 1ml m trn bng votex 6. Trn 1ml dch sau khi ti huyn ph vi 24,5 ml dung dch c pH = 2 (iu chnh dung dch NaCl 0,2 % n pH = 2 bi HCl) 7. Phn phi vo 12 ng eppendoff (1ml dch ti huyn ph/ng eppendoff) 8. Tin hnh cc eppendoff c cha dch trn trn cc mc thi gian khc nhau (0 gi, 1gi, 2 gi, 3 gi) 9. Ly mu sau mi mc gi (mi gi 3 eppendoff), pha long tng eppendoff thnh cc nng khc nhau v chn 3 nng thch hp dn u (50l) ln a thch MRS pH 6,2 (3 a thch cho 1 eppendoff) 10. cc a peptri 370 C trong vng 48 gi 11. Tin hnh m s lng khun lc v tnh kt qu theo cng thc 3.1 3.3.7. Phng php xy dng ng cong sinh trng Cy chuyn ging qua 24 gi nui cy vo cc eppendoff c cha 1ml mi trung MRS lng pH 6,2 sao cho OD ban u t 0,05 bc sng 600 nm. mu trong t n nhit 37oC v ly mu theo thi gian tng gi 1 trong 8 gi u, 2 gi mt ln trong 32 gi tip theo v 8 gi mt ln trong 48 gi sau , mu c o sinh khi thng qua OD bc sng 600nm.

36

3.3.8. Phng php x l s liu [3] S liu c tnh trung bnh, lch chun v x l ANOVA bng chng trnh SAS 9.0.

37

PHN 4 KT QU V BIN LUN 4. 4.1. Kt qu kho st kh nng chu mui mt Havenaar v cng s (1992) [18] ch ra rng probiotic ch pht huy tc dng c li ln vt ch khi chng nh c v tn ti trong rut non. Mi trng rut non cha pancreatine v mui mt l cc yu t c ch sinh trng ca vi sinh vt. Theo Gilliland v cng s (1984) [15], 0,15 - 0,3% c xem l nng quyt nh sng lc cc chng vi sinh vt c kh nng chng chu mui mt. Cc chng kho st c nui cy tng sinh trong mi trng MRS lng trong 24 gi v c cy chuyn vo mi trng MRS c b sung 0,3% mui mt (No.3, Merck). Mu c ly sau khi 370C 0 gi v 4 gi, o OD bc sng 600nm v kt qu c tnh theo cng thc 3.2. Kh nng chu mui mt c nh gi thng qua thi gian tng OD ln 0,3 n v. Thi gian cng ngn th chng t chng c kh nng thch nghi tt v pht trin c trong mi trng c cha mui mt (Hnh 4.1). Kt qu cho thy tt c cc chng kho st u c kh nng tng trng c trong mi trng mui mt 0,3%, trong cc chng DC1, MC9, MC10, DC2, MC5 c kh nng chu mui mt cao nht, tc tng OD ln 0,3 ca chng l khong t 2,34 n 3,24 gi. Cc chng N1, N10, T9, T10, N5 c tc tng OD 0,3 trong mi trng mui mt t trung bnh, t khong 3,66 gi n 4,31 gi. Nhm c kh nng khng mui mt km l N5, NC13 v NC10, cc chng ny cn hn 5 gi gia tng OD ln 0,3 n v trong pha logarit.

38

Hnh 4.1. Thi gian tng OD 0,3 ca cc chng vi khun lactic trong mi trng MRS c 0,3% mui mt
a, b, c, d, e: Cc mc khc bit c ngha khi x l ANOVA. Cc gi tr c cng ch ci xem nh khng c s khc bit mc ngha p = 0,05.

Kt qu ny tng ng vi kt qu ca Liong v cng s (2005) [33]. Nhm tc gi ny tin hnh kho st kh nng tng trng ca vi khun lactic trong mi trng MRS c 0,3% dch mt b, kt qu cho thy thi gian tng trng ca cc chng kho st trong khong 2,36 (Lb. casei ASCC 1520) n 4,36 gi (Lb. casei ASCC 290). Chng DC1, MC9 trong th nghim ca chng ti cng c thi gian tng trng tng ng vi nhm khng mui mt cao nht ca nhm tc gi ny. Lin v cng s (2007) [28] khi nghin cu kh nng chu mui mt b (0,3%) ca cc chng vi khun Lactobacillus fermentum phn lp t ln cho thy sau thi gian tng OD 0,3 sau 4 gi kho st l vo khong t 1,3

39

gi n 4,1 gi, trong s 20 chng c kho st c n 10 chng c tc tng OD ln 0,3 di 2 gi, mt chng tng mnh nht, ch mt trung bnh 1,3 gi tng OD ln 0,3 n v, c hai chng hu nh khng pht trin c trong mi trng c mui mt (OD khng thay i ng k), cc chng cn li c thi gian tng OD 0,3 vo khong 3 n 4 gi . Mota v cng s (2006) [32] cng tin hnh nghin cu kh nng khng mui mt ca 3 chng vi khun Lb. acidophilus phn lp t g, kt qu c 1 chng hu nh b c ch hon ton khi c dch mt b nng 0,3% l Lb. acidophilus 2M14E, hai chng cn li c tc tng trng tng i nhau l Lb. acidophilus 5C14E v Lb. acidophilus 2G14E vi thi gian tng OD 0,3 n v vo khong 2,1 n 3 gi. T cc nghin cu trn, c th nhn thy kh nng khng mui mt ca vi khun lactic bng thng s thi gian tng OD ln 0,3 n v trung bnh vo khong 2 5 gi, nhng chng khng mnh c thi gian khong t 2 n 3 gi, v nhng chng khng km c thi gian trn 4 gi. So snh vi mt s nghin cu nh trn, chng ti nhn thy cc chng DC1, MC9, MC10, DC2, MC5 c kh nng chu mui mt tt. Trong cc chng DC1, MC9 c kh nng khng mui mt cao. Cc chng MC10, DC2, MC5 c kh nng khng mui mt khong gia cao v trung bnh (thi gian gn bng 3). Vi mc tuyn chn dng tim nng protiotic mnh v th chng ti chn cc chng khng cao (DC1, MC9) v cn cao (MC10, DC2, MC5) tip tc kho st kh nng chu axit mc pH 2. 4.2. Kho st kh nng chu axit Chng ti tin hnh kho st kh nng chu axit ca cc chng vi khun DC1, MC9, MC10, DC2, MC5 qua cc mc gi lin tc t 0 gi n 3gi trong dch axit c pH 2. Chng kho st c nui trong mi trng MRS lng trong eppendort 37oC trong 24 gi. Tin hnh ly tm thu sinh khi v vo dch pH 2. Mu c ly vo 0 gi, 1 gi, 2 gi, 3 gi v pha long cc nng khc nhau, dn u cc nng thch hp (50l) ln a thch MRS. Kt qu c xc nh thng qua lng t bo cn sng st sau 3 gi nui cy bng phng php Koch theo cng thc 3.1.

40

S lng khun lc n m c qua 4 mc gi nui cy th hin cc hnh t 4.3 n 4.7 v kt qu th hnh 4.2, cho thy thi gian mu cng lu th t l sng st ca cc chng cng gim nhng tt c u c kh nng chu axit v sng st trong vng 3gi ti pH 2. Trong chng MC9 l chng c kh nng chu cao nht. Lng t bo ban u ca cc chng kho st rt cao (ln hn 108 CFU/ml ) v chnh lch nhau khng ng k. gi nui u tin th lng t bo bt u gim tt c cc chng DC2 cn 7,52 logCFU/ml ( gim 1,269 logCFU/ml), MC5 l 7,67 logCFU/ml ( gim 1,116 logCFU/ml), MC10 l 7,62 logCFU/ml ( gim 1,123 logCFU/ml), gim nhanh nht l DC1 ch cn 6,7 logCFU/ml ( gim 2,207 logCFU/ml ). Trong khi chng MC9 vn ang cn 8, 51 logCFU/ml tc l ch gim c 0,237 logCFU/ml vi t l sng st cao rt nhiu so vi cc chng cn li (Ph luc 1). Tng t mc 2 gi lng t bo vn tip tc gim tt cc chng. Gim nhiu nht l DC1 cn 6,09 logCFU/ml v MC10 cn 6,16 logCFU/ml, t l sng st ca 2 chng ny gim nhanh ch cn 0,15% (DC1), 0,26%( MC10) ( Ph luc 1). Trong khi dn u vn l chng MC9 vi t l sng st sau 2 gi l 4,29% tng ng vi 7,35 logCFU/ml. mc 3 gi cng vy lng t bo ca cc chng gim nhanh nm trong khong 5,12 - 6,57 logCFU/ml. V mc gi ny chng MC9 vn l chng c kh nng chu mnh nht. Nh vy trong c 3gi th chng MC9 l chng c kh nng chu axit tt nht t l sng st l 0.92 %, trong khi cc chng khc t l sng st u rt thp.

41

Hnh 4.2. Kh nng sng st ca cc chng vi khun lactic pH 2 Zhou v cng s (2007) [32] cho rng gi tr pH 2 v pH 3 c xem l gii hn quyt nh trong sng lc cc chng vi sinh vt c tim nng s dng lm probiotic. Kt qu nghin cu ca Kim v cng s (2007) [25] cho thy khi x l bng dch d dy pH = 2,5, c 3/7 chng vi khun c kh nng chu mi trng axit, tuy nhin, t l sng ca cc chng ny gim mnh ch cn 0,8% n 8% sau 30 pht x l v tip tc gim mnh sau 2 gi x l (t 0,04% n 0,2% so vi ban u). Theo nghin cu ca M. T. Liong v N.P. Shah [27] trn 11chng lactic bao gm Lb.acidophilus v Lb.casei kt qu cho thy c Lb. acidophilus v Lb.casei u c kh nng chu axit sau 2 gi nui cy. Trong Lb. acidophilus ATCC 4962, Lb.casei ASCC 290 v Lb.casei ASCC 292 l nhng chng chu tt nht khi gim t khong 1010cfu/ml (10 logCFU/ml) xung khong hn 107 CFU/ml (khong 1000 ln), c mt s chng ch cn 10 4

42

CFU/ml (1 triu ln). Nh vy, kh nng chu axit ca cc chng vi khun lactic thay i rt ln theo chng. Kt qu nghin cu ca Liu v cng s (2007) [29] cho rng Lactobacillus l chi lactic c kh nng chu axit rt tt trong chng Lb. acidophilus GG (ATCC 53103) l chng duy nht c phn lp t phn ngi c kh nng chu axit vi s lng cn li sau 2 gi ti pH t 1-2 l khong 103 CFU (3 logCFU/ml). Maragkoudakis v cng s (2006) [30] cng kho st kh nng chu axit ca mt s chng Lactobacillus, kt qu cho thy cc chng c kh nng khng axit mnh nht l Lb. paracasei subsp. paracasei ACA-DC 130, Lb. plantarum ACA-DC 146, Lb. rhamnosus ACA-DC 112, vi mc gim logCFU/ml t 8,6 xung cn ln lt l 6,8; 5,7 v 7,1 sau 3 gi pH 2, ngoi ra, mt s chng trong nghin cu ca nhm tc gi ny khng c kh nng sng st pH 2 sau 1 gi. Vit Nam Nguyn V Tng Vy v cng s (2007) [9] li khng pht hin c mt chng vi khun lactic no c th chu c pH 2 sau 2 gi. Nh vy theo nh cc nghin trn th cc chng lactic u sng trong iu kin pH d dy v s lng chng gim nhanh theo thi gian, iu ny hon ton ph hp vi cc chng DC1, DC2, MC5, MC9, MC10. i chiu cc nghin cu trn th cc chng ny c kh nng chu axit tt sau 2 gi vn cn tn ti 106 107 CFU vi t l sng st l 0,26 4,29 %, 3gi l 105 106 CFU t l sng l 0,03 0,92 %. Kt hp 2 tiu ch kh nng chu axit v mui mt cho thy c 4 chng c tim nng tt l MC9, MC5, DC1, DC2, cc chng ny c th tip tc tin hnh cc kho st su hn sng lc dng c tim nng probiotic. Qua chng ti nhn thy chng MC9 c kh nng khng li mui mt v axit tt nht. Chng ny c la chn tin hnh cc kho st tip theo v kh nng sinh trng v nh danh.

43

Hnh 4.3 Hnh nh minh ha kh nng chu axit ca chng DC1 trn a peptri
A. Mu 0 gi B.Mu 1 gi C. Mu 2gi D. Mu 3 gi

44

Hnh 4.4 Hnh nh minh ha kh nng chu axit ca chng DC2 trn a peptri
A. Mu 0 gi B.Mu 1 gi C. Mu 2gi D. Mu 3 gi

45

Hnh 4.5 Hnh nh minh ha kh nng chu axit ca chng MC5 trn a peptri
A. Mu 0 gi B.Mu 1 gi C. Mu 2gi D. Mu 3 gi

46

Hnh 4.6 Hnh nh minh ha kh nng chu axit ca chng MC9 trn a peptri
A. Mu 0 gi B.Mu 1 gi C. Mu 2gi D. Mu 3 gi

47

Hnh 4.7 Hnh nh minh ha kh nng chu axit ca chng MC10 trn a peptri
A. Mu 0 gi B.Mu 1 gi C. Mu 2gi D. Mu 3 gi

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4.3. Xc nh kh nng sinh trng v tch ly sinh khi ca MC9 Qu trnh thu sinh khi ca chng vi khun probiotic c ngha rt ln trong sn xut cc ch phm probiotic. Sinh khi ca vi khun thay i theo thi gian, gm 4 pha l pha thch ng, pha logarit, pha n nh v pha suy tn [5]. Vic nm c thi gian ca cc pha c ngha ln trong sn xut, nh xc nh c thi im thu sinh khi cao nht v tit kim c thi gian nui cy v gim chi ph sn xut. Sinh khi ca chng lm probiotic thng c thu vo cui pha logarit, do lng sinh khi cao v t bo pha ny s nhanh chng thch nghi v tng trng khi a vo mi trng mi. tin hnh kho st ng cong tng trng ca chng MC9 v xc nh thi im thu sinh khi ti u, chng ti tin hnh cy mu vo mi trng MRS lng, vi mc pha long nhm t c OD khi im vo khong 0,05. Mu c 37oC trong eppendorf 1ml (khng lc). Tin hnh ly mu mi gi 1 ln trong 8 gi u, 2 gi mt ln trong 32 gi tip theo v 8 gi mt ln trong 48 gi cui. Xc nh sinh khi mu bng cch o OD bc sng 600nm.
7 6 5

OD 600 nm

4 3 2 1 0
0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80 84 88 92 96 100

Thi gian (gi )

Hnh 4.8. ng cong tng trng ca chng MC9

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Kt qu cho thy trong 14 gi u sinh khi tng nhanh mt cch ng k c bit l t 4 gi n 14 gi. cc mc gi cn li th mt t bo tip tc tng chm cho n 96 gi. Nh vy trong khong thi gian t 0 - 4 gi l giai on tim pht ca qu trnh sinh trng v pht trin t bo trong iu kin nui cy tnh v vy trong khong thi gian ny mt t bo c tng nhng cha r rt. Ch khi qu trnh i vo pha logarit (4 gi -14 gi) th vi khun pht trin v sinh trng theo ly tha dn n tc tch ly sinh khi tng nhanh mt cch r rt. T 14 gi n 96 gi sinh khi c tng nhng rt chm, y l pha n nh ca chng MC9 (Hnh 4.8). Kt qu trn kh ng nht vi mt s nghin cu c kho st ng cong tng trng ca Lactobacillus spp., Dumbrepatil (2008) v cng s [13] cng kho st kh nng sinh trng ca Lactobacillus delbrueckii trong 40 gi. Nhm tc gi cng nhn thy rng chng Lactobacillus delbrueckii tc sinh trng tng nhanh trong 12 gi u, n 14 gi i vo pha n nh . Pha n nh trong ng cong tng trng ca vi sinh vt thc cht l mt s cn bng ng khi lng vi khun cht i bng vi lng sinh ra, nn cn bng ny khng ng yn m c s thay i nh, c th tng nh hay gim nh hoc lc gim lc tng ty c im ca chng. Da vo kt qu trn, chng ti xc nh thi im thu sinh khi tt nht ca chng MC9 trong mi trng MRS lng, nui k kh l 14 gi. 4.4. Kt qu nh danh Chng MC9 c nh danh bng phng php gii trnh t mt phn gen m ha trnh t cho tiu phn ribosome 16S (rRNA 16S), kt qu c tra cu trn ngn hng gen ca NCBI thng qua chng trnh search BLAST v da vo s tng ng trong trnh t ca on gen nh danh n loi. Chng c gii trnh t rRNA 16S ti Phng th nghim ca cng ty Nam Khoa, TP H Ch Minh, kt qu trnh by hnh 4.9.

50

Hnh 4.9. Trnh t mt on gen rDNA 16S ca chng MC9 v Lactobacillus fermentum SFCB2-3
Query: chng truy vn: Lactobacillus fermentum SFCB2-3 Sbjct: chng Lactobacillus spp. MC9

51

16s Ribosome RNA gen Chng Lactobacillus MC9

Hnh 4.10. Cy phn loi da trn trnh t rRNA 16S

52

Qua tra cu ngn hng d liu trnh t gen trn NCBI, chng ti nhn thy trnh t gen rRNA 16S ca chng MC9 c s tng ng cao nht vi chng Lactobacillus fermentum SFCB2-3 (99%), mc tng ng th hin trong hnh 4.9. Kt qu so snh trong cy phn loi da trn s tng ng trnh t rRNA 16s cng cho thy chng ny c quan h gn gi v mt di truyn vi nhiu chng Lactobacillus fermentum khc (Hnh 4.10). T chng ti kt lun chng MC9 thuc loi Lactobacillus fermentum.

53

PHN 5 KT LUN V KIN NGH 5. 5.1. KT LUN T nhng kt qu nghin cu, chng ti rt ra nhng kt lun sau - 12 chng vi khun lactic DC1, DC2, MC5, MC9, MC10, N1, N5, N10, T9, T110,NC10, NC13 u c kh nng pht trin trn mi trng MRS c b sung mui mt trong 4 gi, cao nht l DC1 v thp nht l NC10. - 5 chng chu mui mt cao nht trn l DC1, DC2, MC5, MC9, MC10 u c kh nng sng st pH 2 sau 3 gi nui cy. Chng chu axit tt nht l MC9 v km nht l MC10. - Thi im thu sinh khi tt nht ca chng MC9 trn mi trng MRS 37oC l sau 14 gi nui cy. - Chng MC9 thuc loi Lactobacillus fermentum l chng c tuyn chn trong qu trnh nghin cu. 5.2. KIN NGH - Tip tc nghin cu cc tnh cht sng lc chng lm probiotic v nh danh cc chng DC1, DC2, MC5, MC10. - Tin hnh cc th nghim v tc dng ca cc chng trn trn ng vt. - Nghin cu kh nng ng dng chng MC9 vo sn xut.

TI LIU THAM KHO

54

Ting Vit
1. Nguyn Ln Dng, Mt s phng php nghin cu vi sinh vt hc, Nh

2.
3.

4.
5. 6.

7.

8.
9.

xut bn khoa hc v k thut, H Ni, 1978. Nguyn Thnh t, C s sinh hc vi sinh vt, Nh xut bn gio dc, H Ni, 2001. Nguyn Thnh t (Ch bin), Mai Th Hng (2001), Sinh hc vi sinh vt, NXB gio dc. Lng c Phm, Cng ngh vi sinh vt, NXB Nng Nghip H Ni, 1998. L Xun Phng, Vi Sinh Vt Cng Nghip, NXB xy dng, H Ni, 2001 Nguyn Th Trang, Trn nh Mn, Mt s c im phn loi ca hai chng vi khun lactic HN11 v HN34 sinh tng hp L(+)-Lactic axit phn lp ti Vit Nam, Tp ch Cng ngh sinh hc 6(4), 2008,505-511. H Trung Thng, H L Qunh Chu, Nghin cu kh nng sng trong mi trng ng tiu ha ca ng vt ca mt s chng vi sinh vt nhm tng bc chn lc to nguyn liu sn xut probotic, tp ch khoa hc i hc Hu, s 55, 2009. Trn Linh Thc, Phng php phn tch Vi sinh vt trong nc, thc phm v m phm, NXB Gio Dc. Nguyn V Tng Vy, Nguyn Vn Thanh, Trn Thu Hoa (2007), Kho st kh nng chu ng acid, mui mt v khng sinh ca mt s vi sinh vt l nguyn liu sn xut probiotic dng ng ung, Tp Ch Dc hc, (378), tr. 255-263.

Ting Anh

55

10. Analie Lourens-Hattingh, Bennie C. Viljoen, Yogurt as probiotic carrier

food, 2001, International Dairy Journal 11 (2001) 117. 11. Arvola T, Sutas Y, Moilanen E, and Salminen S, Probiotics in the management of atopic eczema, Clin. Exp. Allergy 2000, 30: 1604161. 12. Corzo G., and Gilliland S.E. , Bile Salt Hydrolase Activity of Three Strains, Oklahoma State University, Stillwater 74078. 13. Dumbrepatil A., Adsul M., Chaudhari S., KhireJ. , and Gokhale D. (2008), Utilization of Molasses Sugar for Lactic Acid Production by Lactobacillus delbrueckii subsp. delbrueckii Mutant Uc-3 in Batch Fermentation, Applied and environmental microbiology, Jan. 2008, Vol. 74, No. 1, p. 333335 [ac6] 14. Fuller, R. Probiotics. J. Appl. Bacteriol. Symp. 1986, (Suppl 61), 1S7S. 15.Gilliland, S. E., and D. K. Walker. 1990. Factors to consider when selecting a culture of L. acidophilus as a dietary adjunct to produce a hypercholesterolemic effect in humans. J. Dairy Sci.73:905909. 16. Gunter Klein, Alexander Pack, Christine Bonaparte1, Gerhard Reuterk, Taxonomy and physiology of probiotic lactic axit bacteria, International Journal of Food Microbiology, 1998, 103-125. 17. Gyu Sung Cho and Hyung Ki Do, Isolation and Identification of Lactic Axit Bacteria Isolated from a Traditional Jeotgal Product in Korea, Ocean Science Journal, Vol. 41 No. 2, 2006,113-119. 18. Havenaar, R., Huis int Veld, J.H.J. Probiotics: a general view. In: Wood, B.J.B. (ed) The lactic acid bacteria in health and disease. The lactic acid bacteria, Vol. 1. Chapman and Hall, New York, 1992: 209-224. 19. Ignacio Guerrero Hernndez,1 Aldo Torre Delgadillo,1 Florencia Vargas Vorackova, 1 Misael Uribe, Intestinal flora, probiotics, and cirrhosis, 2008, 120-124. 20. J. B. Brian, Wood, The Lactic Axit Bacteria, The Lactic in Health And Disease. 21. Joint FAO/WHO Expert Consultation, Health and Nutritional Properties of Probiotics in Food including Powder Milk with Live Lactic Acid Bacteria Report, 2001.

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22. Joint FAO/WHO Working Group, Guidelines for the Evaluation of

Probiotics in Food , 2002. 23. John F. T. Spencer, Alicia L. Ragout de Spencer, Food Microbiology Protocols,173- 183. 24. Khalil R., Mahrous H., , Kamaly K. , Frank J., El-Halafawy K. and El Soda M. (2007), Evaluation of the probiotic potential of lactic acid bacteria isolated from faeces of breast-fed infants in Egypt, African Journal of Biotechnology Vol. 6 (7), pp. 935-945, 2 April 2007 . 25.Kim P.I., Jung M.Y., Chang Y.H., Kim S., Kim S.J., Park Y.H. Probiotic properties of Lactobacillus and Bifidobacterium strains isolated from porcine gastrointestinal tract. Appl Microbiol Biotechnol, 74, (2007), 1103-1111. 26.Klaenhammer T.R. and Kullen M.J. Selection and design of probiotics. Int. J. Food Microbiol, 50, (1999), 45-57. 27. Lars Axelsson, Lactic Axit Bacteria: Classification and Physiology, in: Lactic Axit Bacteria, Microbiological and Functional Aspects, 3rd edition, Seppo Salminen, Atte von Wright, Arthur Ouwehand, Marcel Dekker, Inc, New York, USA, 2004, 1-67. 28. Lin W.H.,Yu B., Jang S.H., Tsen H.Y. (2007); Different probiotic properties for Lactobacillus fermentum strains, isolated from swine and poultry; Anaerobe 13 (2007) 107113 . 29. Liu et al, Acid and bile salt resistant Lactobacillus isolates having the ability to lower and assimilate cholesterol, United States Patent, 2007. 30. Maragkoudakisa P. A., Zoumpopouloua G., Miarisa C., Kalantzopoulosa G., Potb B., Tsakalidou E, Probiotic potential of Lactobacillus strains isolated from dairy products, International Dairy Journal 16,2006, p: 189 199 . 31.Martins F.S., Miranda I.C., Rosa C.A., Nicoi J.R., and Neves M.J. Effect of the trehalose levels on the screening of yeast as probiotic by in vivo and in vitro assays Brazilian Journal of Microbiology, 39, (2008), 50-55. 32. Mota R. M., Moreira J. L., Souza M. R., Horta M. F., Teixeira S., Neumann E., Nicoli J. and Nunes A. C. (2006), Genetic transformation of

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novel isolates of chicken Lactobacillus bearing probiotic features for expression of heterologous proteins: a tool to develop live oral vaccines, BMC Biotechnology 2006, 6:2 doi:10.1186/1472-6750-6-2. 33. M. T. Liong and N. P. Shah, Acid and Bile Tolerance and Cholesterol Removal Ability of Lactobacilli Strains, J. Dairy Sci, 2005, 88:5566, American Dairy Science Association. 34. Parvez1 S., Malik2 K.A. , Kang3 S. Ah and H.-Y. Kim, Probiotics and their fermented food products are beneficial for health, Journal of Applied Microbiology ISSN 1364-5072. 35. Suskovic J, Kos B, Matosic S. and Besendorfer V, The efect of bile salts on survival and morphology of a potential probiotic strain Lactobacillus acidophilus M92, World Journal of Microbiology & Biotechnology 16: 673-678, 2000. 36. Tournut, J. Applications of probiotics to animal husbandry. Rev. Sci. Tech. Off. Int. Epiz. 1989, 8, 551566. 37. Yang et al, Bifidobactera strains with acid, bile salt and oxygen tolerance and their culture method, 1998. 38. Yuan Kun Lee And Seppo Salminen, Handbook of probiotics anhd prebiotic, A Jonh Wiley and Sons, Inc., Publication. 39.Zhou X., Pan Y., Wang Y., and Li W. In vitro assessment of gastrointestinal viability of two photosynthetic bacteria, Rhodopseudomonas palustris and Rhodobacter sphaeroides. J. Zhejiang Univ Sci B, 8(9), (2007), 686-692.

PH LC 1 Cc bng s liu ca kho st kh nng chu mui mt

58

Bng 1: Kt qu thi gian OD 0,3 n v ca cc chng vi khun lactic trong mi trng MRS c b sung mui mt STT 1 2 3 4 5 6 7 8 9 10 11
12

Tn chng DC1 DC2 MC5 MC9 MC10 N1 N5 N10 T9 T10 NC10

NC13

Thi gian OD tng ln 0,3 2.345e 3.188cde 3.238cde 2.636 de 3.025cde 3.659 bcd 5.067 a 3.693a 3.835bc 4.307 ab 5.366 a 5.334 a

Bng 2 : Bng phn tch phng sai ca kt qu kho st kh nng chu mui mt Source Model Error Corrected Total DF 11 24 35 Sum of Squares Mean Square 34.38513805 8.34346497 42.72860302 3.12592164 0.34764437 F Value 8.99 Pr > F <.0001

Bng 3: Kt qu o OD ( 600nm) ca kh nng chu mui mt

STT Tn chng 0 Thi gian ( gi ) 4

59

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

DC1 DC2 MC5 MC9 MC10 N1 N5 N10 T9 T10 NC10 NC13

0.33 0.346 0.317 0.318 0.3 0.299 0.339 0.289 0.301 0.26 0.267 0.28 0.334 0.314 0.339 0.276 0.301 0.296 0.381 0.379 0.394 0.315 0.314 0.306 0.274 0.3 0.294 0.393 0.413 0.394 0.327 0.331 0.325 0.283 0.271 0.277

0.331 0.364 0.311 0.34 0.311 0.305 0.34 0.293 0.297 0.251 0.261 0.282 0.328 0.323 0.324 0.282 0.302 0.305 0.385 0.396 0.402 0.301 0.322 0.306 0.264 0.289 0.276 0.396 0.414 0.396 0.331 0.323 0.327 0.287 0.269 0.278

0.355 0.356 0.301 0.321 0.312 0.309 0.339 0.303 0.305 0.262 0.263 0.275 0.333 0.317 0.334 0.281 0.309 0.292 0.386 0.386 0.405 0.317 0.331 0.307 0.283 0.283 0.286 0.403 0.421 0.397 0.336 0.327 0.324 0.279 0.274 0.292

0.812 0.834 0.889 0.683 0.67 0.7 0.654 0.712 0.679 0.612 0.758 0.825 0.779 0.719 0.672 0.637 0.6 0.632 0.676 0.601 0.605 0.579 0.671 0.666 0.641 0.537 0.62 0.7 0.68 0.652 0.52 0.534 0.6 0.498 0.473 0.528

0.823 0.845 0.9 0.694 0.681 0.711 0.665 0.723 0.69 0.623 0.769 0.836 0.79 0.73 0.683 0.628 0.611 0.633 0.687 0.612 0.616 0.586 0.682 0.677 0.652 0.548 0.631 0.711 0.691 0.663 0.531 0.545 0.611 0.509 0.484 0.539

0.818 0.84 0.895 0.689 0.676 0.706 0.66 0.718 0.685 0.618 0.764 0.831 0.785 0.725 0.678 0.633 0.606 0.628 0.682 0.607 0.611 0.585 0.677 0.672 0.647 0.543 0.626 0.706 0.686 0.658 0.526 0.54 0.606 0.504 0.479 0.534

PH LUC 2 Cc bng s liu ca kho st kh nng chu axit

60

Bng 4: S lng t bo m trn a thch ca chng DC1 STT 1 2 3 4 Thi gian (gi) 0 1 2 3 pha long 105 103 103 103 S t bo 400 399 400 249 248 248 63 60 61 40 39 40

Bng 5: S lng t bo m trn a thch ca chng DC2 STT 1 2 3 4 Thi gian ( gi) 0 1 2 3 pha long 105 104 103 103 S t bo 173 575 374 160 173 166 119 287 203 104 96 100

Bng 6: S lng t bo m trn a thch ca chng MC5 STT Thi gian (gi) pha long S t bo

61

1 2 3 4

0 1 2 3

10

104 103 102

206 453 329 149 363 256 211 235 223 270 264 267

Bng 7: S lng t bo m trn a thch ca chng MC9 STT 1 2 3 4 Thi gian ( gi) 0 1 2 3 pha long 105 105 104 103 S t bo 263 309 286 147 177 162 135 91 113 279 264 244

Bng 8: S lng t bo m trn a thch ca chng MC10

62

STT 1 2 3 4

Thi gian (gi) 0 1

pha long 105 105

2 104 3 102

S t bo 283 286 284 227 213 220 74 70 72 71 68 69

Bng 9: S lng t bo ca cc lactic trong kho st kh nng chu axit ( logCFU/ml) S lng t bo ( logCFU/ml) 1gi 2 gi 6.696d 7.517c 7.667b 8.505a 7.623b 6.085c 6.557b 6.642b 7.346a 6.152c

STT 1 2 3 4 5

Chng DC1 DC2 MC5 MC9 MC10

0 gi 8.903a 8.786ab 8.783ab 8.742b 8.755b

3 gi 5.898c 6.279b 5.716d 6.568a 5.134e

Bng 10: T l sng ca cc chng lactic trong mi trng axit

63

Tn chng DC1 DC2 MC5 MC9 MC10

1 gi 0.62c 6.26b 7.705b 58.815a 7.83b

2 gi 0.1533c 0.65bc 0.785b 4.285a 0.26bc

3 gi 0.1017c 0.3867b 0.0983c 0.92a 0.025c

Bng 11: Bng phn tch phng sai ca kho st kh nng chu axit 0 gi

Source Model Error

DF Sum of Squares Mean Square F Value Pr > F 4 40 0.14625980 0.75822550 0.90448530 0.03656495 0.01895564 1.93 0.1244

Corrected Total 44

Bng 12: Bng phn tch phng sai ca kho st kh nng chu axit 1 gi

64

Source Model Error Corrected Total

DF Sum of Squares Mean Square F Value Pr > F 4 40 44 14.83101450 0.44959000 15.28060450 3.70775362 0.01123975 329.88 <.0001

Bng 13: Bng phn tch phng sai ca kho st kh nng chu axit 2 gi Source Model Error Corrected Total DF Sum of Squares Mean Square F Value Pr > F 4 40 44 9.14077530 0.46179300 9.60256830 2.28519382 0.01154483 197.94 <.0001

Bng 14: Bng phn tch phng sai ca kho st kh nng chu axit 3 gi Source Model Error Corrected Total DF Sum of Squares Mean Square F Value Pr > F 4 40 44 10.87816170 1.03759300 11.91575470 2.71954042 0.02593982 104.84 <.0001

Bng 15 : Gi tr OD trung bnh ca kho st ng cong tng trng STT Thi gian ( gi) Ga tr OD trung bnh

65

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

0 1 2 3 4 5 6 7 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 48 56 64 72 80 88 96

0.0494q 0.0773q 0.1215 q 0.2262 pq 0.4003 p 0.7207 o 1.2021n 1.854 m 2.4342 l 3.4026 k 4.1326j 4.6662hi 4.6756 hi 4.5777i 4.5872 i 4.7139 hig 4.9432fgh 4.8527 fghi 4.9874 efg 4.8949fgh 4.9563fg 5.1077def 5.0853def 4.7871hig 4.9099fgh 5.2345cde 5.4105bc 5.313bcd 5.3196bcd 5.4639bc 5.5286ab 5.7317a

66

PH LC 3 Kt qu nh danh chng MC9 theo phng php gii trnh gen 16S

67

68