Trng i Hc Nha Trang Vin cng ngh sinh hc v mi trng C CH TC DNG CA

GVHD: Nguyn Vn Duy Nhm: 3

Nha trang, 26/09/11

Ni dungI. II. Tng quan v probiotic Probiotic chng li mt s vi khun c hi C ch hot ng ca probiotic Cc c ch hiu qu ca probiotic Kt lun

III.IV. V.

probiotic

Vi khun gy hi

I.Tng quan v probiotics1. Khi nim Probiotics l vsv sng c b sung vao thc n mang li nhng tac dung tt cho c th ng vt bng cach duy tri s cn bng vi khun ng rut. Probiotics c cung cp qua sa m, rau, cc sn phm t sa (sa chua,phomats) v cc loi thc phm khc (socola, ng cc...).

I.2 Tiu ch la chn vi sinh vt lm probiotic C kh nng bm dnh vo nim mc ng tiu ha (GI) ca vt ch D nui cy Khng sinh cht c v khng gy bnh cho vt ch C kh nng tn ti c lp trong mt thi gian di C kh nng sinh enzyme hoc cc sn phm cui cng m vt ch c th s dng c Chu c pH thp d dy v mui mt rut non Biu hin hiu qu c li i vi vt ch.

I.3 Cc ch phm probiotic ph bin hin nay Probiotics ph bin l cc ch phm cha vi khun sinh acid lactic (LAB) nh : lactobacilli, bifidobacteria, enterococci. Cc LAB c hiu qu mnh chng li cc tc nhn gy bnh nh: E.coli spp, Samonella spp, Pseudomonas spp, v Campylobacter spp. Mt s chng khc c s dng lm probiotics nh: Bacillus subtilis, mt s cu khun Gram dng: E.faecium, Streptococcus, nm men. Tuy nhin cc chng ny c hiu qu km hn so vi LAB

I.3 Cc ch phm probiotic ph bin hin nay: lactobacillus, streptococus, bifidobacteria, bacillus, sacharomyces boulardii (Nm men) lactobacillus

Streptococcus

Bifidobacteria

Nm men

I.Tng quan v probiotics (tt)

I.4 Li chProbiotics mang li li ch ln cho sc khe: - Bo v nim mc rut khi nhng cht c hi - Ngn chn s pht trin ca vi khun gy bnh trong ng rut (E. coli, Salmonella) - Thc y sc khe ca nim mc rut - Kch thch h thng min dch trong c th nh hng ca probiotic c th l trc tip hoc gin tip thng qua h vi sinh vt ng rut

II. C ch hot ng ca probiotic1. Cnh tranh dinh dng 2. Cnh tranh v tr bm vi vi sinh vt gy bnh 3. Sn xut cc hp cht khng khun 4. c ch h thng quorum sensing 5. Bo v v tng cng chc nng hng ro biu m

II.1 Competing for nutrients Tt c cc vi sinh vt u cn st tng trng Siderophore l mt cht c kh nng gn vi cc ion st, ha tan st d dng cho vi sinh vt Cc VSV v hi sinh siderophore c th s dng nh probiotic cnh tranh st vi cc VK gy hi Loi b c i th cnh tranh dinh dng v nng lng dng cho VK probiotic v cho vt ch

II.2.1 The antagonism of receptor sites on the host epithelium Cc t bo biu m, ng rut, ming v m o, l t bo ch cho hng triu v tr gn th th ca vi khun khc nhau Khi probiotics lin kt vi v tr gn th th trn t bo biu m, lm thay i nim mc ca t bo v t bo ln cn. Do vi khun c hi t c th dnh bm. Probiotics c th tm thy im gn trong rt nhiu v tr, do ngn nga vi khun c hi xm nhp

II.2.1.1 Phng php test s bm dnh ca probioticsMt phng php in hnh nh gi c tnh kt dnh ca VK probiotics l s dng nui cy t bo biu m rut, thng l dng t bo Caco_2, HT_29 hoc t bo tit ra dch nhy HT_29_MTX Tnh dnh bm ph thuc vo:chng ring bit, tui ca t bo nui cy Khng nh hng n s sn xut cytokine ca tb biu m

a. s dnh bm vo dng t bo biu m rut

Kt qu

Vi khun bm dnh trn nim mc rut nh c ch c trng (cht bm dnh v cc phn t th th ca rut) v c ch khng c trng (da vo nhng yu t ha hc) Mt vi thnh phn cu to ca vi khun: protein thnh t bo, carbohydrates, v axit lipoteichoic c xem l c lin quan n tnh dnh bm ca probiotics vo rut

II.2.1.6 Cc nhn t nh hng n c tnh dnh bm ca probioticsc tnh dnh bm ca probiotics l c trng theo chng v nhng nhn t nh: c tnh v thnh phn ca thnh t bo, tng vt ch c th l yu t quan trng quyt nh tnh dnh bm. Ngoi ra mt vi nhn t khc cng gin tip nh hng n nhng c tnh dnh bm

Cc nhn t nh hng (tt)factorpH thp

Study design

effect

references(8, 24) (64)

S dnh bm ca probiotic vo Tng s dnh bm t bo biu m Caco-2 S thay i tnh dnh bm ph thuc vo chng

S c mt ca S dnh bm ca probiotic vo Gim s dnh bm mt nim mc Pha sinh trng S dnh bm ca L.fermentum vo keratinizing biu m d dy chut Tnh dnh bm pha n nh cao hn pha lag

(25,52)

(68)

S dnh bm ca probiotic vo Tnh dnh bm gim pha log, dng t bo biu m Caco-2 tng pha n nh

(8)

Mi trng sinh trng

Cc chng probiotic c kh nng dnh bm vo glycoprotein

Mi trng nui cy c nh hng n s dnh bm

(60)

Cc nhn t nh hng (tt)S c mt ca acid i khng S c mt ca mt i khng VSV ni sinh S dnh bm ca acid lm thay i dch nhy ca Bifidobacterium S dnh bm ca mt i khng lm thay i dch nhy ca Bifidobacterium S c mt ca acid i khng c th lm tng bm dnh S c mt ca mt i khng c th lm tng bm dnh (15)

(25)

S dnh bm vo nim mc H sinh vt ni sinh khng nh trong s c mt ca sinh hng n s dnh bm vo nim vt trong phn hay mng mc trong in vitro nhy S dnh bm ca cc probiotic t hp vi ileostomy glycoprotein S t hp ca nhiu probiotic c th h tr n tnh dnh bm

(51,56)

S c mt ca cc chng probiotic khc

(54)

S dnh bm ca probiotic Cc chng probiotic c th lm vo t bo biu m rut ln gim tnh dnh bm ca cc chng IPEC-J2 khc Lin kt ca c S dnh bm ca t Lactobacillus GG dng t bo biu m Caco-2 S dnh bm gim khi c t lin kt vi t bo

(44)

(39)

Cc nhn t nh hng (tt)Cc enzymes tiu ha S dnh bm ca probiotic vo nim mc S dnh bm ca Propionibacterium acidipropionici vo t bo rut S dnh bm ca probiotic thng mi vo mng nhy S dnh bm ca probiotic v dng t bo biu m Caco-2 phn lp ngi S dnh bm ca probiotic t bo biu m rut ln IPEC-J2 S c mt ca cc ion Zn2+ v Mg2+ S dnh bm ca probiotic t bo biu m rut ln IPEC-J2 Lm gim tnh dnh bm trong (52) hu ht cc trng hp Khng nh hng n tnh dnh (76) bm

S c mt ca lignans S c mt ca Canxi

Tng tnh dnh bm ca mt s chng S dnh bm c hoc khng ph thuc vo Canxi trong mt s trng hp S dnh bm ca hu ht cc chng u tng nh s c mt ca Ca

(43)

(5,6,12,14)

(44)

Mg2+ v Zn2+ khng nh hng (44) n s dnh bm

II.2.1.7 Tnh dnh bm v hn ch nhng c tnh ca probiotic bt hot VK probiotics bt hot bi nhit cng c kh nng bm dnh v nhng c tnh bm dnh lin quan n tnh c trng b mt t bo hn l hot ng TC hay phn chia t bo Nhng cch thc bt hot khc nhau c th gy ra nhng thay i ca loi c th v c tnh kt dnh ca t bo VD: tng nhit c th ci thin cc chng probiotics nhng cng c th gy bt li n cc thuc tnh dnh bm ca cc chng khc

II.2.1.8 Vai tr ca tui v thi gian bnh trong bm dnh S bin i ca cc vi sinh vt ng tiu ha dn n mt s bnh ng tiu ha v cc bnh khc. Hn na, tui c gi l c lin quan n s khc bit trong thnh phn cc vi sinh vt ng tiu ha, c bit l Bifidobacteria Do mt vi nguyn nhn c tnh dnh bm ca nim mc rut c th thay i sut thi gian bnh. Kh nng xm nhim ca mt vi probiotic cc i tng bnh cng c nh gi

Probiotics

Thit k th nghim

Kt qu

Tham kho

Bifidobacteria phn lp t ngi cao tui v ngi trng thnhBifidobacteria thng mi

Trong nghin cu in vitro s dnh bm vo nim mc ngi

S bm dnh ca Bifidobacteria ngi cao tui gim so vi ngi trng thnh

(30)

Trong in vitro s dnh bm vo nim mc tr em, ngi trng thnh v ngi cao tui

S dnh bm vo nim mc ca nhng ngi cao tui gim so vi ngi trng thnh v tr em

(53)

Bifidobacteria Trong nghin cu in S dnh bm vo nim mc t tr b d ng vitro s dnh bm vo tr b d ng gim so vi nim mc nhng ngi nhng tr khe mnh tnh nguyn khe mnh

(27,29)

Cc probiotic thng mi

S dnh bm vo nim mc v m rut kt c ct t nhng bnh nhn b ri lon rut nng

S dnh bm ph thuc vo bnh tt

(57,59)

Cc probiotic thng mi

S dnh bm vo nim S nhim Rotavirus mc tr b nhim khng nh hng ti s Rotavirus bm dnh

(38)

Bifidobacteriu Probiotic c a m infantis vo cc bnh nhn b nhim trng vim kt trng L. plantaum 299v Probiotic c a vo cc bnh nhn nguy kch

Cc chng probiotic (75) c khi phc t cc sinh thit cho thy c s xm nhim tm thi Chng probiotic c (42) khi phc t 3/9 cc sinh thit, c s xm nhim tm thi

II.2.2 Probiotics t hp, s dnh bm v kt t mm bnha. Kt tu cc hiu ng biu hin c li, vi khun probiotic cn phi kt hp to thnh mt khi. Do , kh nng kt t ca probiotic l mt c tnh mong mun.

Cc sinh vt c kh nng ng kt t vi cc vi khun khc c th l li th hn sinh vt khng c ng kt t.

b. dnh bmo S dnh bm cc biu m ng rut v cht nhy c lin quan n s kch thch ca h thng min dch (80, 114), v s dnh bm vo nim mc rut cng rt quan trng cho xm nhim tm thi (114),l mt iu kin tin quyt quan trng ca probiotic kim sot s cn bng ca h vi sinh vt ng rut .o Cht nhy ng rut c mt vai tr kp, n bo v nim mc ca cc loi vi sinh vt ch trong khi cung cp mt vi tr lin kt ban u, ngun dinh dng, v l mi trng thun li cho vi khun sinh si ny n.

b. dnh bm (tt)o S dnh bm l mt qu trnh phc tp bao gm c ch khng c hiu (k nc) v th th c hiu. o S dnh bm ca cc t bo vi khun thng lin quan n c im b mt t bo. Nhiu tc gi bo co rng kh nng ng kt t ca cc loi Lactobacillus c th kch hot n to thnh mt ro cn ngn chn s xm nhp ca vi khun gy bnh.

d. Yu t quyt nh s dnh bm

Cc c ch dnh bm lin quan n yu t th ng, lc tnh in v k nc, acid lipoteichoic, v cc cu trc c trng nh l polysaccharides v lectin. Thnh phn protein c lin quan n s dnh bm ca cc chng probiotic ln cc t bo rut.

e. S t hp trong probioticS t hp gia cc chng probiotics khc nhau c th hiu qu hn so vi cc chng n l. Tuy nhin, hiu qu c th b v hiu nu cc chng khng c chn lc mt cch khoa hc, cht ch. c mt chng minh rng: s kt hp ca cc chng probiotic c th b sung hoc ci thin hot tnh cho tng chng ring bit trong t hp .

e. S t hp trong probiotic (tt) Khi s dng probiotics t hp gip tng cng kh nng dnh bm ln nim mc rut ca tng chng probiotics ring r. V vy, cn thit nghin cu thm v nh hng ca cc sn phm nui cy v trao i cht trong dnh bm ca cc chng probiotic tm ra cc t hp tt nht s dng trong cc sn phm thc phm chc nng.

III.Production of Antimicrobial Compounds LAB cnh tranh vi cc vi sinh vt khc trong sinh thi c trng bng cch sn xut cc hp cht i khng v thay i mi trng ca chng thng qua vic sn xut cc hp cht trao i th cp Cc hp cht khng khun in hnh gm: hp cht trng lng phn t thp < 1,000da nh cc acid hu c, reuterin, reutericyclin, hydro peroxide, acetoin, diacyl v nhm cc bacteriocin c trng lng phn t cao > 1,000da Mi cht khng khun c kh nng nhn bit vi khun ch gy hi. S c mt ca VK sinh cc cht khng khun trong rut ca vt ch, trn b mt hoc trong mi trng nui to ra mt hng ro ngn chn s sinh si ca cc mm bnh.

Bng II: Tm tt cc hp cht khng khun c sn xut bi cc loi LactobacillusKiu hp cht Hp cht Tn gi khc/ Ngun vi sinh ng dung tn thng vt mi Lactobacillus plantarum Thuc phng bnh, bo qun, khng khun, ph gia thc phm -

Acid lactic Acid huc Acid acetic Acid 3- Phenyllactic

-

Lactobacillus plantarum (MiLAB 393)

Acid 4-hydroxyphenyllactic v tetrameric derivative

Reutericyclin Lactobacillus plantarum (MiLAB 393) Reuterin Lactobacillus reuteri

-

Acetaldehyde Aldehyds 3- hydroxypropionaldehyde (3- HPA)

-

Bacteriocins

Nicin PlantiricinL-

-

Lactobacillus plantarum Lactobacillus pentosus Lactobacillus plantarum (MiLAB 393) Lactobacillus plantarum Lactobacillus plantarum -

Bo qun, phngbnh Ph khng khun rng Khng Nm Khng Nm

Phe-L- Pro

Cyclic dipeptides

Cyclo- Gly- L-Leu

-

L-Phe-trans-4-OH-L-Pro

-

Hydrogen peroxide Tetrahydro -4- hydroxy-4methyl-2H-pyran-2-one 5-methyl-2,4imidazolidinedione Diacyl

Mevalonolactone Methylhydantoin -

Ph khng khun rng Khng Nm -

Khc

III.1 Sn xut cc hp cht khng khun trng lng phn t thpa. Production of Organic Acids Trong qu trnh tng trng k kh (anaerobic growth) vi khun ln men trong ng tiu ha to ra cc acid hu c Hm lng v loi acid hu c c sn xut ph thuc vo loi v iu kin tng trng ca vi khun Theo cch ln men carbohydrate Lactobacilli c chia ra ba nhm: ln men ng hnh (homofementative), ln men d hnh ty tin (facutatively heterofermentative) v d hnh bt buc (obligatively heterofermentative)

Ln men ng hnh: lactobacilli ln men hexose sinh cc acid lactic thng qua con ng glycolysis Ln men d hnh ty tin: lactobacilli ln men hexose to cc acid lactic, acid acetic, ethanol v acid formic Ln men d hnh bt buc: - Lactobacilli ln men hexoses to cc acid lactic,acid acetic, ethanol, CO2.Trong khi pentoses c ln men to acid lactic v acid acetic - Cc lactobacilli c sinh mt loi kh t s phn gii cacbohydrate hoc lactobacilli sn xut mt s cht l (exotic) nh 1,2 propanediol.

Hnh thc ln men n gin nht l ln men ng hnh LAB, ch ln men ng hexose to lactate. Trong iu kin hn ch ng hoc oxy th c kh nng ln men d hnh to ra lactate, acetate v mt lng nh ethanol. Cc acid hu c c hiu qu khng khun trn mm bnh .Cn butyrate, propionate, formate c th iu chnh c vi khun c kh nng gy bnh

Mt s cc tc nhn gy bnh s dng cc acid bo bay hi nh cht ch th chn lc preferential xm nhp. Vd:- S.Typhimurium c hai thnh phn BarA/SirA gip iu chnh mi trng ng rut v iu chnh s biu hin ca TTSS-1(Type Three Secretion System 1) - H thng tit ny m ha cho cc c quan, tc ng cc protein cn thit cho Salmonella xm nhp vo cc t bo biu m khng thc bo Acetate v formate c hot tnh kch thch v to iu kin cho s xm nhp trong S.Typhimurium Butyrate v propionate lm gim kh nng gy bnh ca SPI-1 (Salmonella pathogenicity island 1) m ha TTSS-1.C kh nng bin i mi trng gip gim s xm nhp v pht tn bo t Salmonella trong sn xut thc n ng vt

Cc acid hu c to pH ca ng rut gip hiu qu khng khun mnh - Ti pH thp acid hu c phn ly lin tc.pH nh hng trc tip n tng s acid khng phn ly khuch tn qua mng vi khun. - S phn ly cao ca cc proton v anion hu c bn trong t bo lm yu lc di chuyn ca proton qua mng vi khun.v khng c gradien proton c lc ATPase F1/F10 nn lm gim kh nng sn xut ATP ca vi khun Acid hu c lm thiu ht ATP ni bo,gy ra s thm qua mng, gii phng LPS ra khi mng t bo vi khun.Lm cho mng b mn cm vi cc tc nhn nh: bacterioxin, cht ty ra, mt.

a.1 Lactic acid Sn phm ln men chnh ca LAB l L- lactate,c s dng bo qun cc sn phm tht v kim sot mm bnh trn cc sn phm v b mt tht Acid lactic c kh nng thm qua mng mnh (membrane permeabilizer), l nguyn nhn gy tn thng, ph hy mng t bo E.coli. Theo tnh thm, hot ng ca axit lactic c th to iu kin thun li cho cc hot ng ca cc yu t khng khun khc

C ch tc ng khng khun ca lactate l lm gim pH ca mt t bo vi khun thng qua s phn ly cc acid hu c v lc cc cation ha tr hai chui LPS mi n nh

b. Hydrogen Peroxide Lactobacilli sn xut hydro peroxide khi trong mi trng nui c mt oxy Hydro peroxide l phn t quan trng iu tit h vi sinh vt m o (vaginal) v gy c cho cc mm bnh C hot tnh khng khun mnh trong c invitro v invivo nh tc dng oxy ha mnh m trn t bo vi khun (hydroxyl trit ) Nhiu loi lactobacilli c kh nng sinh hydro peroxyde trong ng rut, c bit l trong rut non

c. CO2 CO2 ch yu c hnh thnh t ln men lactic d hnh CO2 c th trc tip to ra trong mi trng k kh v c hi i vi mt s vi sinh vt hiu kh thng qua hot ng ca chng trn trn mng t bo Hot ng khng khun ca phn t CO2 c cho l do s c ch ca enzyme decacboxyl v s tch ly ca CO2 trong mng lipit gy ra ri lon tnh thm ca mng CO2 c th c hiu qu chng li s pht trin ca vi sinh vt gy h hng thc phm, khng vi khun gram m, c hiu qu v phng din thng mi ha p dng cho cc sn phm lm lnh.

c. Reuterin and Reutericyclin Reuterin l hp cht khng khun c trng c sn xut bi Lactobacillus spp Reuterin, - hydroxypropionaldehyde l mt ph khng khun rng c sn xut bi mt s chng L.reuteri Sn xut reuterin trong iu kin k kh ti u trn 1 hn hp ca ng v glycerol hoc glyceraldehydes Sn xut reuterin ph thuc vo s c mt ca glycerol dehyratase,co-enzyme B12 Reuterin l mt aldehyde tan trong nc,tn ti trong hn hp phc tp gm monomeric,hydrated monomeric v cyclic dimeric nhng cu trc ca hydroxypropionaldehyde

Reuterin hot ng trong phm vi pH rng,c kh nng chu c protease v lipases, v c hot tnh khng khun khng li cc mm bnh c trong thc phm Trong giai on tng trng, Reuterin khng sn xut do s gim trao i cht ca ng. Khi cc t bo tham gia vo giai on tng trng, Reuterin bt u tch ly. Hn na, Reuterin tch ly khi tip xc vi cc t bo mc tiu

Reutericyclin l mt dn xut acid tetrameric sn xut bi chng L.reuteri .Bao gm mt u a nc mang in tch m v hai chui bn k nc Reutericyclin c cho l to nn cc knh,l trn mng vi khun,tuy nhin cc nghin cu s dng thuc nhum hunh quang (fluorescent dyes) cho thy proton in th xuyn mng,hnh thnh l v in th kali xuyn qua mng. Reutericylin khng to nn cc l mng nhng lm mt kh nng proton xuyn mng chn lc gy phn tn PMF (proton motive force)

d. Other Secondary Metabolites Nhiu lactobacilli ln men to cc sn phm ph l cc hp cht c hng thm nh: Diacetyl v acetoin.ngoi ra cn c acetaldehide, ethanol - Diacetyl c hnh thnh khi t bo c hot tnh acetaldehyde t pyruvate v thiamine pyruvate - Acetoin c hnh thnh bng cch decarboxylation - acetolatate, hoc bng cch gim diacetyl bi diacetyl reductase Vi khun Gram m, nm men, nm mc nhy cm hn vi diacetyl so vi vi khun gram dng v phng thc hot ng ca n c cho l do s can thip vi vic s dng khong cht

III.2 Sn xut cc hp cht khng khun trng lng phn t cao :Bacteriocin Bacteriocins l cc peptide khng khun,bn cht protein c tng hp qua ribosome vi khun - Cc bacteriocin c hot tnh khng cc vi sinh vt cnh tranh trong cng sinh thi v thng khng cng loi (ph hp) hoc qua chi (ph rng) - Cc bacteriocin c hot tnh cao,do cc vi sinh vt sn xut da vo cc protein min dch t bo v trnh cc bacteriocin ring ca chng Cc bacteriocin c hot tnh mnh nht vi khun Gram dng v LAB c trng khc

Classification of BacteriocinsVic phn loi l quan trng thit k peptide khng sinh mi.c chia ba nhm chnh Class I : Lanthionine lp I cha cc bacteriocin c gi l lantibiotic Class I lantibiotics: lantibiotics c gc acid amin khng c bn cht l protein v methyllanthionine,c cc lin kt ng ha tr gia cc acid amin. Hai lantibiotic ph bin l nisin v mersacidin. Nisin l bacteriocin lp I tiu chun bao gm mt peptide amphiphilic, hnh thnh nn cc l trn mng vi khun.Lm phn tn PMF v l mt nguyn nhn gy thot mt s cht trao i cht t t bo

Class II: Non- lanthionine cha bacteriocin ,ph bin hn lp I nh t yu cu sa i sau dch m hn Bacteriocin lp II l cc peptide nh (