B GIO DC V O TO

TRNG I HC NHA TRANG VIN CNG NGH SINH HC V MI TRNG

N TT NGHIP

PHN LP, TUYN CHN V NH GI MT S C TNH CA MT S CHNG LACTOBACILLUS TRN C CHIM VY VNG

Ging vin hng dn : Th.s L NH C Sinh vin thc hin: L THANH HUN Lp : 49 CNSH Kho : 49

ii

Nha Trang, thng 7 nm 2011

B GIO DC V O TO

TRNG I HC NHA TRANG VIN CNG NGH SINH HC V MI TRNG

N TT NGHIP

PHN LP, TUYN CHN V NH GI MT S C TNH CA MT S CHNG LACTOBACILLUS TRN C CHIM VY VNG

Ging vin hng dn : Th.s L NH C Sinh vin thc hin: L THANH HUN Lp : 49 CNSH Kho : 49

ii

Nha Trang, thng 7 nm 2011

LI CM N Trc tin, ti xin chn thnh cm n cc thy c gio Vin Cng ngh sinh hc v Mi trng, trng i hc Nha Trang lun quan tm, ch bo v ging dy nhit tnh, gip cho ti c c nhng kin thc qu bu trong sut thi gian hc tp ti trng. Ti xin dnh li cm n su sc nht n Thy L nh c, B mn Cng ngh sinh hc, Vin Nghin cu Cng ngh sinh hc v mi trng, trng i hc Nha Trang nh hng, du dt v tn tnh hng dn ti trong sut thi gian ti thc hin n tt nghip ny. Ti xin chn thnh cm n n ch Nguyn Minh Nht, cn b qun l phng th nghim Cng ngh sinh hc, to mi iu kin v thi gian ti hon thnh ti. Ti xin gi li cm n n cc bn sinh vin lp 49SH, cng ton th cc bn sinh vin thc tp ti phng th nghim nhit tnh gip ti. Cui cng, ti by t lng bit n chn thnh n gia nh, bn b, nhng ngi lun quan tm gip , ng vin, ng thi l ch da tinh thn rt ln gip ti hon thnh tt mi cng vic c giao trong sut thi gian hc tp v thc hin n va qua.

Nha Trang, thng 6 nm 2011 Sinh vin L Thanh Hun

GVHD: Th.s L nh c

SVTH: L Thanh Hun

ii

MC LC

GVHD: Th.s L nh c

SVTH: L Thanh Hun

iii

DANH MC BNG Bng 3.1: Hot tnh khng 7 chng Vibrio ca 5 chng Lactobacillus trn mi trng MRS, nhit 37oC.................Error: Reference source not found Bng 3.2: Kt qu th cc c tnh ca hai chng L1.2 v L1.3..................................52

GVHD: Th.s L nh c

SVTH: L Thanh Hun

iv

DANH MC HNH V V TH Hnh 1.1 : C chim vy vng...........................Error: Reference source not found Hnh 3.1: Kh nng i khng 7 chng Vibrio (V 2.1, V 2.2, V 2.3, V 2.4, C1, C7 v C23) ca 6 chng Lactobacillus trn mi trng MRS, c xc nh bng ng knh vng khng khun (D-d) sau 1-2 ngy nui 37oC.....Error: Reference source not found Hnh 3.2: Vng khng Vibrio ca 2 chng Lactobacillus la chn sau 24h nui cy trn mi trng MRS, lc 180 vng/pht, nhit 28 - 30oCError: Reference source not found Hnh 3.3: Hnh thi khun lc chng L1.2 trn sau 24h trn mi trng MRS nui 340C.............................................Error: Reference source not found Hnh 3.4: Hnh thi t bo ca chng L1.2 khi soi ti vt knh 100XError: Reference source not found Hnh 3.5: Hnh nh nhum gram ca chng L1.2. .Error: Reference source not found Hnh 3.6: Hnh thi khun lc chng L1.3 trn sau 24h trn mi trng MRS nui 340C.............................................Error: Reference source not found Hnh 3.7: Hnh thi t bo ca chng L1.3 khi soi ti vt knh 100XError: Reference source not found Hnh 3.8: Hnh nh nhum gram chng L1.3.........Error: Reference source not found Hnh 3.9: Kh nng di ng ca chng L1.2 v L1.3...Error: Reference source not found Hnh 3.10: Kh nng ln men cc loi ng ca chng L1.2 v L1.3...Error: Reference source not found Hnh 3.11: Mi tng quan gia thi gian v OD600 nm ca chng L1.2..Error: Reference source not found

GVHD: Th.s L nh c

SVTH: L Thanh Hun

Hnh 3.12: Mi tng quan gia thi gian v OD600 ca chng L1.3.....Error: Reference source not found Hnh 3.13 : nh hng ca nhit nui cy ln s sinh trng v pht trin ca chng L1.2 v L1.3..........................Error: Reference source not found Hnh 3.14: Mi tng quan gia thi gian nui cy v mt t bo sng ca hai chng L1.2 v L1.3 OD600 nm.........Error: Reference source not found Hnh 3.15: nh hng ca pH ln s sinh trng v pht trin ca chng L1.2 v L1.3.....................................................Error: Reference source not found Hnh 3.16: nh hng ca nng mui NaCl n s pht trin ca chng L1.2 v L1.3.....................................................Error: Reference source not found K HIU CC CM T VIT TT

GVHD: Th.s L nh c

SVTH: L Thanh Hun

LI M U Trong nhng nm gn y, nghnh nui trng thy sn nc ta ang pht trin nhanh chng. Theo cng b ca FAO nm 2006, tng sn lng nui trng thy sn ca Vit Nam l 1,67 triu tn, xp v tr th 6 Chu . Hin nay i tng nui trng ch yu l cc loi thy hi sn nc mn nh: c m (Epinephelus spp), c gi (Rachycentron canadum), c chm (Lates calcariper), c cam (Seriola spp), c hng (Lutijanus erythropterus), c chim (Trachinotus blochii), c hng (Babylonia areolata), vm xanh (Perna viridis), tm s (Penaeus monodon),... trong i tng nui mi l c chim vy vng ang c ch pht trin nui bi v tht c thm ngon hp dn, hm lng dinh dng cao, rt c a chung. C chim vy vng (Trachinotus blochii Lacepede, 1801) l loi phn b tng i rng vng bin nhit i, Ty Thi Bnh Dng, Nht Bn, i Loan, Indonesia, min Nam Trung Quc. Nc ta c phn b ch yu vnh Bc B, min Trung v Nam B. C c kch c thng mi 0,8 -1 kg/con, gi tr kinh t cao vi gi bn 100.000 VN/kg, th trng xut khu: Nht Bn, i Loan, Trung Quc, Hng Kng, M, Singapore. y l mt i tng nui mi Vit Nam, cha c nghin cu nhiu nn ngi nui vn gp kh khn do t l c cht cao, sc chng chi trc cc iu kin bt li ca mi trng v cc vi khun gy bnh cn thp c bit l cc bnh do Vibrio gy ra. khc phc tnh trng ny, ngi nui dng cc loi ha cht kh trng, cc cht khng sinh phng bnh. Tuy nhin hiu qu ca cc phng php ny khng cao, ngc li cn gy nh hng n mi trng, nguy him hn nu lm dng cht khng sinh s gy ra tn d khng sinh v hin tng khng khng sinh ca cc vi khun gy bnh trn thy sn. V vy, vic tm ra mt gii php thch hp gii quyt vn ny l rt quan trng. Phng php s dng ch phm sinh hc c cha nhng vi sinh vt mang nhng c tnh: i khng vi vi khun gy bnh, sinh cc enzyme tiu ha, phn hy cc cht hu c tha c p dng. Cc ch phm sinh hc khng nhng tng

GVHD: Th.s L nh c

SVTH: L Thanh Hun

kh nng sinh trng, kh nng khng bnh cho vt nui m cn hn ch c ti a kh nng s dng khng sinh trong vic phng v tr bnh thy sn. Nhiu nhm vi sinh vt mang cc c tnh probioic c p dng, trong c nhm vi khun Lactobacillus. Vi l do nh vy nn chng ti tin hnh thc hin ti Phn lp, tuyn chn v nh gi tim nng probiotic ca cc chng Lactobacillus phn lp trn C Chim vy vng Mc tiu ca ti l phn lp, tuyn chn mt s chng Lactobacillus c hot tnh khng Vibrio b sung vo ch phm probiotic nui c chim vy vng nhm mc ch tng tc tng trng, kh nng khng bnh, m bo s pht trin bn vng ca ngh nui c chim vy vng. Cc ni dung nghin cu ca ti: - Phn lp cc chng Lactobacillus v Vibrio trong rut c chim vy vng. - Tuyn chn cc chng Lactobacillus c hot tnh khng Vibrio. - Nghin cu c im sinh hc ca cc chng Lactobacillus. - Nghin cu iu kin nui cy thch hp cho cc chng Lactobacillus.

GVHD: Th.s L nh c

SVTH: L Thanh Hun

Chng 1: TNG QUAN TI LIU 1.1. Tng quan v probiotic 1.1.1. Gii thiu v Probiotics Theo T chc nng nghip v lng thc ca Lin hp quc (FAO) Probiotic l nhng vi sinh vt sng, nu c b sung vi liu lng hp l s mang li li ch cho vt ch. Nghin cu ng dng probiotic mi c ch trong 20 nm tr li y, nhng tc dng ca n c nhn thy t lu. Elie Metnhicoff l ngi u tin t nn mng cho vic s dng probiotic (Metnhicoff, 1908). Nm 1908, ng ngh s dng vi khun lactic (Lactobacterium delbruekii spp bulgaricus) ko di tui th con ngi. Ngy nay ch phm probiotic c s dng kh hiu qu phng bnh cho ngi v vt nui. Ch phm probiotic ang c s dng nh mt loi thuc phng v iu tr bnh cho ngi v vt nui. Cc nghin cu cho thy ch phm ny mang li nhiu li ch cho ng rut nh: cn bng h vi sinh vt khu tr trong ng rut, kch thch tiu ha, tng kh nng min dch v phng nga nhim trng cho c th. Ngoi ra, ch phm probiotic s dng trong y hc cn c kh nng gim vim, gim cholesteron, tng qu trnh hp thu khong, ngn nga s pht trin vi khun gy bnh vim rut, iu ho trng hp khng dung np lactose v phng c ung th kt trng. Mt s chng vi sinh vt c s dng trong ch phm probiotic cho ngi nh: Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus johnsonii, Bacillus subtilis Nhiu chng vi khun cng c s dng sn xut ra cc ch phm ng dng rt hiu qu nhm kim sot cn trng gy hi cho cy trng nh vi khun Bacillus thuringiensis, nm Beauveria bassiana, Metarrhizium anisopliae, virus NPV.v.v. Ngoi ra cc ch phm vi sinh cn c s dng lm phn bn vi sinh nhm phn gii cc cht hu c lm giu cho t, phn gii ln kh tiu thnh ln d tiu cy

GVHD: Th.s L nh c

SVTH: L Thanh Hun

trng hp thu c. Cc ch phm vi sinh s dng trong cc h thng x l rc thi v nc thi, lm sch du m trong cc v trn du. Mc d vy, vic s dng cc ch phm vi sinh trong nui thy sn (tm, cua, c, nhuyn th) vn cn kh mi m, mi bt u trong hn thp k gn y. i vi mi loi thy sn khc nhau cn c ch phm probiotic vi nhng chng vi sinh vt thch hp ring. Phng thc s dng ch yu l b sung trc tip vo thc n hoc thm vo mi trng nc nui. Nhiu nh nghin cu trong lnh vc ny u nhn thy ch phm Probiotic to ra nhng thay i ng k v khu h vi sinh trong ng rut theo hng cn bng c li trong cc vi khun c ch tng ng k, s lng cc vi sinh vt c kh nng gy bnh nh E.coli, Samomella, Listeriagim mnh. Nhng pht hin mi v kh nng tng cng min dch, iu chnh nhng sai lch bt li h vi sinh vt ng rut do bnh nhim khun, virus, d ng thc n ang m ra trin vng p dng ch phm ny trong nui trng thy sn, cn bng sinh thi v bo v mi trng. * Thnh phn ch phm probiotic Vi sinh vt trong ch phm Probiotic thng gm 4 nhm sau: - Nhm vi khun lactic: Thng chn cc chng vi khun lactic in hnh thuc ging Lactobacillus (Lactobacillus acidophillus, Lactobacillus plantarum, Lactobacillus casei ). Trong hot ng sng vi khun lactic chuyn ng thnh axit lactic, ngoi ra n c th sinh ra Baterioxin mt loi hp cht c hot tnh khng khun v c ph c ch vi sinh vt kh rng. Loi cht khng sinh ny khng gy ra tnh khng khng sinh cc vi khun gy bnh. Axit lactic v Bacterioxin c kh nng c ch vi khun gy bnh, gii c cho ng rut. - Nhm vi khun d dng hoi sinh: Chn cc chng vi khun c hot tnh amylase v protease. Cc chng ny khng gy c, gy bnh cho ngi v vt nui. Nhm vi khun ny c tc dng phn gii cc hp cht hu c b nhim trong mi trng. Thng chn cc chng vi khun thuc ging Bacillus (Bacillus subtilis, Bacillus megaterium, Bacillus lichenifomis, ). Ngoi kh nng sinh

GVHD: Th.s L nh c

SVTH: L Thanh Hun

amylase v protease chng cn sinh ra Bacterioxin. Cc vi khun ny ch yu tham gia vo qu trnh lm sch mi trng v tham gia vo qu trnh u tranh sinh hc, c ch vi khun gy bnh, hn ch dch bnh cho vt nui. - Nhm vi khun quang t dng ( Photoautotrop): Gm cc vi khun ta v vi khun khng ta c kh nng ng ha H2S v CO2 xy dng t bo v khng sinh ra O2. Trong khi phn hy H2S hoc mui sulfua kim loi gy ra, lm gim tnh c cho mi trng. Khi cho thm vi khun quang t dng vo thc n hoc mi trng nc nui c th loi b nhanh chng NH3, H2S, axit hu c, nhng cht c hi, cn bng pH, ci thin cht lng nc. - Nhm nm men: Gm mt s loi thuc ging Sacaromyces, s dng nm men s c tc dng tn dng lng ng c to thnh trong mi trng nc ln men ru nh, hn ch dinh dng ca cc loi vi sinh vt gy bnh khc, ci thin mi v mu ca nc. 1.1.2. C ch tc ng ca probiotic 1.1.2.1. Sn sinh ra cc cht c ch Cc vi khun probiotic sinh cc cht c ch: bacteriocin, siderophores, lysozym, protease, H2O2, cc axit hu c, amoni, diaxetylcc cht ny c sinh ra bn trong ng rut v bn ngoi mi trng nui s c tc dng c ch s pht trin ca cc vi khun gy hi khc. (Saurabh S, Choudhary AK v Sushma GS, 2005).V d: Lactobacillus sp sn sinh ra bacteriocin l cht c ch sinh trng ca cc vi khun gy bnh. Alteromonas sp dng B-10-31 sn sinh monostatin lm c ch hot tnh protease ca Aermonas hydrophyla v Vibrio anguillarum. 1.1.2.2. Cnh tranh c cht, nng lng vi nhng vi khun khc Cnh tranh dinh dng ca cc vi sinh vt probiotic c vai tr quan trng trong vic m bo s n nh ca h vi sinh vt bn trong ng rut v bn ngoi mi trng nui. Probiotic c kh nng cnh tranh v mt dinh dng i vi cc vi khun ly bnh v s hn ch c s pht trin, ly lan ca cc chng vi sinh vt . Vi

GVHD: Th.s L nh c

SVTH: L Thanh Hun

khun c hi b loi b c ngha l loi b c i th cnh tranh cht dinh dng v nng lng dng cho vi khun probiotic v cho vt ch. V d: s hp th cc monosaccarit ca cc vi sinh vt probiotic c th lm gim s pht trin ca Clostridium difficile gy ra bnh tiu chy trn vt nui bi v s pht trin ca Clostridium difficile ph thuc vo cc monosaccarit (Kenneth, H.Wilson v Fulvio Periniz, 1998). Ngoi ra cc vi khun sinh siderophore c s dng lm probiotic cnh tranh Fe vi cc vi khun gy hi. V siderophore l cht c khi lng phn t thp, n c kh nng gn vi cc ion Fe. Siderophore c th ho tan Fe kt ta thnh dng d s dng cho vi sinh vt, do n l cng c thu lm Fe trong mi trng. 1.1.2.3. Cnh tranh v tr bm dnh vi vi khun gy bnh Cnh tranh v tr bm dnh trn ng rut l mt tiu ch quan trng dnh gi hiu qu ca vi sinh vt probiotic (Ringo E v Gatesoupe FJ, 1998). Vi khun probiotic cnh tranh v tr bm dnh trn thnh rut hay trn cc biu m khc c vai tr ngn cn s khu tr v pht trin ca cc vi khun gy bnh .Ngi ta chng minh kh nng bm dnh v pht trin trn b mt rut ca Lactobacillus GG v Lactobacillus plantarum 299V ngn cn s pht trin v ly lan ca vi khun Escherichia coli 0157H7 gy bnh tiu chy ngi v ng vt nui (Mack DR, Michail S, Wei S, Macdougal L, Hollingsworth MA, 1999). Ngoi ra mt s vi khun thuc ging Lactobacillus cng c bit c th kha cht cc th th b mt trong ng rut hn ch c s bm dnh ca cc vi sinh vt gy bnh khc. (Bernet MF, Brassart D, Neeser JR, Servin AL, 1994 ). 1.1.2.4. Tng cng p ng min dch Ch phm probiotic c tc ng tch cc ln h thng min dch ca c th. Nhiu nghin cu cho thy cc vi khun trong ch phm probiotic c kh nng tng cng qu trnh sn xut cc khng th v gii phng cytokin gy ra p ng min dch cho c th. (Jon A. Vanderhoof, M.D., Series Editor, 2005). Probiotic

GVHD: Th.s L nh c

SVTH: L Thanh Hun

cn tc ng ln cc t bo tua, t cm ng t bo limpho T dung np/iu ha v kch thch cc p ng min dch. Ngoi ra, khng nguyn ca vi khun probiotic kch thch t bo nim mc rut sn sinh khng th chng li tc nhn gy bnh, cc chht c ch ca probiotic tip tc nng cao hiu qu khng th ca vt ch. Ngi ta thy rng b sung vi khun lactic lm tng kh nng chng li bnh truyn nhim ng rut. Mt s nghin cu khc cng cho thy probiotic c nh hng quan trng i vi cc p ng min dch thch ng nhng bnh nh d ng sa b tr em, vim khp t min ngi ln. 1.1.2.5. Ci thin cht lng nc Ch phm probiotic gip gim nng cc cht hu c trong nc, gim hm lng BOD, gim c do amoni, nitrat v kh hydrosunfua, khng ch c vi khun gy bnh. Cc vi sinh vt trong ch phm probiotic c th ci thin cht lng nc bi chng c th s dng cc cht hu c d tha hoc cc cht kh nh H 2S lm cht dinh dng thng qua cc enzyme ngoi bo. (Prieur v cng s,1990). 1.1.3. ng dng ca probiotic 1.1.3.1. ng dng ca ch phm Probiotic trong y hc, trong trng trt, trong bo v mi trng. - Trong y hc: cc ch phm probiotic khi c b sung vo ng rut s gip lm sch ng rut, c ch cc vi sinh vt gy bnh, cn bng h sinh thi ca cc vi sinh vt trong ng rut, loi b cc qu trnh ln men bt li do cc vi sinh vt c hi ny gy nn, lm cho cc chc nng ca ng rut c hot ng tt hn, tng h s hp thu v s dng cc cht dinh dng trong thc n. Ngoi ra, cc hot cht sinh hc t ch phm probiotic nh axit amin, cc enzyme, cc nucleotit, cc axit nucleic, cc vitamin, c bit l biotin c tc dng tng cc qu trnh chuyn ha ca t bo, kch thch v tng cng kh nng min dch, tng sc khng chng li s xm nhp ca cc vi khun c hi.

GVHD: Th.s L nh c

SVTH: L Thanh Hun

- Trong trng trt: Ch phm probiotic c tc dng vi nhiu loi cy trng (bao gm cy lng thc, cy n qu, cy hoa mu) v mi giai on sinh trng pht trin khc nhau (V Th Hnh v cng s, 2004). Nhng nghin cu v tc dng ca probiotic vi cy trng cho thy chng c th: + kch thch s ny mm, ra hoa, kt qu v qu trnh chn ca qu, + ci thin h vi sinh vt t, ngn chn cc mm bnh, + tng cng kh nng hp th dinh dng ca cy trng v ko di c thi gian bo qun, tng cht lng cc sn phm ti sng, lm cho hoa tri ti lu. Dng ch phm probiotic trong t c th ti lp qun th h vi sinh vt mi c li cho cy trng, c bit l h vi sinh vt vng r. Cy trng s pht trin tt t, ni m cc vi sinh vt c ch chim vai tr ch yu, gip cho cy trng nng cao c hiu sut quang hp v s dng phn bn, c bit l phn bn hu c (V Th Hnh v cng s, 2005).
- Trong bo v mi trng: Do c tc dng tiu dit cc vi sinh vt gy thi

(sinh ra cc loi kh H2S, SO2, NH3) nn khi phun ch phm probiotic vo rc thi, cng rnh, chung tri chn nuis kh mi hi mt cch nhanh chng. ng thi s lng rui, mui, ve, cc loi cn trng bay khc gim hn s lng. Rc hu c c x l EM ch sau mt ngy c th ht mi v tc mn ho din ra rt nhanh. Trong cc kho bo qun nng sn, s dng EM c tc dng ngn chn c qu trnh gy thi, mc. Cc nghin cu cho bit ch phm EM c th gip cho h vi sinh vt tit ra cc enzym phn hu nh lignin peroxidase, enzyme amylase, protease, cellulase v c bit l cellulase, hemicellulase. Cc enzym ny c kh nng phn hu cc ho cht nng nghip tn d, thm ch c dioxin. Belarus, vic s dng EM lin tc c th loi tr nhim phng x (Teruo Higa, 2002). 1.1.3.2. ng dng ca ch phm Probiotic trong nui trng thy sn

GVHD: Th.s L nh c

SVTH: L Thanh Hun

Mc d ch phm probiotic mi c ng dng vo nui trng thy sn trong thi gian 10 nm tr li y nhng hiu qu ca ch phm ny l rt to ln. Hin nay cc loi vi sinh vt: Bacillus, Lactobacilus, nhm vi khun quang dng c s dng ch yu sn xut cc ch phm ny. Nhng nghin cu cho thy rng cc loi vi khun ny u khng c hi, d nui cy, d tn ti trong mi trng nc. Ci thin mi trng nc nui: Ch phm probiotic c b sung vo mi trng nc nui thy sn c tc dng ci thin cht lng nc. Trong nui trng thy sn, lng thc n d tha do ng vt thy sn hp th khng ht chim s lng rt ln, y l nguyn nhn chnh gy nhim mi trng nc. c bit trong nui tm, tm ch hp th c di 1/3 tng lng dinh dng u t vo ao nui (Briggs v Funge-Smith, 1994) v phn cn li b mt vo h thng ao nui (Wu, 1995 v Piedrahita, 2003). Hn na, cc cht bi tit t cc loi thy sinh vo mi trng nc chim khong 70 80% lng protein chng tiu ha, phn ln trong s (80%) di dng d ha tan trong nc, c bit l ammoniac (Porter v cng s, 1987). Cc cht thi ny, bao gm thc n d tha v cc sn phm bi tit, c th ph nhng cho ao nui v kt qu l s pht trin bng n ca to c cng nh gy ra hin tng thiu -xy trong nc. Ch phm probiotic c b sung vo mi trng nc c cha cc vi khun c kh nng sinh ra cc enzyme ngoi bo nh protease, amylase c th phn gii cc cht hu c v cc cht bi tit thnh CO2 v nc, chuyn cc cht c hi nh NH3, H2S, NO2 - thnh cc cht khng c nh NO3-, NH4+, gip gim mi hi ca mi trng nc, n nh pH v mu nc ao (V Th Hnh v cng s, 2005). Cc chng vi sinh thng c s dng thc hin chc nng ny gm Bacillus sp, Nitrosomonas, Nitrobacter.. nh vy cc vi khun trong ch phm probiotic co tc dng - Lm n nh cht lng nc v nn y trong ao nui tm c. - Gim thiu nhim mi trng ao nui v xung quanh do nui trng thu sn gy nn.

GVHD: Th.s L nh c

SVTH: L Thanh Hun

10

Nhiu loi ch phm vi sinh c s dng ci thin mi trng nc nui trng thy sn nh: ch phm EM, BIOnAQUA 1-MV( cng ty Mai Vit), BZT Aquaculture (USA), BIO II (V Th Hnh v cng s, 2005). Tng tc sinh trng, kh nng khng bnh cho i tng nui: Ngh nui trng thy sn ang i mt vi cc loi bnh dch, nguyn nhn ch yu l do cc loi vi khun Aeromonas.sp, Pseodomonas.sp, Vibrio sp. Tuy nhin , chng ta hn ch s dng khng sinh phng bnh nn ch phm probiotic c vai tr v cng quan trng. Hin nay trong nui trng thy sn cng phn lp c nhiu loi probiotic nh Carnobacterium piscicola (Hius, 1984), Lactobacillus plantarum (Schroder, 1984), Pediococcus acidilactici (Pucci, 1988 ). Cc chng vi sinh vt c th cnh tranh v tr bm dnh v dinh dng bn trong ng rut nh kh nng chu mn, chu kim, chu axit ca ng rut. Bn cnh , cc chng probiotic ny cng c ch hn nh kh nng sinh ra cc cht c ch (bacteriocins, sideropheres, lysozymes, hydrogen peroxides..) v tng hp c cc hp cht khng sinh tng ng nh: piscicolin, plantarin, pediococin (PA - 1) c tnh st khun cao, c bit i vi vi khun gy bnh nh: Listeria monocytogenes (Pucci, 1988), Shigella spp. v Salmonella spp V vy khi b sung ch phm probiotic c cha cc vi sinh vt ny vo thc n s km hm s pht trin ca cc vi khun gy bnh, m bo s n nh ca h vi sinh vt ng rut tm c, gip i tng nui tng trng v pht trin tt, chng chi c vi cc loi bnh dch. S dng probiotic trong nui trng thy sn s hn ch dng mt lng ln cht khng sinh v ha cht vo ao nui thy sn. c bit l hn ch ng k kh nng gy bnh ca mt s loi vi khun c hi trn i tng nui y l bin php tng hiu qu sn xut c ngha thc tin (Xiang-Hong v cng s, 1998). Bn cnh ch phm probiotic s gip tm c sinh trng mnh hn do cc vi khun trong ch phm probiotic chnh l ngun cung cp cc enzyme tiu ha, mt s vitamin v axit bo c tc ng tch cc n qu trnh chuyn ha ca c tm. (Sakata,

GVHD: Th.s L nh c

SVTH: L Thanh Hun

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1990). Nh tm c s hp th thc n tt hn, c sc chng chu vi cc iu kin mi trng, tng sc khng. Ngoi ra, ch phm probiotic cn c bit vi kh nng khng virus. Mt s nghin cu cho thy cc chng vi sinh vt trong ch phm probiotic c kh nng khng mt s tc nhn virus gy bnh trn ng vt thy sn, trong kh nng khng virus gy bnh trn trn tm s nh IHNV (Kamei v cng s, 1988) v OMV (Direkbusarakom v cng s, 1998). Nh vy, ch phm probiotic cn c th gip cho nui trng thy sn chng chi vi tc nhn gy bnh nguy him nht l virus. y c th l mt u th ln cho vic s dng ch phm probiotic vo nui trng thy sn trong tng li. 1.1.4. Cng ngh sn xut ch phm probiotics. Quy trnh to ch phm probiotic :

Nguyn liu

Chng ging

Nhn ging

Ln men

Sinh khi Cht ph gia

Ch phm dng bt

Ch phm dng dch

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1.1.4.1. Nguyn liu

Mi trng dng nui thu sinh khi vi khun cn m bo thnh phn cht dinh dng, khong, cht kch thch sinh trng, VTM. V mt yu cu i vi nguyn liu dng sn xut cng nghip l nguyn liu phi r tin, d kim. i vi chng ging vi sinh vt, cn la chn nhng chng c y cc c tnh m ta cn sn xut ch phm m ta mong mun.

1.1.4.2. Nhn ging

Chng ging c bo qun trong t lnh nhit 4oC. V vy, khi s dng ta cn hot ha ging, bng cch cy chuyn sang ng thch nghing, t trong t m 320 sau 24-48h.
Nhn ging cp1: Chng sau khi pht trin tt trong ng thch nghing c

cy chuyn tip sang ng nghim cha 10ml mi trng MRS, LB, EB i vi cc chng vi khun lactic, bacillus, vi khun quang dng. Nui 370C trong 24h.
Nhn ging cp 2: Cc chng ging cp 1 pht trin tt c cy chuyn

sang mi trng lng vi t l 10% ging, trong bnh tam gic. Nui 370C trong 24h.
Nhn ging cp 3: Ging cp 2 c nhn tip sang thng nha c th tch ln

hn vi t l ging 10%, nui 370C. Ging cp 3 c dng ln men trong nhng thng ln men ln thu sinh khi.

1.1.4.3. Thu sinh khi

Sau ln men, dch ln men c x l thu sinh khi. C cc phng php thu sinh khi nh: - Ly tm vi tc 10000 vng/pht, nhit 40C trong 10 pht. Phng php ny ch c p dng trong phng th nghim. - Dng mng siu lc. Nhng mng ny c th lc c vi khun. Hin nay phng php ny c p dng nhiu trong cng nghip.

1.1.4.4. To ch phm

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Dch sinh khi sau khi x l c trn vi cht mang. Cht mang gip vi khun c th tn ti lu, n nh cht bo qun ch phm. Cht mang thng l kh u tng, kh lc, cm go, bt go, ng lactose, sucrose Sau khi trn cht mang ta tin hnh sy kh ch phm n m 8-12% bo qun. C th dng cc phng php sy:
Sy nhit:

Cho ch phm vo thit b sy bnh thng nhit 40-500 C trong 4-5h. u im: - Tn t chi ph cho thit b - Hao ph cho nng lng thp Nhc im: - Thi gian sy ko di - Khng th t ng ha c - Cht lng sy km Sy ht chn khng + Nguyn l lm vic: Di mi trng p sut chn khng, cc khay cha nguyn liu c quay o bn trong bung sy nhm tng hiu sut sng cao tn. Hm m trong nguyn liu c gia nhit nhanh v bay hi thot ra ngoi b mt. tng tc sy so vi kiu gia nhit truyn dn. + u im: Hiu sut sy cao, tiu hao nng lng t, nhit sy thp nn thch hp vi sn phm nhy nhit. + Nhc im: Thit b t tin. Sy chn khng thng hoa + Nguyn l hot ng: Giai on 1: lm lnh ch phm n -180 C kt tinh lng nc trong v trn b mt nguyn liu chuyn t lng sang rn. Giai on 2: To iu kin chn khng kt hp vi vic nng nhit t t (ln n 250 C) nc trong nguyn liu chuyn t rn sang hi.

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+ u im: Nhit sy thp nn bo ton c gi tr ca sn phm. + Nhc im: Chi ph cho thit b v nng lng cao.
1.1 . Tng quan v c chim vy vng

1.2.1. Gii thiu chung v c chim vy vng C chim vy vng (Trachinotus blochii Lacepde 1801) phn b min Nam Nht Bn, Ty Thi Bnh Dng, n Dng, i Ty Dng. Vng bin nhit i v nhit i, vng bin ng Nam v vng bin Ty Chu Phi. Trung Quc chng phn b vng bin ng Hi, Nam Hi, Hong Hi, vng bin Qung ng, Qung Ty, Hi Nam, Phc Kin. C chim vy vng cng tm thy vng bin nc ta. Vit Nam, c chim vy vng c tm thy trn vnh Bc B, min Trung v Nam B.

Hnh 1.1 : C chim vy vng C chim vy vng c c th hi trn v b bn dp chnh gia lng hnh vng cung, b mt da c mu trng xm v vy lng c mu nh bc vng. C chim vy vng c kch thc tng i ln, kch thc c th t 45 - 60 cm. C sinh trng nhanh, trong iu kin nui bnh thng 1 nm t 0,5 - 1 kg/con. T nm th 2 tr i mi nm khi lng tng tuyt i l 1 kg. C chim vy vng l loi c nc m, c tp tnh di c, sng tng gia v tng mt. giai on c ging hng nm sau ma ng c thng tp trung thnh n sng

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vng vnh, ca sng. C trng thnh sinh trng tt nht iu kin nhit 22 280C, l loi rng mui 3 - 33 ppt. Di 20 ppt c sinh trng nhanh, trong iu kin mn cao tc sinh trng chm li. Nhu cu hm lng oxy ha tan ln hn 2,5 mg/L. L loi c n tp, thin v ng vt, c c th kim thc n trong ct, c trng thnh c th bt nhng ng vt v cng nh: ngao, cua, c. Giai on c ging thc n l ng vt ph du v ng vt y, ch yu l lun trng, nauplius ca Artemia. C con n tm c nh, hai mnh v nh. Thc n chnh ca c trng thnh l cc loi tm c nh. C trng thnh n tm nh v thc n cng nghip hoc hon ton thc n cng nghip trong nui thng phm. C Chim vy vng (Trachinotus bloochi) c tht thm ngon, c kh nng chng chu dch bnh tt. C chim vy vng l loi c bin c gi tr kinh t cao, c nui nhiu vng bin min Nam Trung Quc v mt s quc gia trn th gii, c ang c tiu th mnh Nht Bn, i Loan, Hng Kng, Trung Quc, M, Singapore. Nc ta c vng bin m, nhiu vng vnh, m ph, eo bin, bin ni a t sng gi. Bn cnh , l cht lng nc tt, mn nc bin tng i n nh. y l iu kin v cng thun li pht trin ngh nui c chim vy vng Vit Nam. 1.2.2. Tnh hnh nghin cu c chim vy vng trn th gii v trong nc 1.2.2.1. Tnh hnh nghin cu c chim vy vng trn th gii Nm 1986, i Loan, Lm Lit ng thu gom 126 con c chim vy vng loi nh, loi va v ln nui chung vi nhau. Nm 1989, Lm Lit ng ln u tin sinh sn nhn to c chim vy vng thnh cng. Nm 1991, Lm Lit ng tng thm n b m cho tin hnh sinh sn nhn to. Nm 1993, Trung tm chuyn giao cng ngh Trng i hc Trung Sn kt hp vi Tri Nghin cu ging Thy sn Qung ng Trung Quc nghin cu sinh sn nhn to thnh cng ging c chim vy vng quy m nh (ng nui u trng trong b xi mng). Nm 1998, Trung tm kt hp vi Cng ty trch nhim hu hn

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ging thy sn Thng Li Hi Nam Trung Quc nghin cu sn xut nhn to thnh cng trn quy m ln (ng nui u trng trong ao t). Bn cnh , Trung tm pht trin nui bin Batam Indonesia (Nur. Muflich Juniyanto, Syamsul Akbat v Zakimi) thnh cng trong vic sn xut ging nhn to c chim vy vng, v vy con ging c th c sn xut a phng vt mc v lm gim p lc khai thc ging ngoi t nhin. C chim vy vng v ang c nui nhiu vng bin min Nam Trung Quc v mt s quc gia trn th gii, c ang c tiu th mnh Nht Bn, i Loan, Hng Kng, Trung Quc, M, Singapore. 1.2.2.2. Tnh hnh nghin cu c chim vy vng Vit Nam Nm 2006, Trung tm Khuyn nng Quc gia phi hp vi Trng Cao ng Thy sn, Vin Nghin cu Nui trng Thy sn I thc hin d n nhp cng ngh sn xut nhn to c chim vy vng do Trung tm chuyn giao cng ngh Trng i hc Trung Sn Trung Quc chuyn giao. Nm 2009 Khoa Nui trng Thy sn Trng i hc Nha Trang bt u th nghim cho nhn to c chim vy vng ti B Nghin cu Thc nghim - Khoa Nui trng Thy sn Trng i hc Nha Trang ti Vng Ngn Nha Trang Khnh Ha, sau p trng v ng ging thnh cng ti Tri Sn xut Ging c Bin Khoa Nui trng Thy sn Trng i hc Nha Trang, ti ng Nha Trang. Vi ngun ging v k thut nui c ch ng sn xut, mt s vng nui nc ta c chuyn giao cng ngh nui thng phm c chim vy vng. u nm 2011, Trung tm ging Hi sn tnh Nam nh nui th nghim ging c chim bin vy vng trong ao t v c hiu qu cao. Bn cnh , Qunh Lu Ngh An ang xy dng m hnh nui thng phm c chim vy vng. Hin nay c chim vy vng ang c nui th nghim Ph Yn, Vng Tu, Nha Trang v bc u mang li hiu qu. Trn th gii v trong nc mi ch tp trung vo vn sinh sn nhn to v k thut nui c chim trng vy vng, trong khi c chim trng vy vng nui lng

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bin vn c th i din vi nguy c bnh dch v b nh hng bi cc tc ng t mi trng t nhin. Tuy nhin t trc n nay, vn nghin cu v thc n v bnh dch trn c chim trng vy vng vn cn b ng, cha c mt nghin cu no v lnh vc ny c cng b.
1.2.3. Tnh hnh bnh dch trn c nui nc mn ni chung v c chim vy vng

ni ring Nui c bin l ngh pht trin mnh Vit Nam t 30 nm nay v mang li hiu qu kinh t to ln nhng trong thi gian gn y ang c du hiu dng li do dch bnh xy ra nhiu, nguyn nhn gy bnh bao gm: 1.2.3.1. Bnh do nm, k sinh trng Nm l nguyn nhn gy nn nhiu bnh c nh: Bnh nm thy mi gy ra do do 4 ging nm Leptolegnia, Aphanomyces, Saprolegnia v Achlya; Bnh nm mang - gy ra bi Branchiomyces. Cc loi nm gy hi nhiu i vi nhiu loi c nui giai on c con, c tht v trng c. Nm gy bnh trn c lm cho c b nga ngy, l lot, km n. Bin php phng v tr hin nay thng dng mui n ( NaCl) nng 2-3% sulfate ng ( CuSO4) nng 0,5g/m3 nc tm cho c nui. K sinh trng cng l tc nhn gy bnh trn c bin nui. Nguyn Th Mui v Th Ha (1978-1980) pht hin c 80 loi k sinh trng k sinh trn c bin. Nhiu loi k sinh trng nh: k sinh trng n bo (Amyloodinium), k sinh trng bnh xe (Trichodiniosis), v cc loi sn l n ch (Monogeneansis, Neobenedenia girellae, Benedenia epinepheli v Benedenia sp) cng l nguyn nhn gy ra mt s bnh c nui. Mc d khng gy tn tht ln nhng n lm cho c chm ln, gim cht lng tht c, to c hi cho vi khun v virus tn cng. Bin php phng nga cc bnh do k sinh trng thng l gi v sinh ao c nht l ao ng, ci to ao nui k cng, th c vi mt va phi v ty giun sn nh k cho c nui bng thuc.

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1.2.3.2. Bnh do virus Virus l tc nhn gy bnh nguy him trn c bin nui lng b. Tuy nhin Vit Nam tnh hnh nhim bnh do virus trn c nui vn cha nghim trng, cc tc nhn virus gy bnh thng gp ch yu tm nui nh bnh m trng ( WSSV), bnh u vng (YHV), bnh hoi t c quan to mu (IHHNV), hi chng Taura (TSV) trn c bin nui, tc nhn virus gy bnh c ghi nhn nhiu l virus gy bnh t hoi thn kinh (VNN). Bnh ny gy ra trn nhiu loi c bin v phn b rng ri nhiu vng a l khc nhau (Takana v cng s 2003, 2004; Curtis v ctv 2001...). Bnh ny c pht hin trn cc loi c m (Epinephelus spp.) c chm (Lates calcarifer) v c bp (Rachycentron canadum) nui Khnh Ha, Vit Nam (Trn V Hch v cng s, 2008). Mt s bnh do virus cng gy thit hi ng k cho ngh nui c bin trn th gii nh virus IHN gy hoi t c quan to mu c hi vn, iridovirus gy ra hin tng hoi t mang v da c tm trng. Ngoi ra, mt s bnh khc do virus cng c ghi nhn cc loi c khc nh bnh nhim trng xut huyt do vi rt do virus VHS gy nn c hi, c trch, c bn; bnh do RSIV c vc, c trp v c m song. Hin nay cha c bin php cha tr cho bnh bi virus trn c nui nn gim thiu nguy c mc bnh virus th phi m bo ngun ging sch virus v hn ch nhp, xut khu cc loi c c nguy c nhim virus bit. 1.2.3.3. Bnh do vi khun Hin nay, bnh do vi khun gy thit hi rt ln cho ngh ng nui c thng phm. Nhiu bnh trn c nui lng b trn bin do vi khun c ghi nhn nh: bnh m trng thn trn c gi nui thng phm, bnh Vibriosis, bnh mn vy ct ui v bnh xut huyt nhim trng mu c m, c gi, c chm ( Th Ha v cng s, 2008). Theo thng k Khnh Ha, c khong 30% h nui c bin b chu tc hi ca bnh do vi khun. Bnh c th xy ra nhiu loi c bin nui nh c m, c chm, c

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hng, c bit giai on c nh (5-20cm), c nui lng thng chu tc hi nng hn giai on c ln vi t l cht c th t 50-100%, y l bnh khng c ma v r rng ( Th Ha, 2008). Cc virus gy bnh trn c bin c bit nh : Vibrio spp, Aeromonas spp, Flexibacter sp, Pseudomonas fluorescents, Pseudomonas putida , Photobacterium damsela, ( Th Ha, 2008). Trong , nhm vi khun Vibrio spp gy bnh ang c ch hn c v tc ly lan v mc nh hng nghim trng ca chng trong ngh nui trng thy sn hin nay. Vibrio nhm vi khun gy bnh in hnh ng vt thy sn Bnh vibriosis l tn gi chung cho cc bnh khc nhau ng vt thy sn do vi khun Vibrio spp. gy ra. Trong bnh vibriosis, vi khun Vibrio c th l tc nhn s cp hoc tc nhn th cp (tc nhn c hi, k sinh trng k sinh hay cc tc ng mi trng nh c hc, ha hc) c th ng cc vai tr quan trng trong cc dch bnh vibriosis ng vt thy sn ( Th Ha v cng s, 2004). Vibrio l tc nhn gy bnh quan trng i vi ng vt nui thy sn. V. anguillarum, V. salmonicida, v V. vulnificus l ba trong s nhng tc nhn gy bnh chnh cho vi loi c ( Bi Quang T , Phan Th Vn v cng s, 1998). S lng cht gy ra bi Vibrio trn c v cc loi s hn l rt ph bin trong giai on u trng sm v c th xut hin t ngt, i khi dn n cht ton b (Thompson v cng s, 2004). Trong nhng nm gn y, ngh nui c lng trn bin pht trin mnh, bnh vibiosis tr thnh cc bnh thng gp v gy nhiu tc hi cho ngh nui thy sn ( Th Ha v cng s, 2004). Bnh do Vibrio gy ra c th quan st c khp mi ni c ngh nui ng vt thy sn nc l v nc mn, s phn b ca bnh ny rng khp th gii, tp trung chu , Phi v M. Nhiu loi c bin c gi tr kinh t cao ang c nui ph bin nhiu quc gia chu , nh c m (Epinephelus spp.), c chm (Lates calcarifer) thng b bnh ny, c bit l hnh thc nui lng b trn bin (Liopo v cng s, 2001). Bnh

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thng th hin cc du hiu: trn thn xut hin cc m nh, ti vy c b trc v rng i, sau mt thi gian to nn cc vt lot nh, su. Gii phu bn trong cho thy hin tng xut huyt ni tng, v xut huyt trong c ca c. C b bnh c th gy cht hng lot khi b cp tnh, gy cht ri rc khi cc th th cp tnh ( Th Ha v cng s, 2004). T c bnh Vit Nam ngi ta phn lp c mt s loi vi khun nh Vibrio parahaemolyticus, V. alginolyticus, v V. anguillarum (Phan Th Vn v cng s, 2000). Ngoi ra c nhng thng bo khc v bnh do Vibrio c nh vi khun V. anguillarum, V. vulnificus gy bnh nhim khun mu c trnh, V. salmonicida gy bnh c vng nc lnh ( Th Ha v cng s, 2004). Tnh hnh bnh dch v nguyn nhn gy bnh trn cc loi c nui thng phm nh c m, c chm, c gi c nhiu nghin cu cng b. Tuy nhin do c chim vy vng mi c a vo nui thng phm trong thi gian gn y nn cha c nghin cu c th v dch bnh c cng b, nhng qua thc tin nui c chim trng vy vng lng b trn bin, chng ti quan st thy s lng c b hao ht do bnh tt l tng i ln. Chng ti cng tin hnh thu mu c chim trng vy vng b bnh em v phn lp cc chng gy bnh, kt qu l c rt nhiu vi khun thuc Vibrio spp c phn lp, y l mt c s kim tra v xem xt cc tc nhn vi khun gy bnh trn loi c ny trong tng lai. lm sng t vn ny th cn cc nghin cu c th nh gi cng nh a ra cc bin php khc phc ngh nui thng phm c chim trng vy vng c pht trin mnh.
1.2 . Tng quan v vi khun lactic 1.3.1.

Gii thiu v vi khun lactic 1.3.1.1. Gii thiu chung T lu vi khun lactic c con ngi ng dng rng ri ch bin cc loi thc n chua (sa chua, mui da, mui c, ), chua thc n cho gia sc hoc sn xut axit lactic v cc loi mui ca axit lactic. Ngay t nm 1780 nh ha hc ngi Thy in Scheele ln u tin tch c axit lactic t sa b ln men chua. Nm 1875, L.Pasteur chng minh c rng vic lm chua l kt qu hot ng ca mt

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nhm vi sinh vt c bit gi l vi khun lactic. 21 nm sau (1878) Lister phn lp c vi khun lactic v t tn l Bacterium lactic (ngy nay gi l Streptococus lactic ) v ngnh cng nghip ln men nh vi khun lactic hnh thnh t nm 1881. Nhng vi khun gy ln men lactic c gi l vi khun lactic, chng c th ln men c cc loi ng monosacarit, disacarit nhng khng ln men c tinh bt. Vi khun lactic thng c dng hnh cu ( hoc oval) v hnh que, ng knh ca cc dng lactic thng t 0.5 1.5 m, cc t bo hnh cu xp thnh cp hoc chui c chiu di khc nhau. Kch thc t bo trc khun lactic khong 1 8 m, trc khun ng ring r hoc kt thnh chui. Tt c cc vi khun lactic u khng chuyn ng, khng sinh bo t, G+, k kh ty tin vi hiu kh. Trc khun thng nhy cm hn so vi lin cu khun. Vi khun lactic ln men c monosaccarit v disaccarit nhng khng phi tt c cc vi khun lactic u ln men c bt k loi disaccarit no, mt s khng ln men c saccaroza, mt s khng s dng c latose, cc vi khun lactic khng ln men c tinh bt ( tr chng Lactobacillus delbruceckii) v cc polysaccarit khc. Kh nng to thnh axit lactic ca cc loi vi khun lactic khc nhau thi rt khc nhau v nh vy bn vi axit ny cng rt khc nhau. a s cc trc khun lactic ng hnh to c axit lactic cao, cc trc khun ny c th pht trin pH = 3,8 4 cn cu khun khng th pht trin trn mi trng ny, hot lc ln men tt nht ca trc khun l pH = 5 6. a s vi khun lactic c bit l trc khun lactic ng hnh rt kn chn thnh phn dinh dng trong mi trng v ch pht trin c trong mi trng c tng i y cc axit amin hoc cc hp cht Nit phc tp hn. Ngoi ra chng cn c nhu cu v vitamin ( B1, B6 , PP, cc axit pantotenic, axit folic ). V vy mi trng nui cy vi khun lactic kh phc tp. Chnh cc c im dinh dng v axit amin v vitamin cho nn nhiu chng vi khun lactic c dng trong phn tch hai dng hp cht ny cc c cht khc nhau.

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Vi khun lactic c hot tnh protease phn hy protein thnh peptit, axit amin, hot tnh ny cc loi khc nhau th khc nhau, thng trc khun l cao hn. Vi khun lactic chu c trng thi kh hn, bn vng vi CO2 v etylic, nhiu loi vn sng c trong mi trng 10 15% cn hoc cao hn, mt s trc khun bn vi NaCl, c th sng trong mi trng t 7 10 % NaCl. Vi khun lactic a m c nhit sinh trng ti thch trong khong 25 350C, nhm a nhit c nhit ti thch 40 450C, nhm a lnh pht trin tt nhit tng i thp ( <= 50C). Khi gia nhit n 60 800C th hu ht b cht sau 10 30 pht. Mt s vi khun lactic c kh nng to thnh mng nhy, mt s khc c kh nng i khng vi th hoi sinh v cc vi sinh vt gy bnh hoc lm thi ra thc phm. Nh vy ngoi kh nng to ra axit lactic, cc loi ny cn sinh ra cc hp cht c hot tnh khng sinh ( Bacterioxin). Trong t nhin, vi khun lactic thng gp trong khng kh, t, nc nhng ch yu l thc vt v cc sn phm thc phm thc phm (rau qu, sa, tht,) mt s tm thy trong ng tiu ha ca ngi v ng vt. 1.3.1.2. Phn loi vi khun lactic Ngi ta da vo qu trnh ln men chia vi khun lactic thnh hai loi: vi khun ln men lactic ng hnh v d hnh. 1.3.1.2.1. Ln men ng hnh - Lactobacterium casei: y l nhng trc khun rt ngn gy chua sa t nhin. Ym kh tu tin, ln men tt glucose, maltose, lactose to ra mi trng c t 0,8 1% axit lactic. iu kin bnh thng gy chua sa trong vng 10 n 12 gi. Ngun nit cho vi khun ny l peptone. Nhit ti thiu cho chng pht trin l 100C, ti u l 350C v ti a l 450C, chng thu phn cazein v gelatin rt yu. - Streptococcus cremoris: Thng to thnh chui di, thng pht trin nhit thp hn Lactobacterium casei, ti u t 250C 300C, ln men glucose, galactose.

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23

- Lactobacterium bulgaricus: y l trc khun rt di, nhit pht trin ti u l 200C, c kh nng ln men glucose, lactose. C kh nng to axit cao (3,7% axit lactic). - Lactobacterium delbruckii: thng gp trn ht i mch, y l trc khun ln. Trong qu trnh pht trin ca mnh chng c kh nng to thnh si. Nhit ti u cho chng pht trin t 45 500C, khc vi cc loi khc chng khng c kh nng ln men ng lactose v vy chng khng c dng trong ch bin sa. - Lactobacterium cueumeris fermenti: thng tm thy chng trong sa chua. L trc khun khng chuyn ng, thng to thnh t bo n v c khi to thnh chui. Thng chng to thnh chui trong qu trnh ln men. Kh nng to axit ti a trong mi trng t 0,9 1,2%. 1.3.1.2.1. Ln men d hnh - Streptobacterium hassice fermentatae: thng thy chng trong cc dch ln men chua rau ci. Chng tn ti tng t bo ring bit hoc ghp thnh tng i, hoc chui ngn c khi ghp thnh tng chui di hnh si. Khi ln men rau ci chua to thnh axit lactic, axit axetic, ru etylic v CO2. Ln men ng saccarose tt hn ln men ng lactose. - Lactobacterium lycopersici: l trc khun gram dng, sinh hi, t bo to thnh chui hay n, c khi ghp thnh i mt. Khi ln men chng to thnh axit lactic, ru tylic, axit axetic v CO2. Chng c kh nng to bo t, t bo sinh dng thng cht nhit 800C. 1.3.2. Gii thiu v ging Lactobacillus Lactobacillus l vi khun Gram dng, kch thc ln (0,5 - 0,7 2 - 8 m), i b phn cc loi khng di ng, khng sinh bo t, c dng hnh que hay hnh cu. Chng c nhng nhu cu v dinh dng phc tp v lm ln men hon ton, hiu kh hay k kh, a axit.( De Man v cng s, 1960). Nhit pht trin ti thch thng l 30 350 C. Khun lc ca Lactobacillus thng c hnh trn, mu trng hay c sa.

GVHD: Th.s L nh c

SVTH: L Thanh Hun

24

Lactobacillus c c trng bi kh nng sn xut axit lactic l mt sn phm ph ca qu trnh chuyn ha glucose. -Lactobacillus c tm thy trong cc mi trng sng c cha nhiu cacbohydrat v phn b rng khp trong thc n, thc n chua, phn bn, sa v cc sn phm sa v trong ng rut ngi, ng vt, thy sn. -Nhiu loi Lactobacillus c s dng thng mi trong vic sn xut cc loi sa chua, pho mt v sa chua, v h c mt vai tr quan trng trong sn xut rau qu ln men (da chua v da ci bp), ung (ru vang v nc tri cy), bnh m bt chua, v mt s xc xch . Ngy nay Lactobacillus c s dng trong ch phm probiotic cho c con ngi v vt nui. c bit, trong lnh vc nui trng thy sn ch phm probiotic ang c pht trin v ng dng ngy cng rng ri. 1.3.2.1 Ging Lactobacillus cc c tnh ca vi khun probiotic 1.3.2.2.1 Kh nng sinh ra cc cht khng khun v i khng vi cc vi khun gy bnh Lactobacillus c kh nng sinh ra cc cht khng khun v i khng vi nhiu vi khun Gram (+), Gram (-) v k c nm. Lactobacillus c kh nng sinh ra cc cht khng khun bao gm: cc axit hu c (axit lactic v axetic axit), hydrogen peroxit, cacbondioxit, v diaxetyl cng nh bacterioxin v cc hp cht ging bacterioxin ( Mishra v Lambert, 1996; Ouwehand v cng s, 1999). C axit lactic v axit axetic u c kh nng hn ch s pht trin ca cc vi sinh vt khc bi chng lm gim pH bn trong ng rut v chnh iu ny nh hng n qu trnh trao i cht ca cc vi sinh vt khc (Mishra v Lambert, 1996). Hydrogen peroxit c ch c s pht trin ca c vi khun Gram(+) v Gram(-) (Hollang, Knapp, v Shoesmith, 1987; Mishra v Lambert, 1996). Diacetyl tc ng c ch ln s tng trng bng cch can thip s dng arginine (Jay, 1986) Ngoi ra Lactobacillus cn c kh nng sinh ra bacterioxin, mt loi protein c kh nng tiu dit cc vi khun khc do s to thnh cc knh lm thay i tnh thm ca mng t bo, nhiu loi bacterioxin cn c kh nng phn gii ADN, ARN v tn

GVHD: Th.s L nh c

SVTH: L Thanh Hun

25

cng vo peptidoglycan lm suy yu thnh t bo. Bacterioxin s tn cng cc vi khun gy bnh v c ch s pht trin ca chng, trong c cc vi khun gy bnh nh: E.coli; Samonella; Vibrio; Campylobacter, Shigella, Vibrio, Clostridium, Candida albicans, v mt s virus khc na (OSullivan v Kullen, 1998). 1.3.2.2.3 Kh nng chu mn Kh nng chu mn ca Lactobacillus c vai tr quan trng v y l yu t u tin quyt nh s tn ti ca probiotic trong mi trng nc bin. Kh nng chu mn l c tnh qu khi s dng cc chng ny lm ch phm cho NTTS cc vng khc nhau. Mt s chng Lactobacillus c bit vi kh nng chu mn cao, cao hn rt nhiu so vi mn trung bnh t 3,1 ti 3,8 ca nc bin nh: L. amylovorus DCE 471c th tn ti v sinh ra bacterioxin trong mi trng 3 % (w/v) NaCl (Patricia Neysens v cng s, 2003) Lactobacillus sakei c th sng trong mi trng c mn rt cao 9% NaCl (Chaillou v cng s, 2005). 1.3.2.2.4 Kh nng tn ti trong ng tiu ha Trong cc iu kin invitro, nhiu chng Lactobacillus c tuyn chn vi kh nng tn ti trong iu kin bt li ca ng rut ng vt thy sn nh mi trng axit HCl d dy, mi trng kim ca rut, lyzozyme, dch ty, dch mt. (Srikanjana Klayraung v cng s, 2008 ). Kh nng tn ti trong cc iu kin khc nghit ca ng tiu ha gip cho cc vi sinh vt probiotic c th cnh tranh c v tr bm dnh v cc ngun dinh dng, nng lng vi cc vi khun gy hi. Mt s chng Lactobacillus c kh nng tn ti tt trong ng tiu ha c phn lp nh: L. curvatus, L. reuteri, L.plantarum, L. parapentarum, L. pentosus, L. keferi, L. fermentum, L. animalis, L.mucosae, L. aviaries ssp. aviaries, L.salivarius ssp. salicinus, L. salivarius ssp.L.salivarius, L. hilgardii, and L. panis (Srikanjana Klayraung v cng s, 2008 ).

GVHD: Th.s L nh c

SVTH: L Thanh Hun

26

1.3.2.2. Cc yu t nh hng ti s pht trin ca vi khun lactic ni chung v Lactobacillus ni ring.

1.3.2.1.1. nh hng ca cc cht dinh dng

Trong cng nghip, vt liu dng lm mi trng cho vi sinh vt pht trin cn m bo cc yu t: y cht dinh dng, khng c c t, cho hiu sut thu hi l ln nht v gi thnh r (Lng c Phm, 2004). Mi ngun dinh dng cung cp khng ch nh hng n s pht trin ca vi khun trong qu trnh nui cy m cn nh hng khng nh n qu trnh thu hi v bo qun ch phm sinh khi sau ny.
nh hng ca ngun cacbon

Cacbon l thnh t chnh trong cc hp cht hu c xy dng ln c th ca mi loi sinh vt. V vy s chuyn ho ngun dinh dng cacbon thnh cc cht cn thit cho t bo vi sinh vt chim v tr hng u trong qu trnh dinh dng ca t bo vi sinh vt. Vi khun lactic s dng c rt nhiu loi hydratcacbon, t cc hexose (glucose, fructose, manose, galactose), cc ng i (saccarose, lactose, maltose) cho n cc polysaccarit (tinh bt, dextrin). + Glucose dng D- glucose, l loi monosaccarit hp th d dng nht. Chng c vi sinh vt s dng u tin ri mi n cc loi kh chuyn ho hn. V vy trong qu trnh sn xut ngi ta thng a v loi ng ny cho vi sinh vt d s dng. + Lactose l disaccarit, n l ng c trong sa ngi v ng vt. Mt s chng vi khun lactic c kh nng s dng lactose lm thc n v chng c kh nng sinh enzym -galactosidase. Enzym ny thu phn lactose thnh glucose v galactose, gip cho qu trnh trao i cht ca vi khun c d dng hn. + Sucrose cng l mt disaccarit. Trong qu trnh ln men di tc dng ca enzym sucrose hydrolase b thu phn thnh glucose v fructose. Vi loi lactococci, saccarose c vn chuyn bi h enzym sucrose phosphotransferase v mt enzym

GVHD: Th.s L nh c

SVTH: L Thanh Hun

27

c hiu sucrose-6-phosphate hydrolase phn ct sucrose-6-phosphate thnh glucose-6phosphate v fructose. Nhng enzym ny ch c tit ra khi xut hin sucrose trong mi trng (Thompson v Chassy, 1981). + Maltodextrin l sn phm trung gian khi thu phn tinh bt thnh ng, c 320 chui. Nhng chui ny c to bi vi gc dextroza lin kt vi nhau bi lin kt hidro yu. Ngun nng lng quan trng nht cho vi khun lactic l monosaccrit v disaccarit. Cc ngun cacbon ny c dng cung cp nng lng, xy dng cu trc t bo v sinh ra cc axit hu c nh axit citric, malic, pyruvic, fumaric, axetic Mt vi loi vi khun lactic ln men d hnh phn lp t cc sn phm thc phm, c th s dng cc axit gluconic v galacturonic to thnh CO 2, axit axetic v axit lactic. Trong qu trnh ln men cc c cht cha cacbon, vi khun lactic c th s dng c cc axit amin nh axit glutamic, arginin, tirozin lm ngun cung cp nng lng. Khi to ra qu trnh cacboxyl v to ra CO2. Cc loi vi khun khc nhau th i hi ngun cacbon khc nhau. Mt s loi vi khun lactic c th s dng c dextrin v tinh bt (William v Wilkins, 1986). S pht trin ca vi khun lactic vi mi loi ng khc nhau s to ra cc t bo c c im hnh thi v sinh l khc nhau v v vy cng s c kh nng chng chu khc nhau trc nhng p lc ca cc qu trnh x l sau ny. Nhm cc nh khoa hc ca Carcalho khng nh rng kh nng sng st ca L. bulgaricus trong v sau sy ng kh ph thuc vo loi ng c b sung trong qu trnh nui cy v thu hi ch phm, nu ln men t manose th t l t bo cht nhiu hn hn so vi ln men t fructose v lactose (Carvalho v cng s, 2004). Tuy nhin, vic la chn loi ng no cng cn quan tm n vn kinh t nhm gim thiu chi ph u vo.
nh hng ca ngun nit

Nit cng l mt nguyn t cn thit cho s sng ca tt c sinh vt. Vt cht c bn ca t bo (protein, axit nucleic) u cha nit, v vy nit ng vai tr ht sc

GVHD: Th.s L nh c

SVTH: L Thanh Hun

28

quan trng trong qu trnh sinh trng v pht trin ca vi khun. Vi khun lactic i hi rt nhiu axit amin khc nhau do chng cn mi trng c sn ngun nit nhm m bo s pht trin ca mnh. Axit amin c th c ng ho di dng peptit nh vo tc dng ca enzym protease ngoi bo hay ni bo. Mi loi vi khun khc nhau li c nhu cu v ngun nit khc nhau. Phn ln vi khun lactic khng th sinh tng hp c cc cht hu c phc tp c cha nit nn chng i hi ngun nit c sn trong mi trng. Ch c mt s t loi vi khun lactic c kh nng sinh tng hp cc hp cht hu c t ngun nit v c nh L. helveticus, chng c th b kch thch bi s c mt ca mui amoni trong mi trng (Chopin, 1993). sinh trng v pht trin bnh thng, ngoi nit di dng hn hp cc axit amin, vi khun lactic cn cn nhng hp cht hu c cha nit nh cc sn phm thu phn protein t lactanbumin, casein, pepton, peptit, dch nm men thu phn, dch chit tht, tryptony cng l ngun nit thng xuyn c s dng chun b mi trng nui cy. Tuy nhin quy m cng nghip ta cn nghin cu nhng ngun nit thch hp sn xut gip gim gi thnh sn phm m nng cao c hiu qu sn xut. Trong dch nm men thu phn c s dng kh nhiu (Lars Axelsson, 2004).
nh hng ca cc mui v c v cht kch thch sinh trng

Cc mui v c v cc cht khong ch vi lng rt nh nhng li c nh hng rt ln n s sinh trng v pht trin ca vi khun. Chng hn i vi Lactobacillus Mn2+, Mg2+, Fe2+ lm tng cng s pht trin ca vi khun lactic, hay Ca2+ tham gia vo cu trc enzym protease thu phn mt s protein l ngun dinh dng nui t bo. Nhn chung mangan v magie l nhng cht ng cc vai tr ch yu sau: + Tham gia cu trc v m bo chc nng hot ng ca enzym. + Gii c cho t bo khi s c mt ca oxy.

GVHD: Th.s L nh c

SVTH: L Thanh Hun

29

+ n nh cu trc t bo. Mg2+ l cht hot ng trong qu trnh ln men lactic bng cch gip vi khun lactic s dng tt hn cc loi ng. Carvalho v cng s nhn thy l khi b sung NaCl v saccarose vo mi trng MRS khi nui cy L. bulgaricus em li nhng kt qu khc nhau trong qu trnh to sinh khi v t l sng st ca t bo trong khi sy v bo qun sau ny (Carvalho v cng s, 2004). Cc cht cha axit bo c mt trong mi trng cng c nh hng khng nh n qu trnh sinh trng v pht trin ca vi khun. Chng khng nhng kch thch sinh trng m cn ng vai tr trong qu trnh lnh ng sau ny. V d Tween 80 s lm thay i mt s axit bo trong t bo vi khun lactic, s thay i ny nh hng n kh nng chu lnh v kh nng chng chu mui mn ca vi khun lactic (Ho Phu Ha v Michelle Cartherine Adams, 2007).

13.2.2.2. nh hng ca cc iu kin nui cy

nh hng ca t l tip ging

T l tip ging c nh hng khng nh n s pht trin ca vi khun. T l tip ging qu thp s ko di thi gian nui cy, d nhim tp, hiu sut thu hi sinh khi thp. T l tip ging qu cao, mc d thi gian nui cy rt ngn nhng hm lng sinh khi khng cao do vi khun pht trin nhanh qu lm ngun lm thc n nhanh chng cn kit, v chng sinh ra mt s sn phm gy c ch qu trnh sinh trng. V vy chn t l tip ging thch hp s tit kim canh trng ging, m bo qu trnh ln men hiu qu, rt ngn thi gian ln men.
nh hng ca pH

Trong qu trnh ln men, vi khun lactic sinh axit lm pH mi trng gim, khi pH mi trng gim n mt mc no n s c ch chnh s pht trin ca vi khun lactic. V vy trong qu trnh nui ngi ta phi lun iu chnh pH v khong ti thch cho vi khun pht trin. Mi mt loi vi khun lactic c mt khong pH ti thch khc

GVHD: Th.s L nh c

SVTH: L Thanh Hun

30

nhau, dao ng trong khong 4,5-6,5, nhng cng c mt s chng c th pht trin pH=9,6 v mt s hot ng pH=3,2 nh Lactobacillus fermentum c th chu c pH=3. Theo Giraud v cng s i vi vi khun L. plantarum A6, s gim pH s lm gim s chuyn ho c cht. Ngoi ra Silva v cng s tm ra mi lin h gia pH cui cng ca mi trng nui cy chng L. bulgaricus lin quan n qu trnh sy phun v bo qun kh sau ny. H nhn nh c t bo s chu c qu trnh x l nhit tt hn nu trong qu trnh nui cy khng iu chnh pH (Lars Axelsson, 2004).
nh hng ca nhit

Nhit nh hng trc tip n qu trnh sinh trng v pht trin ca vi khun. Nhit nh hng n cc phn ng enzym ca t bi vi sinh vt. Nhit nui cy qu cao hay qu thp u c th gy c ch cc enzyme, lm nh tr cc phn ng trao i cht v do nh hng n qu trnh sinh trng v pht trin ca vi khun. V d: vi khun Lactobacillus fermentum l loi a m, pht trin tt nhit c th ngi 37oC. 1.3.3. Tnh hnh nghin cu s dng Lactobacillus b sung vo ch phm probiotic trong nui trng thy sn Nhng nghin cu nc ngoi: mt s nc Chu u, nhiu chng Lactobacillus c la chn lm ch phm vi sinh trong nui c. Trong mt nghin cu ca Nikoskelainen v cng s (2001) cho thy kh nng gim t l cht ca c hi ca hai chng Lactobacillus rhamnosus v Lactobacillus bulgaricus vi liu lng b sung vo thc n ca c l 1012 CFU/g . Bn cnh , Carnevali v cng s (2004) cho thy kh nng ci thin sc khe ca c trp bin khi b sung Lactobacillus plantarum vi nng 104 CFU/g (Carnevali v cs, 2004).

GVHD: Th.s L nh c

SVTH: L Thanh Hun

31

n , theo nghin cu ca Mohanty v cng s (1996) cho thy ch phm vi sinh s dng Lactobacillus v nm men Saccharomyces cerevisiae c kh nng kch thch s tng trng ca c chp. Thi Lan, Jiravanichpaisal v cng s (1997) s dng Lactobacillus trong nui tm s (P.momodon Fabrius) v gim c t l tm cht do dch bnh gy ra bi nhm vibrio v bnh m trng. Ngoi ra c rt nhiu ch phm s dng Lactobacillus nh BZT AQUA, BZT DIGESTER (Bio-Form,L.L.C.,Tulsa,Oklahoma,USA); Aqua Ron (International Biologicals, n ); EPICIN-Pond (Epicore BioNetworks Inc - USA ). Cc ch phm ny c tc dng lm n nh cht lng nc v nn y trong ao nui tm c; nng cao sc kho v sc khng tm c nui; gim thiu nhim mi trng ao nui v xung quanh do nui trng thu sn gy nn; nng cao hiu qu s dng thc n ca thy sn. Nhng nghin cu trong nc: Vit Nam, nhng nghin cu v vic s dng cc ch phm vi sinh v Lactobacillus . spp vo ch phm ci thin mi trng nui thy sn ni chung v nui tm ni ring cn tng i t (Nguyn Hu Phc v Nguyn Vn Ho, 1998) Theo V Th Th v cng s, (2004) th nghim men vi sinh Biochie x l nc nui tm s ging v tm tht ti Sn, Hi Phng v H Ni cho kt qu kh tt thng qua mi trng c ci thin, c bit rt c hiu qu i vi nui tm ging nh gim chu k thay nc v gim mi hi, tng t l sng v tng trng ca tm. M hnh nui tm s bng ch phm vi sinh (ES-01 v BS-01 ca Trung tm nghin cu ng dng sinh hc phc v nui trng thy sn Sc Trng) gp phn a nng sut tm nui nhiu trang tri t ti 12 tn/ha/v. Nhiu h nui tm c x l ch phm vi sinh cho thy mi trng nc lun n nh, tm pht trin nhanh khc phc c nhiu kh khn v thi tit, mi trng, chi ph u t, dch bnh. C Mau, vic p dng m hnh nui tm bng ch phm EM.ZEO bc u mang li hiu qu kh quan, gi cho mi trng ca ao lun sch, tm kho mnh m hon ton

GVHD: Th.s L nh c

SVTH: L Thanh Hun

32

khng s dng cc loi ho cht c hi, khng sinh. Trong sut qu trnh nui, tm pht trin tt v khng b nhim bnh (http://www.fistenet.gov.vn). Nghin cu ca ng Th Hong Oanh v cng s, (2000) tm hiu tc dng ca men vi sinh Bio-dream ln cc yu t v sinh v hu sinh trong ng nui u trng tm cng xanh vi liu lng 1g/m3 v cho thy hiu qu tch cc trong vic gim mt Vibrio tng s v n nh c mi trng nc. Nghin cu ca Nguyn Thanh Phng (2007) s dng 3 loi men vi sinh Ecomarine, Bio-dream, BZT trong ng nui u trng tm cng xanh theo m hnh nc xanh ci tin, cho thy cc yu t mi trng ph hp cho s pht trin ca u trng, men vi sinh gp phn hn ch s lng vi khun Vibrio spp trong mi trng b ng, vi t l sng ca ng u trng tm cng xanh kh cao, dao ng t 59,176,6%. Cc ch phm vi sinh nh Biochie ES-01 v BS-01 EM.ZEO Bio-dream Ecomarine, BZT u cha hai chng vi sinh ch yu l Bacillus .spp v Lactobacillus spp. Ngoi ra nc ta cn nhiu ch phm vi sinh khc s dng Lactobacillus acidophilus, Lactobacillus prorogenes, Lactobacillus plantarum nh TP- 05- Super; ZeoBac BIO- ZEOGREEN EMUNIV.S Biosure

GVHD: Th.s L nh c

SVTH: L Thanh Hun

33

Chng 2: I TNG V PHNG PHP NGHIN CU 2.1. i tng nghin cu 2.1.1. Mu c Chng ti s dng cc mu c vy vng (Trachinotus blochii Lacepede, 1801) khe, tri qua mt s dch bnh. C chim c ly v t tri c Trng i hc Nha Trang (Vng Ngn Nha Trang Khnh Ha). Mu phn lp l ton b rut c. 2.1.2. Mi trng nghin cu + Alkaline Peptone Water (APW): Peptone NaCl Nc ct + MRS: Casein peptone, tryptic digest 10, cao tht - 10, cao nm men - 5, glucose -20. Tween 80, K2HPO4 - 2, Na-acetate - 5, (NH4)2 citrate - 2.00, MgSO4 . 7 H2O - 0.20, MnSO4 . H2O - 0.05, nc 1000 ml, pH to 6.2 - 6.8. Kh trng 1210C trong 15 pht. iu chnh pH mi trng t 7,5, sau thm agar vo. Hp kh trng 121oC trong 15 pht. + Thiosunfate Citrate Bile Salts Sucrose (TCBS): Cao nm men Peptone Sucrose= Sodium thiosulfate.75H2O Sodium citrate.72H2O Sodium cholate 5g 10g 20g 10g 10g 3g 10g 10g 1 lt

iu chnh pH mi trng t 8,5 0,2. Hp 1210C trong 10 pht.

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SVTH: L Thanh Hun

34

Oxgall NaCl Ferric citrate Bromothymol blue Thymol blue Agar

5g 10g 1g 0,04g 0,04g 15g

Nc ct 1 lt Cho cc cht vo nc ct lm m v un nng ha tan. Ch va si ri nhc ra ngay. Khng hp kh trng. ngui n 50oC ri a.

GVHD: Th.s L nh c

SVTH: L Thanh Hun

35

2. 2. Ni dung nghin cu Quy trnh nghin cu c s ha trong hnh 2.1 Mu ni tng c chim vy vng

Phn lp Lactobacillus
Tuyn chn cc chng Lactobacillus

c hot tnh khng Vibrio spp.

Hnh thi khun lc Xc nh c tnh sinh l sinh ha

Kh nng sinh axit lactic Kh nng di ng, phn ng catalase Kh nng ln men ng

Soi ti

Nhum Gram

Kh nng chu mn Kh nng sinh enzyme amylase, protease Xc nh cc c tnh probiotic v iu kin nui cy thch hp

Nhit pH

Thi gian Kt lun

Hnh 2.1:S cch tip cn cc ni dung nghin cu ca ti

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SVTH: L Thanh Hun

36

2. 3. Phng php nghin cu 2.3.1. Phng php phn lp, tuyn chn 2.3.1.1. Phn lp Lactobacillus Phn lp cc chng Lactobacillus trn ni tng ca c chim vy vng trn mi trng MRS. Cn 5g mu ni tng ca c chim vy vng cho vo ti nilon, b sung 45 ml canh thang tng sinh c pha long 10-1, ng nht bng my dp mu Stomacher trong 1 pht. Sau cho vo bnh tam gic v qua m nhit phng. Sau 24h em pha long thnh cc nng 10-2 n 10-7. Ht 0,1 ml mu (Trn Linh Thc, 2008) t ba nng 10-5, 10-6, 10-7 cho vo mi trng thch dinh dng chun b trong cc a petri v trng v dng que cy trang u ln b mt a thch. Cc thao tc pha long, a thch v cy mu c lm trong t cy v trng. Sau cc a petri cy mu vo t m 370C trong 1 2 ngy. Quan st hnh thi, mu sc khun lc la chn s b cc loi thuc chi Lactobacillus . Chng c tch ra cy ria nhiu ln chn cc dng thun chng, sau cy chuyn vo mi trng thch nghing trong ng nghim gi ging (Lng c Phm, 1998). 2.3.1.2. Nui cy v bo qun cc chng Lactobacillus Trong sn sut, nghin cu, vic hot ho ging v thng xuyn kim tra cht lng ca ging l ht sc cn thit. hot ha ging ngi ta thng s dng mi trng nui cy giu cc cht kch thch sinh trng nh: cao nm men, nc chit c chua, hn hp vitamin, axit bo, V vy vic chon phng php gi ging c vai tr quan trng trong duy tr c nhng hot tnh u vit ca chng, chng thoi ho ging v khng lm mt hot tnh. Qu trnh tin hnh gi ging: - Sau khi phn lp c nhiu chng Lactobacillus chng ti s tin hnh gi ging trn mi trng thch nghing: chn cc a petri c cha khun lc thun chng, dng que cy ria chn ly cc khun lc v cy ria trn mi trng agar MRS

GVHD: Th.s L nh c

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trong cc ng thch nghing c chun b trc. Cc ng c bo qun nhit 4 6oC. nh k cy truyn ging, 2 3 thng cy truyn li mt ln. - Sau khi xc nh c cc c tnh probiotic ca cc chng va phn lp c, chng ta s tin hnh gi ging trong mi trng MRS lng c cha t 30 50 % glycerol v th tch: Cc chng Lactobacillus sau khi tuyn chn s c nui cy mi trng MRS lng, 28 -300C, lc 180 vng/pht cho n khi t n thi gian gia pha Logarit ca ng cong sinh trng . Ht dch nui cy cho vo ng eppendoff c cha glycerol vi t l t 30 50 % th tch ca ng, votex cc ng ht, cho vo t lnh 4 -6 0C khong 30 pht trc khi em bo qun t ng su 700C. Phng php bo qun trong glycerol ny cho php chng ta c th gi ging trong thi gian di t 6 thng n 1 nm. 2.3.1.3. Tuyn chn cc chng Lactobacillus khng Vibrio Cc chng Vibrio c s dng c ly t b su tp ca phng th nghim v phn lp t trn i tng c chim vy vng. T mi trng gi ging, cc chng Lactobacillus v Vibrio c a vo mi trng lng tng ng (Lactobacillus nui trn mi trng MRS, Vibrio nui trn mi trng APW) v nui hot ha qua m 37 0C. Khi mt t bo ca Vibrio t khong 104 CFU ml-1 v mt t bo Lactobacillus t khong 105 CFU ml-1 th Vibrio c trang u trn b mt a thch cha mi trng LB chun b sn (Ravi v cs, 2007). Sau c cc l thch ng knh khong 5 mm, ht 50 l dch nui cy cc chng Lactobacillus cho vo cc l khoan (Sarker v cs, 2008). em cc a cy vo t m 370C, sau 1 2 ngy quan st cc vng khng khun v xc nh ng knh ca n. chn la c cc chng Lactobacillus khng li Vibrio c tin cy cao th chng ta tin hnh qua 2 3 vng th i khng. Vng 1 tin hnh th i khng tt c cc chng Lactobacillus vi 2 -3 chng vibrio tuyn chn s b c cc chng c kh nng khng. Tip theo s dng cc chng tuyn chn ln 1 th kh

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nng i khng vi 3-4 chng vibrio, t chn ra cc chng c hot tnh khng mnh lm cc bc tip theo. 2.3.2. Quan st c im hnh thi v c tnh sinh ha 2.3.2.1. Quan st c im hnh thi 2.3.2.1.1. Quan st t bo vi khun bng knh hin vi - Chun b mu t bo vi khun. Cc chng Lactobacillus la chn c nui cy trn mi trng MRS, lc vi tc 200 vng/pht, nhit 28 30 0C. Sau 24h nui cy, canh trng c thu nhn lm tiu bn quan st t bo vi khun ( trng thi sng v nhum Gram). - Chun b tiu bn Phin knh (lame) v l knh (lamelle) c ra sch vi x bng, lm kh v ngm trong cn 950. To tiu bn git p - quan st vi sinh vt trng thi sng: nh 1 git canh trng vi sinh vt ln phin knh. t l knh ln git nc (cn thn khng to bt nc bng cch nghing l knh mt gc 450 v t t h xung). - Soi knh hin vi Trng hp quan st vi sinh vt trng thi sng (s dng vt knh 10X v 40X): t tiu bn ln bn mu. H t quang, ng bt chn sng. Chn vt knh 10X, dng nt chnh th nng bn mu sao cho vt knh tip xc vi phin knh. Chnh t t theo chiu ngc li cho n khi thy nh vi sinh vt trong th trng. Dng b phn di chuyn bn mu sao cho vng mun quan st nm gia th trng. Chuyn sang vt knh 40X, iu chnh nt chnh tinh tm nh. 2.3.2.1.2. Nhum Gram Vi khun Lactic Gram (+), c hnh trc khun ngn hoc di, dng n, i hoc xp thnh chui. Ngoi ra vi khun Lactic cn c hnh cu hoc cu trc khun, dng n, i, m hoc xp thnh chui.

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Tin hnh: Bc 1: Cho mt git canh trng cha vi khun ln phin knh sch, dn u v kh t nhin hoc h qua trn ngn la n cn (trnh khng cho ngn la trc tip trn vt mu). Bc 2: Nh mt git cht nhum Violet ln vt mu c c nh trong vng 1 pht, ri ra li bng nc ct. Bc 3: Nh mt git dch cm mu lugol ln vt mu trong vng 30 giy n 1 pht, sau ra bng nc ct.
Bc 4: Dng cn 950 tia qua li trn vt mu v phin knh n khi ht mu

(khong 15 giy), sau ra ngay bng nc ct. Bc 5: Nh mt git cht mu fucshin ln vt mu trong khong 1 pht, sau ra bng nc ct. Dng giy thm kh v em mu i quan st di knh hin vi vt knh du. S dng vt knh 100X, t tiu bn nhum Gram ln bn mu v nh mt git du ln vt nhum. Nng t quang, m chn sng. Nhn vo mu (t ngoi) v h t t vt knh 100X sao cho u vt knh chm trong git du. Nhn vo th knh, dng nt chnh th iu chnh n khi thong nhn thy nh th dng li. Sau , dng nt chnh tinh cho n khi nhn thy nh r nt. 2.3.2.2. Quan st c tnh sinh ha 2.3.2.2.1. Kh nng sinh acid lactic

Phng php nh tnh: c 2 cch Nui vi khun trong hp petri trn mi trng MRS c b sung CaCO 3 vi lng

10 g/l. Khi khun lc mc, nu chng c sinh axit lactic th xung quanh khun lc c vng phn gii. Ngc li l chng khng sinh axit lactic. Cho vo mi ng nghim 0,5 ml phenol, thm t t tng git FeCl 3 1% cho n khi dung dch c mu tm ca phc phenol-st. Sau , cho vo dch nghin cu. Nu mu tm chuyn thnh mu vng th trong dch nui c mt axit lactic.

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2.3.2.2.2. Phn ng catalase - Nguyn tc: Nhm xc nh s c mt ca men catalase. - C s sinh ha: Do oxy c th to ra hng lot nhng dn xut mang tnh c i vi vi sinh vt, khng c g ngc nhin l vi sinh vt cng c th t gii c bng cch to ra nhng men c kh nng ph hy mt s sn phm cha oxy nh vy. in hnh ca loi men ny l catalase. Men catalase c hu ht cc vi sinh vt hiu kh v k kh ty tin cha cytochrome. - Phng php th: Th trn lam: Dng que cy ly tm khun lc thun nui trn mi trng MRS c t ln lam knh sch. Nh mt git H2O2 30% ln khun lc nm trn lam knh. Nu thy cc bt kh xut hin chng t c enzym catalase trong t bo. - Lu : + Khi th nghim trn lam khng c o trn trnh t tin hnh ( khng a H2O2 ln lam knh trc vi sinh vt) bi c st ln platinum co trong que cy u c th gy phn ng dng tnh gi. + Nn dng cc khun lc nui trong thi gian 18 24 gi v nhng khun lc gi hn c th gy mt hot tnh catalase v gy phn ng m tnh gi. + H2O2 30% rt khng bn v d b phn hy di tc dng ca nh sng nn cn gi lnh dung dch v cn kim tra hot tnh trc khi dng. 2.3.2.2.3. Kh nng di ng Chng Lactobacillus c cy m su trong mi trng thch ng. Chun b cc ng nghim cha mi trng MRS c, cao 5 - 6cm. Dng que cy u nhn chm vo dch nui ri m su thng xung ng thch ng cha mi trng MRS rn. Nui 30 350C trong 2 ngy em ra quan st. Nu vi khun mc dc theo vt cy v mc lan ra xung quanh, chng t chng c kh nng di ng. Vi khun ch mc dc theo vt cy chng t chng khng c kh nng di ng v h hp tu tin.

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Vi khun mc trn b mt ng thch chng t chng h hp hiu kh. 2.3.2.2.4. Kh nng s dng cc loi ng: S dng mi trng c bn gm c:Cao tht- 3 g; Pepton- 10 g; NaCl- 5 g; phenol 0,03g; thm nc ct n 1000 ml. B sung ng vi nng 0,5% v chnh pH n 7,2 0,2. Phn mi trng vo cc ng nghim, mi ng 5ml. t vo mi ng nghim 1 ng nh (ng Durham) ln ngc u hng kh CO 2 sinh ra nu vi khun c kh nng ln men ng. Kh trng trong 10 pht 121 0C. ng arabinose, xylose, v cc ng kp cn kh trng ring bng mng lc ri mi b sung vo mi trng. Cy vi khun mi hot ho vo cc ng nghim, t 36 0C, theo di hin tng sinh axit sau 1-3 ngy. Nu vi khun c kh nng ln men ng (sinh axit) cht ch th s chuyn mu vng lc. Cc loi ng c kim tra l: glucose, lactose, mantose, manitol, saccarose, sucrose. 2.3.3. Xc nh cc iu kin nui cy 2.3.3.1. Xc nh kh nng sinh trng Chng Lactobacillus c nui cy trn mi trng MRS lng 20 -300C, lc 180 vng/pht. Tin hnh ly mu lc u v sau 3h nui cy xy dng ng cong sinh trng bng phng php o c (OD600). Nguyn tc: Khi pha lng c cha nhiu phn t khng tan th s hnh thnh mt h huyn ph v c c bi cc phn t hin din trong mi trng lng cn nh sng, lm phn tn chm nh sng ti. T bo vi sinh vt l mt thc th nn khi hin din trong mi trng cng lm mi trng tr nn c. Gi tr OD (optical density, mt quang) cng cao th c cng cao, chng t vi khun sinh trng cng mnh. V vy c th xc nh kh nng sinh trng ca vi khun thng qua o c bng my so mu cc bc sng t 500 610 nm.

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Cch tin hnh: o c ca dch nui cy Lactobacillus bng my quang ph. Cho mi trng vo cuvet s 1 lm i chng, cho vo my o OD 600 nm, a v gi tr bng 0. Ly dch nui cy Lactobacillus cho vo cuvet s 2 v cho vo my o, c kt qu hin trn mn hnh. 2.3.3.2. Xc nh nhit thch hp Chng Lactobacillus c nui trn mi trng MRS lng. Lng mi trng chim 2/3 th tch bnh nui cy, t l tip ging 10 %. Tin hnh nui cy hai chng Lactobacillus cc mc nhit 300, 330, 370, 400. C sau 12 gi v 24 gi nui cy, tin hnh o pH v OD600 nm. 2.3.3.3. Xc nh thi gian nui cy Sau khi chn c nhit thch hp cho tng chng Lactobacillus ta tin hnh th nghim xc nh thi gian nui cy thu sinh khi ti u cho tng chng. Th nghim c tin hnh trn mi trng MRS lng. Lng mi trng chim khong 2/3 bnh nui cy, t l tip ging 10%. Tin hnh o pH v OD600 sau 0, 8, 12, 16, 18, 22, 26, 30, 34, 38, 42 gi nui cy. 2.3.3.4. Xc nh pH thch hp Chng Lactobacillus c nui trn mi trng MRS lng vi lng mi trng khong 2/3 th tch bnh nui cy. Chnh pH mi trng v cc mc pH = 4, 5, 6, 7, 8 v cho 10 % ging vo nui cy nhit v pH thch hp c chn. Tin hnh o pH v OD600 sau 0, 4, 8, 12, 16, 20, 24, 28, 32h nui cy. 2.3.4. Xc nh cc c tnh probiotic 2.3.4.1. Xc nh kh nng sinh enzyme tiu ha Lactobacillus c nui cy trn mi trng MRS lng, lc 180 vng/pht, sau 16 24 h tin hnh thu dich lc bng phng php ly tm tc 8000 vng/pht trong 15 pht v xc nh hot tnh cc enzyme theo phng php khuch tn trn mi trng thch cha c cht c trng (casein vi enzyme protease v tinh bt vi

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enzyme amylase. Chun b mi trng trong nc ct ri phn phi vo a peptri. Sau khi thch ng, khot nhng l nh ng knh 0,5 cm trn mt thch, cho vo 0,1 ml dung dch lc, gi trong t m. Sau 24 h lugol v o ng knh phn mi trng trong sut khng bt mu vi lugol. Biu din hot tnh tng i ca enzyme bng s mm ng knh vng thy phn. Hot tnh tng i enzyme (H) xc nh theo cng thc: H = D d (cm) D: ng knh vng phn gii cng ng knh l khoan. d: ng knh l khoan. 2.3.4.2. Xc nh kh nng chu mn Lactobacillus c nui cy trn mi trng MRS lng cc nng mui NaCl khc nhau, lc 180 vng/pht, 370C trong 24h. kim tra kh nng sng st ca hai chng Lactobacillus bng gi tr OD600 nm.

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Chng 3: KT QU V THO LUN 3.1. Kt qu phn lp tuyn chn 3.1.1. Phn lp Lactobacillus t ni tng c chim vy vng Cc mu c chim vy vng c t tri c Trng i hc Nha Trang ti Vng Ngn (Nha Trang Khnh Ha), chng ti phn lp v thun khit c 11 chng Lactobacillus. Phn lp v thun khit Lactobacillus c tin hnh trn mi trng MRS, nhit 37oC. Cc chng Lactobacillus c t tn theo th t L1.1, L1.2, L1.3, L1.4, L1.5, L2.1, L2.2, L2.3, L2.4, L2.5, L2.6. 3.1.2. Kt qu tuyn chn cc chng Lactobacillus c hot tnh khng Vibrio Cc chng Lactobacillus c nui trn mi trng lng MRS, lc vi tc 180 vng/pht, nhit phng (28-30oC). Cc chng Vibrio c tin hnh ng thi, nui trn mi trng APW, lc 150 vng/pht, nhit phng (28-30oC). Tin hnh th hot tnh khng Vibrio ca Lactobacillus sau khi cc chng Lactobacillus v Vibrio c nui khong 18 22 gi. Trang u 0,1ml chng Vibrio ln b mt a thch MRS, c cc l c ng knh 5 mm ln mi trng cy Vibrio v cho 50 l dch nui cy cc chng Lactobacillus vo l thch. Cc a petri c nhit 37oC trong t m. Tnh i khng ca cc chng Lactobacillus c nh gi thng qua kch thc vng khng khun (D d, mm), trong D l ng knh vng khng khun, d l ng knh l thch. Sau 1 2 ngy nui cy, cc a nui cy c em ra c kt qu. Kt qu c th hin t bng 3.1 v hnh 3.1:

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Bng 3.1: Hot tnh khng 7 chng Vibrio ca 5 chng Lactobacillus trn mi trng MRS, nhit 37oC D d (mm) V 2.1 L 1.2 L 1.3 L 1.4 L 1.5 L 2.2 15 18 11.5 10 13 V 2.2 14 16 16 14.5 14 V 2.3 20 14 13 16.5 16 V 2.4 18 22 12 10 12.5 C1 15 10 10 12 13 C7 22 24 13.5 11 10 C 23 20 21 17 18.5 10.5

2 5 Hot tnh khng Vibrio

2 0 D - d (mm) 1 5

1 0 5

0 L1 .2 L1 .3 C g hn
V2 .1 V2 .2 V2 .3

L1 .4 Lc bc s a to a illu
V2 .4 C1

L1 .5

L2 .2

C7

C2 3

Hnh 3.1: Kh nng i khng 7 chng Vibrio (V 2.1, V 2.2, V 2.3, V 2.4, C1, C7 v C23) ca 6 chng Lactobacillus trn mi trng MRS, c xc nh bng ng knh vng khng khun (D-d) sau 1-2 ngy nui 37oC

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Hnh 3.2: Vng khng Vibrio ca 2 chng Lactobacillus la chn sau 24h nui cy trn mi trng MRS, lc 180 vng/pht, nhit 28 - 30oC . Kt qu t bng 3 cho thy c 5 chng Lactobacillus c hot tnh khng Vibrio, chim 45,45%. Qua bng 3, hnh 3.1 v hnh 3.2 cho thy chng Lactobacillus L1.2 v L1.3 c kh nng khng Vibrio mnh nht, th hin qua ng knh vng khng khun cao nht. T kt qu trn chng ti chn hai chng L1.2 v L1.3 lm cc nghin cu tip theo. 3.2. c im hnh thi v c im sinh ha 3.2.1. c im hnh thi 3.2.1.1. c im hnh thi ca chng L1.2 Chng L1.2 c cy trang trn mi trng MRS b sung agar 2%, trong t m 370C. Sau 24h nui, ly a petri ra quan st hnh thi khun lc.

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L1.2

Hnh 3.3: Hnh thi khun lc chng L1.2 trn sau 24h trn mi trng MRS nui 340C L1.2

Hnh 3.4: Hnh thi t bo ca chng L1.2 khi soi ti vt knh 100X

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Hnh 3.5: Hnh nh nhum gram ca chng L1.2 Kt qu cho thy: Chng L1.2 c khun lc trn, c nh nhn, mu trng nht, khun lc sau 24 h nui cy c kch thc khong 2mm. Khi lm tiu bn soi ti v nhum gram quan st hnh thi t bo ca chng L1.2, kt qu trn hnh 3.4 v hnh 3.5: t bo c dng hnh cu, ng n, xp chui, t thnh m. T bo bt mu tm ca thuc nhum gram, iu chng t chng LP2 l vi khun gram dng. 3.2.1.2. c im hnh thi ca chng L1.3 Chng L1.3 c cy trang trn mi trng MRS b sung agar 2%, trong t m 370C. Sau 24h nui, ly a petri ra quan st hnh thi khun lc.

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L1.3

Hnh 3.6: Hnh thi khun lc chng L1.3 trn sau 24h trn mi trng MRS nui 340C

L1.3

Hnh 3.7: Hnh thi t bo ca chng L1.3 khi soi ti vt knh 100X

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Hnh 3.8: Hnh nh nhum gram chng L1.3 Kt qu nui cho thy: Chng L1.3 c khun lc trn, c mu trng c sa, b mt trn bng, kch thc khun lc sau 24 gi nui cy khong 2,2 mm. Chng L1.3 T bo c dng hnh que di 1-2 m, ng n, xp i, to chui, bt mu tm ca thuc nhum gram. Kt lun chng L1.3 l trc khun, gram dng.

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3.2.2. c im sinh ha Song song vi cc th nghim xc nh c im hnh thi ca 2 chng, chng ti ng thi tin hnh kim tra kh nng sinh axit lactic, kh nng to catalase, kh nng di ng ca 2 chng L1.2 v L1.3. Th catalase bng cch nh H2O2 vo khun lc, khng thy si bt l catalase m tnh. Th kh nng sinh axit lactic bng phng php cho to phc vi phenol. Kim tra kh nng di ng bng phng php cy m su trn mi trng thch ng. Kt qu th nghim kh nng di ng c th hin trn hnh 3.9; hnh 3.10 v kh nng ln men cc lai ng bng 3.2:
L1.2 L1.3

Hnh 3.9: Kh nng di ng ca chng L1.2 v L1.3

L1.2 L1.3

Hnh 3.10: Kh nng ln men cc loi ng ca chng L1.2 v L1.3

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Bng 3.2: Kt qu th cc c tnh ca hai chng L1.2 v L1.3 Chng Kh nng sinh axit lactic Gram Kh nng di ng Hot tnh catalase Kh nng s dng cc ng Tinh bt L1.2 + + + 0 L1.3 + + + 0

Kt qu th nghim cho thy L1.2 v L1.3: l nhng vi khun c sinh axit lactic, gram dng, catalase m tnh, khng di ng, khng to bo t. Theo kha phn loi Bergey c th kt lun 2 chng L1.2 v L1.3 u l nhng vi khun lactic.

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Hai chng L1.2 v L1.3 c la chn ny s em i l cc th nghim tip theo. 3.3. c tnh nui cy v c tnh probiotic 3.3.1. ng cong sinh trng ca chng L1.2 v L1.3 Xc nh ng cong sinh trng ca cc chng gip ta kim sot qu trnh nui cy v xc nh thi gian thch hp nht cho qu trnh thu sinh khi. Chng ti tin hnh xc nh ng cong sinh trng mi trng MRS lng, pH 6,5 v nhit nui cy l 370C 2. c xc nh n 30h, c 3h ly mu mt ln o OD.

a)Chng L1.2
3 2.5 OD (600 nm) 2 1.5 1 0.5 0 0 5 10 15 20 25 30 35 Time (h) 0.83 0.34 2.43 2.46 2.44 2.42 2.35 2.41 2.31 2.13 1.71

Hnh 3.11: Mi tng quan gia thi gian v OD600 nm ca chng L1.2

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Nhn vo hnh 3.11 c th thy chng L1.2 t n pha cn bng 18h. Mt t bo t gi tr ln nht 21 24h. 27h 30h t bo bt u gi v cht dn. Nh vy ta c th thu sinh khi 21 24h. b)Chng L1.3
3 2.5 2 OD (600) 1.5 1 0.5 0 0 5 10 15 20 T im e (h ) 25 30 35 0.47 1.88 1.03 2.23 2.45 2.47 2.5 2.53 2.51 2.49

2.41

Hnh 3.12: Mi tng quan gia thi gian v OD600 ca chng L1.3 Nhn vo hnh 3.12 c th thy chng L1.3 t n pha cn bng 15h. Mt t bo t gi tr ln nht 18h 21h. 27h 30h t bo bt u gi v cht dn. Nh vy ta c th thu sinh khi 18 21h. Qua kt qu trn chng ta thy c 2 chng u b qua giai on thch ng. iu ny d gii thch bi mi trng hot ha ging khng khc nhiu vi mi trng ln men. Giai on pht trin logarit ngn, iu ny c li cho qu trnh sn xut thu sinh khi, rt ngn c thi gian ln men. Giai on cn bng di chng t chng c kh nng duy tr pht trin tt. iu ny thun li cho qu trnh sn xut x l thu sinh khi. 3.3.2 nh hng ca nhit n s pht trin ca 2 chng L1.2 v L1.3 Nhit c nh hng rt ln i vi hot ng sng ca vi sinh vt. nhit thp s ko di thi gian sinh trng do ko di thi gian thu sinh khi. Cn nhit qu cao chng s b c ch. Vi mc ch thu sinh khi ln trong thi gian ngn ta

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cn tm nhit ti thch nht cho chng pht trin t yu cu mong mun ca nh sn xut. tm khong nhit ti thch chng ti tin hnh ln men trong mi trng MRS lng ti cc khong nhit c kho st l: 300C; 340C; 370C v 400C. Vi pH l 6,5. Kt qu c o sinh khi bng phng php o mt quang OD bc sng = 600nm.. Kt qu c trnh by hnh 3.13:
3

OD 600 nm

2.5

L1.2 L1.3

2 25 27 29 31 33 35 37 39 41 43 45 Nhit

Hnh 3.13 : nh hng ca nhit nui cy ln s sinh trng v pht trin ca chng L1.2 v L1.3 Kt qu nghin cu cho thy nhit nh hng n s pht trin ca hai chng L1.2 v L1.3, hai chng u l cc vi khun a m i vi chng L1.2 nhit ti thch cho s pht trin ca chng l 35 -370C mnh nht 370C , nhit t 37 40oC chng b c ch bi nhit . Do vy p ng mc tiu ca sn xut l sinh khi chng ti chn nhit 37 0C lm nhit thch hp cho chng L1.2 pht trin. Chng L1.3 pht trin tt 33 - 350C v mnh nht 340C, sau 350C th chng L1.3 pht trin km dn. Chng ti chn nhit 340C cho mc tiu sn xut thu sinh khi ca chng L1.3.

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3.3.3. Thi gian nui cy Trong nghin cu sn xut ch phm vic xc nh thi im thu nhn sinh khi c vai tr rt quan trng, n khng ch c ngha trong vic rt ngn thi gian sn xut m cn nh hng ti cht lng ch phm sau ny. Vic nghin cu thi gian nui cy c thc hin trn mi trng MRS vi t l tip ging l 10%. Thc hin qu trnh nui cy tnh trong t m, chng L1.2 nui 370C v chng L1.3 nui 340C , theo di kt qu o OD600nm sau thi gian t 8h n 40h.
3 2.5 OD 600 nm 2 1.5 1 0.5 0 0 10 20 30 40 50 Time (h) L1.2 L1.3

Hnh 3.14: Mi tng quan gia thi gian nui cy v mt t bo sng ca hai chng L1.2 v L1.3 OD600 nm. Kt qu nghin cu cho thy chng L1.2 pht trin v cho sinh khi ln nht trong khong t 24 - 28 h nui cy, chng L1.3 cho sinh khi ln nht trong khong thi gian t 20 24h nui cy. Thi gian ny chm so vi ng cong sinh trng khong 3h nui. Nguyn nhn dn n s chnh lch ny l do qu trnh nui ly sinh khi ny c thc hin trn th tch ln hn rt nhiu ,so vi th tch nui xc nh ng cong sinh trng l 200 ml th th tch nui sinh khi l 1 L. khi th tch nui cy ln th s nh hng ca cc cht c ch nh axit ln vi sinh vt s yu hn v vy thi gian pht trin thu c sinh khi ln nht s ko di hn.

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T kt qu trn, chng ta s xc nh thi gian nui cy thu nhn sinh khi trn quy m ln hn trn quy m cng nghip. 3.3.4. pH nui cy Tin hnh nghin cu nh hng ca pH n s pht trin ca 2 chng L1.2 v L1.3 trong mi trng MRS lng. Di pH c kho st t 4 8, chng L1.2 c nui cy nhit 370C v chng L1.3 nui 340C trong 48h. C 3h ly mu o OD mt ln. Kt qu c biu din trn hnh 16, 24h.

2.4 2.3 OD 600nm 2.2 2.1 2 2 3 4 5 pH 6 7 8 9 L1.2 L1.3

Hnh 3.15: nh hng ca pH ln s sinh trng v pht trin ca chng L1.2 v L1.3 T hnh 3.15 ta thy rng pH nh hng ln n qu trnh sinh trng ca c 2 chng. Chng L1.2 pht trin ti thch pH = 6 7 , tt nht pH = 7. pH = 8 chng bt u c du hiu pht trin km hn Khong pH t 5 6 chng vn pht trin c nhng pH t 4 4,5 s pht trin ca chng b c ch. Chng L1.3 th pht trin tt pH = 6 7.5 v ti thch pH = 6.5. pH = 8 chng vn pht trin nhng km hn, pH = 4 6 chng pht trin yu dn.

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Qua kho st ta thy rng hai chng pht trin tt pH trung tnh v hi kim. iu chng t chng rt thch hp pht trin trong mi trng nc bin. Da vo s kho st pH chng ti chn pH = 6,5-7 lm pH mi trng cho qu trnh ln men thu sinh khi 2 chng L1.2 v L1.3. 3.3.5. Kh nng chu mn ca hai chng L1.2 v L1.3 Vi mc ch ng dng cc chng vi khun lactic lm ch phm probiotic cho nui trng thu sn. Do vy, ta cn kim tra kh nng chu mn ca cc chng L1.2 v L1.3. Th nghim c kim tra trong mi trng MRS vi mn khc nhau. Kt qu cho trn hnh 3.16.
3 2.5 OD 600nm 2 1.5 1 0.5 0 1 2 3 4 5 Nng mui NaCl (%) L1.2 L1.3

Hnh 3.16: nh hng ca nng mui NaCl n s pht trin ca chng L1.2 v L1.3 Chng L1.2 c kh nng pht trin nng mui 5%. S pht trin ca chng t l nghch vi mn ca mi trng. Chng L1.3 c kh nng chu mn yu hn, mt t bo gim dn cc nng mui cao hn v n 5% mui th s pht trin ca chng L1.3 rt yu ( OD 600 = 0.95). Tuy nhin nng mui 1% th chng pht trin yu hn nng mui 2%, iu ny chng t nng mui NaCl c nh hng n s pht trin ca L1.3. Chng ta nhn thy 2 chng u c kh nng chu mn kh tt, c bit chng L1.2 c kh nng chu mn tng i cao 5%( so vi mn trung bnh ca nc bin

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l khong 3%). y l c tnh qu khi s dng cc chng ny lm ch phm cho NTTS cc vng khc nhau.

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KT LUN V KIN NGH

Kt lun

1.

T cc mu c chim vy vng c ly t Tri c Trng i hc thy sn Nha

Trang (ti Vng Ngn Nha Trang Khnh Ha) phn lp c 5 chng Lactobacillus c hot tnh khng 7 chng Vibrio spp.
2.

Trong nm chng c hot tnh khng Vibrio spp , hai chng L1.2 v L1.3 c Cc iu kin thch hp cho 2 chng pht trin: Chng L1.2: nhit 370C, pH= 6.5 - 7 , thi gian thu sinh khi 24 Chng L1.3: nhit thch hp 340C, pH= 6 - 7, thi gian thu sinh 28h, kh nng chiu mn n 5%. khi 20 24h, kh nng chu mn n 5%.

hot tnh khng mnh nht sau 24h nui cy.

3.

4.

Hai chng L1.2 v L1.3 c nghin cu c cc c im sinh hc ca chi vi

khun Lactobacillus gm: hnh que, khng di ng, sinh axit lactic, ln men cc loi ng (glucose, saccharose, sucrose, mantose, manitol, sorbitol). Kin ngh Hng nghin cu tip theo: 1. S dng hai chng L1.2 v L1.3 vo ch phm probiotic th nghim invivo trn c chim vy vng.
2. Nghin cu su hn v c ch khng khun ca cc chng Lactobacillus 3. Gii trnh t gene hai chng Lactobacillus ni trn

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TI LIU THAM KHO Ti liu Ting Vit: 1. B thy sn (2004), S tay kim nghim vi sinh vt thc phm thy sn, d n ci
thin cht lng v xut khu thy sn, NXB nng nghip H Ni, H Ni, 296 tr.

2. Bi Trng Khim, (2008) Tm hiu k thut ng ging c chim vy vng (Trachinotus blochii Lacepede, 1801) ti Tri Thc nghim sn xut Hi sn Vnh Ha - Nha Trang. 41 tr 3. Nguyn Vn Sn, (2008) K thut nhn to sn xut ging c chim vy vng (Trachinotus blochii Lacepede, 1801) ti tri thc nghim Trng Cao ng Thy Sn Yn Hng Qung Ninh. tr 6 9. 4. Trn Duy Thit, (2004) Nghin cu ng dng chng Lactobacillus acidopillus trong sn xut ch phm sinh hc (BIOF) dng trong phng v tr bnh cho tm c. 54 tr 5. Th Ha, Bi Quang T, Nguyn Hu Dng v Nguyn Th Mui (2004), Bnh hc thy sn, NXB Nng nghip Tp. H Ch Minh, Tp. H Ch Minh, tr. 224 231. 6. Th Ha, Trn V Hch, Nguyn Th Thy Giang, Phan Vn t, Nguyn Th Nguyt Hu Cc loi bnh thng gp trn c bin nui Khnh Ha Tp ch Khoa hc v Cng ngh Thy sn s 02/2008 i hc Nha Trang, tr. 16 24. 7. Trn V Hch, Phm Th Duyn Bnh t hoi thn kinh trn c bin nui ti Khnh Ha Tp ch Khoa hc Cng ngh Thy Sn s 01/2008 i hc Nha Trang, tr 19 24. 8. Lng c Phm (1998), Cng ngh vi sinh vt, Nh xut bn nng nghip, H Ni, 358 tr. 9. Trn Linh Thc (2007), Phng php phn tch vi sinh vt trong nc, thc phm v m phm, Nh xut bn gio dc, 232 tr

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1.

Bernet MF, Brassart D, Neeser JR, Servin AL, (1994) Lactobacillus acidophilus LA 1 binds to cultured human intestinal cell lines and inhibits cell attachment and cell invasion by enterovirulent bacteria Gut 35, pp 483-489.

2.

Briggs, M. R. P. & Funge-Smith, S. J.,(1994) A nutrient budget of some intensive marine shrimp ponds in Thailand Aquaculture and Fisheries Management. 25, pp 789-811.

3.

Carvalho, A.S. Silva. J, Ho. P. Teixeia, F. X. Gibbs, (2004). Relevant factor for the preparation of freeze-died lactic acid bacteria. International Dairy Journal, 14, 835-847, Elsrier Science B.V.

4.

De Man. J.C., Rogosa, M and Sharpe, M.E. (1960) A medium for the cultivation of Lactobacilli. Journal of applied bacteriology. 23: pp 130-135. Direkbusarakom, S., Yoshimizu, M., Ezura, Y., Ruangpan, L., Danayadol Y., (1998) Vibrio spp. the dominant flora in shrimp hatchery against some fish pathogenic viruses J. Mar. Biotechnol. 6, pp 266267.

5.

6.

Ho Phu Ha and Michelle Cartherine Adams, (2007). Selection and identifinication of a novel probiotic strans of Lactobacillus fermentum isolated from Vietnamese fermented food. School of Enviromental and Life Science, Faculty of Science and Information Technology, The University of Newcastle, Australia.

7.

Hollang, K. T., J. S. Knapp, and J. G. Shoesmith. (1987) Anaerobic Bacteria 1st ed. Blackie and Son, Ltd., London. Kamei, Y., Yoshimizu, M., Ezura Y., Kimura, T., (1988) Screening of bacteria with antiviral activity from fresh water salmonid hatcheries Microbiol Immunol. 32, pp 6773.

8.

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9.

Kenneth H. Wilson and Fulvio Perin2, (1998) Role of Competition for Nutrients in Suppression of Clostridium dijficile by the Colonic Microflora. INFECTION AND IMMUNITY, Oct. 1988, p 2610-2614.

10.

LARS AXELSSON, (2004), Lactic Acid Bacteria: Classification and Physiology. MATFORSK, Norwegian Food Research Institute, As, Norway. Mack DR, Michail S, Wei S, Wei S, Macdougal L, Hollingsworth MA, (1999) Probiotics inhibit enteropathogenic E. coli adherence in vitro by inducing intestinal mucin gene expression Am J Physiol 39, pp 941-950.

11.

12.

Mishra, C. and J. Lambert. (1996) Production of anti-microbial substances by probiotics Asia Pacific J Clin Nutr 5, pp 2024. Nikoskelainen S, Ouwehand AC, Salminen S, Bylund G, (2001) Protection of rainbow trout(Oncorhynchus mykiss) from furunculosis by Lactobacillus rhamnosus Aquaculture 2001b;198:pp 229-236.

13.

14.

OSullivan, D. J. and M. J. Kullen. (1998) Tracking of probiotic bifidobacteria in the intestine Intl Dairy J 8: pp 513525. Patricia Neysens, Winy Messens, Luc De Vuyst, (2003) Effect of sodium chloride on growth and bacteriocin production by Lactobacillus amylovorus DCE 471 International Journal of Food Microbiology 88, pp 29 39.

15.

16.

Porter, C. B., Krom, M. D., Robbins, M. G., Brickel, L. & Davidson, A, (1987) Ammonia excretion and total N budget for Gilthead Seabream (Sparus aurata) and its effect of water quality conditions Aquaculture. 66, pp 287297.

17.

Prieur, G., Nicolas, J.L., Plusquellec, A., Vigneulle M., (1990) Interactions between bivalves molluscs and bacteria in the marine environment. Oceanogr. Mar. Biol. Annu. Rev. 28, pp 227352.

18.

Sakata, T., (1990) Microflora in the digestive tract of fish and shellfish In: Lesel, R. (Ed.), Microbiology in Poecilotherms. Elsevier, Amsterdam, pp 171176.

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19.

Saurabh S, Choudhary AK and Sushma GS (2005) Concept of probiotics in aquaculture. Fishing Chimes 25, pp 1922. Srikanjana Klayraung, Helmut Viernstein, Jakkapan Sirithunyslug, Siriporn Okonogi(2008) Probiotic Properties of Lactobacilli Isolated from Thai Traditional Sci Pharm 76: pp 485503.

20.

21.

Tanaka S, I Kuriyama, T Nakai and Miyazaki (2003) Susceptibility of cultured juveniles of several marine fish to the sevenband grouper nervous necrosis virus. Journal of fish diseases 26, pp 109-115.

22.

Teruo Higa, (2002). Technology of Effective Microorganisms. Concept and Phisiology. Royal Agricultural College, Cirencester, UK. Thompson, J.; Chassy, B.M.(1981) Uptake and metabolism of sucrose by Streptococcus lactis. J. Bacteriol. 1981, 147, pp 543551 Wu, R.,(1995) The environmental impact of marine fish culture: toward a sustainable future Mar Pollut Bull. 31, pp 159-166.

23.

24.

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