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MN: K THUT THC PHM 3 TI:LN MEN B SU- NG HC CA QU TRNH LN MEN GIN ON TP. HCM, THNG 10 NM 2013

1QU TRNH LN MEN???1Ln men l mt qu trnh sinh hc, m phn ng oxy ha kh xy ra trong t bo vsv, trong iu kin khng c cht oxy ha no. Mt phn cht hu c b phn gii thnh cht kh, phn cn li b phn gii thnh cht oxy ha

2Nghin cu qu trnh ln men l NC c im sinh l-ha sinh v hot ng sng ca mt chng VSV3Ty thuc vo c im sinh l ca VSV i vi oxy, ta coi qu trnh l hiu kh hay k kh khng bt bucCC PHNG PHP LN MEN1i vi ln men k kh thc hin theo phng php nui cy chm2Ln men hiu kh c thc hin nh hai phng php c bn: nui cy b mt v nui cy b su (nui cy chm)PHN LOI QU TRNH LN MEN1. Da vo k thut nui cy: Da vo cch np liu + thu sn phm ta c cc loi ln men:Gin onLin tc Bn lin tc Da vo thnh phn ng nht ca mi trng ta c cc loi ln men:B mt (ni) B su (chm) Bn rn

PHN LOI QU TRNH LN MEN2. Da vo sn phm chnh:Ln men etilic Ln men lactic Ln men propionic Ln men formic Ln men butiric v ln men axeton-butanol Ln men metan Ln men axetat Ln men xenlulozo

1LN MEN B SU TRONG MI TRNG LNG u imTn t mt bng trong xy dng v lp t dy chuynChi ph in nng, nhn lc v cc khon ph cho mt n v sn phm thp.D t chc c x nghip c sn lng ln.Cc thit b ln men chm d c kh ho, t ng hoLN MEN B SU TRONG MI TRNG LNG Nhc imi hi trang b k thut cao, d b nhim trng ton bTrong ln men chm cn phi khuy v sc kh lin tcL qu trnh nui cy VSV m mi trng dinh dng ch c cho vo lc u v khng thm cht dinh dng cho n khi kt thc qu trnh nui cy Sn phm ca qu trnh ln men c tch ly dn dn v ch ly ra khi qu trnh ln men kt thcVSV sinh trng n chng no mt thnh phn ch yu ca mi trng dinh dng b gii hnLN MEN GIN ONC IMLN MEN GIN ONGIAI ON TIM PHTGIAI ON LOGARITGIAI ON N NHGIAI ON T VONGTrong qu trnh nui cy, VSV sinh trng v pht trin theo mt quy lut gm 4 giai onNG CONG SINH TRNG

Th hin s sinh trng ca qun th vi sinh vt trong mt mi trng nui cy theo phng php nui cy theo m (batch culture) hoc trong mt h thng kn

GIAI ON TIM PHT (Lag phase) Trong giai on ny t bo cha c s tng ln v s lng nhng th tch v khi lng tng ln r rt do c s tng cc thnh phn mi ca t bo. Do c s thay i v mi trng sng nn t bo cn mt thi gian nht nh tng hp cc enzyme mi nhm s dng c cc cht dinh dng mi. Giai on tim pht di hay ngn lin quan n bn thn tng loi vi sinh vt v tnh cht ca mi trng. Nu tnh cht ha hc ca mi trng mi sai khc nhiu vi mi trng c th giai on tim pht s ko di v ngc li.

GIAI ON TIM PHT (Lag phase)CC YU T NH HNGTui ging cy. V d: pha lag s khng c nu dng ng cy gm cc TB ang pha sinh trng logaritLng ging cy: ni chung lng ging cy nhiu th pha lag ngn v ngc li. Trong cng nghip ln men t l cy ging thng mc 1/10Thnh phn mi trng: mi trng c thnh phn dinh dng phong ph th pha lag ngn.Trong giai on ny vi sinh vt sinh trng v phn ct vi nhp ti a nu gp mi trng v iu kin nui cy thch hp. Do cc t bo sinh ra ch khc nhau rt t cho nn ng cong sinh trng l mt ng trn nhn ch khng gp khc Qun th t bo trong giai on ny c trng thi ha hc v sinh l hc c bn l nh nhau => thng c s dng nghin cu sinh ha hc v sinh l hc ca vi sinh vt. Giai on logarit (Log Phase) hay Pha Ch s (Exponential Phase)Giai on logarit (Log Phase) hay Pha Ch s (Exponential Phase)Sinh trng logarit l sinh trng ng u nu cc thnh phn t bo c tng hp vi tc tng i n nh. Mt khc, nu cn bng dinh dng hay iu kin mi trng thay i s dn n s sinh trng khng ng u.

S sinh trng ca vi sinh vt ph thuc nhiu vo nng cht dinh dng.

Giai on logarit (Log Phase) hay Pha Ch s (Exponential Phase)

(a )- nh hng ca s hn ch cht dinh dng i vi sn lng chung ca vi sinh vt. Lc nng cao th sn lng chung s t ti n nh.(b)- nh hng ca s hn ch cht dinh dng ti tc sinh trng.

S lng t bo sinh ra bng s t bo cht i3GIAI ON N NH(Stationary Phase) 1ng cong sinh trng i ngang2Nng vi khun trong giai on n nh thng v khong 109/mlC IMHn ch cht dinh dng.

Vsv hiu kh thng b hn ch bi nng oxygen.

Tch ly cc sn phm trao i cht c hi.

GIAI ON N NH(Stationary Phase) NGUYN NHNGIAI ON N NH(Stationary Phase) GIAI ON T VONG(Death Phase) S lng t bo c kh nng sng gim theo ly tha

S cht ca t bo c th nhanh hay chm, c lin quan n s t phn hay khng t phnNGUYN NHNiu kin bt li ca mi trng: nng cht dinh dng thp di mc cn thit cho hu qu lm gim trao i cht , phn hy dn dn cc cht d tr v cui cng dn n s cht hng lot ca t bo

Qu trnh bo qun cc chng VSVTNH TON QU TRNH SINH TRNGTNH TON NHP SINH TRNGNghin cu v sinh l hcNghin cu v sinh thi hc VSVGii quyt mt s vn ng dng trong sn xut cng nghipTNH TON QU TRNH SINH TRNGS lng t bo vi khun tng theo phng thc 2n

Ta hy tng tng trong mt bnh kn cha mt lng ln mi trng dinh dng, ta cy vo mt t bo vi khun. Nu thnh phn mi trng hon ton ph hp vi nhu cu ca t bo, vi khun s sinh trng, tng khi lng v th tch,n kch thc nht nh ri phn chia cho 2 t bo. Hai t bo ny tip tc sinh trng v phn chia cho 4, 8, 16, 32,.

TNH TON QU TRNH SINH TRNGThi gian S ln phn ct 2n S lng(N02n )lg10Nt0020 = 110,00020121 = 220,30140222 = 440,60260323 = 880,90380424 = 16161,204100525 = 32321,505120626 = 64641,806TNH TON QU TRNH SINH TRNG

Sinh trng th h ca VSVTNH TON QU TRNH SINH TRNGS lng t bo thi gian t:Nt = N0 2nTrong : Nt: s TB sau n ln phn chia trong thi gian t N0: l s lng t bo ban u. n: l s th h hay s ln phn chia.Logarit 2 v:logNt = logN0 + nlog2

S th h n:TNH TON QU TRNH SINH TRNGTrong nui cy gin on, giai on logarit: Tc sinh trngTc sinh trng bnh qun k=

k: s th h sinh ra trong n v thi gian Cn c trc tip vo th bn logaritDa vo hng s tc sinh trng Xc nh thi gian th h g TNH TON QU TRNH SINH TRNG26TNH TON QU TRNH SINH TRNGThi gian th h g l thi gian tnh t khi mt t bo sinh ra n khi t bo phn chia hoc s t bo trong qun th tng gp i.

Nu thi gian t = thi gian th h g(t=g) th Nt = 2N0Thay vo cng thc tnh k ta c:

TNH TON QU TRNH SINH TRNGNh vy:Thi gian th h l nghch o ca hng s tc sinh trng bnh qun

Thi gian th h thay i theo chng loi VSVTNH TON QU TRNH SINH TRNG

Xc nh thi gian th hPHNG PHP NH LNG VI SINH VTS hin din ca VSV c th c nh lng bng nhiu phng php nh:

m s lng t bo trc tip trn bung m hng cu bng knh hin vi quang hc hoc bng knh hin vi hunh quang.

m gin tip bng s lng khun lc mc trn mi trng xc nh hoc o c ( mt quang) ca t boGII THIU CC PHNG PHP NH LNG VI SINH VTPHNG PHP NH LNG VI SINH VTM TRC TIP BNG BUNG M HNG CU Vi sinh vt n bo c kch thc ln nh: nm men, to c th c nh lng bng cch m trc bng bung m trn knh hin vi.u im:Xc nh c ngay lng t bo trong mu.Nhc im:Khng xc nh c t bo cn sng hay cht, d lm t bo VSV vi cc ht vt th khc c trong mi trng v dch huyn ph VSV phi c mt t bo phi ln.PHNG PHP NH LNG VI SINH VT

m trc tip bng bung m hng cuPha long mu cn m sao cho trong mi nh ca bung m c khong 5 - 10 t bo VSV.

Sau nh 1 git mu ln bung knh v y li bng l knh, quan st di lnh hin vi.

PHNG PHP NH LNG VI SINH VTPHNG PHP NH LNG VI SINH VTM TRC TIP BNG KNH HIN VI HUNH QUANGu im:Kt qu phn nh ng vi sinh khi.

Nhum VSV bng cc cht pht hunh quang nh: acridin cam (AODC), 4,6-dianidino-2-phenyl indol (DAPI), flourescent isothiocyanate (FITC).

PHNG PHP NH LNG VI SINH VTPHNG PHP M KHUN LC Phng php m khun lc c thc hin bng k thut hp hay hp tri vi cc thit b h tr (my m khun lc, bt m khun lc) c kt qu. Phng php: Chun b dch pha long muChun b cc chui pha long muCy mu vo mi trng, mum s khun lc hnh thnh.u im:Cho php xc nh s t bo sng.nh lng chn lc vsv.

PHNG PHP NH LNG VI SINH VT

My m khun lc t ng C 2 phng php m khun lca. Phng php cy b mt:Mi trng phi chun b trc 1-2 ngy kh mt.u im: nh lng c cc vsv nhy nhitC th nhn dng c khun lc c trngD dng lm thun chng vsv mc tiuNhc im: Ch cy c th tch mu nhCh cho m s khun lc thpPHNG PHP NH LNG VI SINH VTPhng php a:u im: - Cy c th tch mu ln, xc nh c cc vsv cn dinh dng tip xc t nhiu pha Cho php m c mt vsv cao, khong 150-300 khun lcNhc im: - Khng nh lng c nhng vsv qu nhy- Khng xc nh c hnh dng khun lc nt nh- Kh lm thun mt dng vsv.

PHNG PHP NH LNG VI SINH VTPhng php lc mng xc nh s lng vi sinh vtPHNG PHP MNG LCXc nh s lng vi sinh cc pha long khc nhau. Mu c lc v mng lc c t trc tip ln mt mi trng thch thch hp.=> Phng php ny thng p dng vi mu nc v nc thiu im: Xc nh c mt vsv c thNhc im:Khng thch hp phn tch mu thc phm rnPHNG PHP NH LNG VI SINH VT

PHNG PHP NH LNG VI SINH VT Cc loi khun lc mc trn mng lc.

Theo sch ca Prescott,Harley v Klein (2005)(a)- Tng s vi khun mc trn mi trng tiu chun, Dng ch th mu nhum khun lc cho d im;(b)- Dng mi trng thch hp kim tra nhm vi khun coliform c ngun gc t phn (khun lc bt mu xanh);(c)- Dng mi trng thch m-Endo xc nh vi khun E.coli v cc Coliform khc- khun lc c mu lc;(d)- Nm si v nm men mc trn mi trng Thch - Mch nha.

PHNG PHP NH LNG VI SINH VT XC NH KHI LNG T BO XC NH TRNG LNG KH CA T BOThch hp xc nh khi lng ca t bo nm, nhng khng thch hp i vi vi khun.Nhc im: Phng php ny tn thi giano s lng vi sinh vt bng phng php o c.

O C c ca dch t bo c th m bng quang ph k v c biu din bng n v hp th. S lng t bo lin quan mt thit vi c ca dch vi khun.T bo phi c khuy trn k trc khi a vo quang ph ko c ca dch treo t bo. y l pp rt thun li. trong thc ta thng o mt quang hc ca dch treo (dch huyn ph).

O MT QUANG (OD) O MT QUANG (OD)

Thng qua vic o hp th nh sng c th xc nh c sinh khi vi sinh vt O MT QUANG (OD)Cng thc:

Vi: A : l mt quang ti bc sng .I0 : l cng sng trc khi qua mu.I : l cng sng sau khi qua mu.

A = ln (I0 / I) O MT QUANG (OD) XC NH NNG T BOCng thc:

C(mg/ml)= A* k * n

Vi:C: nng t bon: h s pha longk: h s chuyn i bng 5x108u im: C th thc hin trong thi gian ngn v khng gy ph hy t bo