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B GIO DC V O TOTRNG 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 CHNGLACTOBACILLUS

TRN C CHIM VY VNG

Ging vin hng dn : Th.s L NH CSinh vin thc hin: L THANH HUNLp : 49 CNSHKho : 49

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Nha Trang, thng 7 nm 2011

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B GIO DC V O TOTRNG 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 CHNGLACTOBACILLUS

TRN C CHIM VY VNG

Ging vin hng dn : Th.s L NH CSinh vin thc hin: L THANH HUNLp : 49 CNSHKho : 49

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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.


Sinh vin

L Thanh Hun

GVHD: Th.s L nh c SVTH: L Thanh Hun

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MC LC


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DANH MC BNG

Bng 3.1: Hot tnh khng 7 chng Vibrio ca 5 chngLactobacillus 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


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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 chngLactobacillus 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 chngLactobacillus la chn sau 24h

nui cy trn mi trng MRS, lc 180 vng/pht, nhit 28 - 30oCError:


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:


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 trngMRS nui 340C............................................. Error: Reference source not found

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


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


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



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Hnh 3.12: Mi tng quan gia thi gian v OD600 ca chng L1.3.....Error:


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


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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 quytvn 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


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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 chngLactobacillus v Vibrio trong rut c chim vy vng.

- Tuyn chn cc chngLactobacillus c hot tnh khng Vibrio.

- Nghin cu c im sinh hc ca cc chngLactobacillus.

- Nghin cu iu kin nui cy thch hp cho cc chngLactobacillus.


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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 thcon 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


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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


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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 trongng 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

caAermonas 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


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http://opt/scribd/conversion/tmp/scratch6128/t?nh%20h?nh%20nghi?n%20c?u%20v?%20probiotic%20trong%20nuoi%20trong%20th?y%20san/4_probiotics_in_aquaculture_importance_and_future_perspectives_794.pdfhttp://opt/scribd/conversion/tmp/scratch6128/t?nh%20h?nh%20nghi?n%20c?u%20v?%20probiotic%20trong%20nuoi%20trong%20th?y%20san/4_probiotics_in_aquaculture_importance_and_future_perspectives_794.pdf

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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 khunEscherichia 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


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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 hmlng 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. (Prieurv 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.


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- 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 cli 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 this 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


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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 gmBacillus sp, Nitrosomonas, Nitrobacter.. nh vy cc vi khun trong chphm 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.


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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,


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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 :


Chng gingNguyn liu

Nhn ging

Ln men

Sinh khi

Ch phm dngdch

Cht ph gia

Ch phm dngbt

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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 vinguyn 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 vtrn 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, nhiuvng 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 kthp 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 nayc 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 IHNgy 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 VHSgy nn c hi, c trch, c bn; bnh doRSIV

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 vyct 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 chtgy 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 nyrng 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 loithc 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 lBacterium 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 trngnui 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

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-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 gingLactobacillus

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.


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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 caLactobacillus 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 Klayraungv 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 ).


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c hiu sucrose-6-phosphate hydrolase phn ct sucrose-6-phosphate thnh glucose-6-

phosphate 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 3-

20 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 CO2, 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


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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.


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+ 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, khipH 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


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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 nhLactobacillus fermentum c th chu c

pH=3. Theo Giraud v cng s i vi vi khun L. plantarumA6, 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 vikhun. 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 khunLactobacillus fermentum l loi a m, pht trin tt nhit

c th ngi 37oC.

1.3.3. Tnh hnh nghin cu s dngLactobacillus b sung vo ch phm probiotic

trong nui trng thy snNhng 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).


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n , theo nghin cu ca Mohanty v cng s (1996) cho thy ch phm

vi sinh s dngLactobacillus 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.momodonFabrius) v gim c t l tm

cht do dch bnh gy rabi nhm vibrio v bnh m trng.

Ngoi ra c rt nhiu ch phm s dngLactobacillus 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 khcphc 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


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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 59,1-

76,6%.

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 vLactobacillus

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


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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

10g

10g

1 lt

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

+ 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

20g10g

10g

3g


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Oxgall

NaCl

Ferric citrate

Bromothymol blue

Thymol blue

Agar

Nc ct

5g

10g

1g

0,04g

0,04g

15g

1 ltCho 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.


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2. 2. Ni dung nghin cu

Quy trnh nghin cu c s ha trong hnh 2.1

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


Kh nng chu mn

Kh nng sinh axitlactic

Phn lpLactobacillus

Mu ni tng c chim vy vng

Kh nng di ng,phn ng catalase

Kh nng ln menng

Tuyn chn cc chngLactobacillusc hot tnh khng Vibrio spp.

Xc nh c tnh sinh l sinh ha

Xc nh cc c tnhprobiotic v iu kin nui

cy thch hp

Kt lun

Hnh thi khunlc

Soi ti

Nhum Gram

Nhit

pH

Thi gian

Kh nng sinhenzyme amylase,

protease

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2. 3. Phng php nghin cu

2.3.1. Phng php phn lp, tuyn chn

2.3.1.1. Phn lpLactobacillus

Phn lp cc chngLactobacillus 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 btrong 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 chngLactobacillusTrong 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 giging 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


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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 chngLactobacillus 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 chngLactobacillus 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 caoth 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 chngLactobacillus 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, ihoc 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 ntchnh 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

ChngLactobacillus 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 30 350

C 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 trngChngLactobacillus c nui cy trn mi trng MRS lng 20 -30 0C, 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

ChngLactobacillus 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 cySau 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 enzymeprotease v tinh bt vi


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enzymeamylase. 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 chngLactobacillus 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 lpLactobacillus 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 khitLactobacillus c tin hnh trn mi trng

MRS, nhit 37oC. Cc chng Lactobacillusc 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 chngLactobacillus c hot tnh khng Vibrio

Cc chng Lactobacillusc 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 caLactobacillussaukhi cc chngLactobacillusv

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 Lactobacillusvo 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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0

5

10

15

20

25

L 1.2 L 1.3 L 1.4 L 1.5 L 2.2

Chng Lactobacillus

Hottnh

khng

Vibri

D-

d(mm)

V2.1 V2.2 V2.3 V2.4 C 1 C 7 C 23

Bng 3.1: Hot tnh khng 7 chng Vibrio ca 5 chngLactobacillus trn

mi trng MRS, nhit 37oC

D d (mm)

V 2.1 V 2.2 V 2.3 V 2.4 C 1 C 7 C 23

L 1.2 15 14 20 18 15 22 20

L 1.3 18 16 14 22 10 24 21

L 1.4 11.5 16 13 12 10 13.5 17

L 1.5 10 14.5 16.5 10 12 11 18.5

L 2.2 13 14 16 12.5 13 10 10.5

Hnh 3.1: Kh nng i khng 7 chng Vibrio (V 2.1, V 2.2, V 2.3, V 2.4, C1, C7v C23) ca 6 chngLactobacillus 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 chngLactobacillus la chn sau 24h nuicy 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 chngLactobacillus

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

nui 340C

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


L1.2

L1.2

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Hnh 3.5: Hnh nh nhum gram ca chng L1.2Kt 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 L1.3c 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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Hnh 3.6: Hnh thi khun lc chng L1.3 trn sau 24h trn mi trng MRS

nui 340C

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


L1.3

L1.3

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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:

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

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


L1.2 L1.3

L1.2 L1.3

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Bng 3.2: Kt qu th cc c tnh ca hai chng L1.2 v L1.3

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.


Chng L1.2 L1.3Kh nng sinh axit lactic + +Gram + +Kh nng di ng - -Hot tnh catalase - -Kh nng s dng cc ng + +Tinh bt 0 0

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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)ChngL1.2

0.34

2.312.442.43

2.352.422.462.41

2.13

1.71

0.83

0

0.5

1

1.5

2

2.5

3

0 5 10 15 20 25 30 35

Time (h)

OD(600nm)

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)ChngL1.3

0 .4 7

1 .0 3

2.472 .4 5

2 .2 3

1.88

2. 5 2 .5 3 2 .5 1 2 .4 92 .4 1

0

0. 5

1

1. 5

2

2. 5

3

0 5 1 0 1 5 20 25 30 35

T ime (h

OD

(600)

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:

2

2.5

3

25 27 29 31 33 35 37 39 41 43 45

Nhit

OD60

0nm

L1.2

L1.3

Hnh 3.13 : nh hng ca nhit nui cy ln s sinh trng v phttrin 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 -37 0C

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 37 0C v chng L1.3 nui 340C ,

theo di kt qu o OD600nm sau thi gian t 8h n 40h.

0

0.5

1

1.5

2

2.5

3

0 10 20 30 40 50

Time (h)

OD600nm

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 xcnh 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

2.1

2.2

2.3

2.4

2 3 4 5 6 7 8 9

pH

OD600nm

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.

0

0.5

1

1.5

2

2.5

3

1 2 3 4 5

Nng mui NaCl (%)

OD600nm

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 ( OD600 =

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

hot tnh khng mnh nht sau 24h nui cy.

3. 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 -

28h, kh nng chiu mn n 5%.

- Chng L1.3: nhit thch hp 340C, pH= 6 - 7, thi gian thu sinh

khi 20 24h, kh nng chu mn n 5%.

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

khun Lactobacillusgm: 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 chngLactobacillus

3. Gii trnh t gene hai chngLactobacillus 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 cithin 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. ThHa, 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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2. Briggs, M. R. P. & Funge-Smith, S. J. ,(1994) A nutrient budget of some

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Management. 25,pp789-811.

3. Carvalho, A.S. Silva. J, Ho. P. Teixeia, F. X. Gibbs, (2004).Relevant factor

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cultivation ofLactobacilli.Journal of applied bacteriology. 23: pp 130-135.

5. 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.

6. Ho Phu Ha and Michelle Cartherine Adams, (2007). Selection andidentifinication of a novel probiotic strans of Lactobacillus fermentum

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7. Hollang, K. T., J. S. Knapp, and J. G. Shoesmith. (1987) Anaerobic

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