TRN QUC VIT - Phn lp , tuyn chn v nh gi .

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PHN LP, TUYN CHN V NH GI CC C TNH PROBIOTIC CA MT S CHNG VI SINH VT HU CH SN XUT CC CH PHM

PROBIOTIC DNG TRONG CHN NUI

Trn Quc Vit1, Bi Th Thu Huyn1, Dng Vn Hp2 v V Thnh Lm2 1Vin Chn nui - Thy Phng - T Lim - H Ni

2 Vin Vi sinh vt v Cng ngh sinh hc - i hc Quc gia - H Ni.

*Tc gi lin h: Trn Quc Vit - B mn Dinh Dng, Thc n v ng c Vin Chn nui - Thy Phng - T Lim - H Ni

Tel: (04) 38.386.126 / 0982.011.584; Fax: (04) 38.389.775; Email: hainiah2008@gmail.com

ABSTRACT

Isolation, selection and evaluation of probiotic characteristics of some kinds of useful microorganism for probiotic producton

Sixty four strains of microorganism were isolated from the intestinal content of pigs, chickens and from commercial probiotics. Amongs them, 4 strains including two strains of lactic bacteria and two strains of yeast were selected for futher investigation of their characteristics. Based on metabolic characteristics and taxonomic key of Bergey and frequency of rADR 16S analysis, two strains of lactic bacteria were belong to Lactbacillus fermentum named NC1 and Lactobacillus casei named NC2 and two strains of yeast were classed as Saccharomyces cerevisiae-SC and Saccharomyces boulardii-SB. All of strains selected were useful microorganisms with a high toleran to low and high pH environments and to a high level of bile salt. These strains were also highly compatible with some active components commonly used in diets for pigs and chickens such as some kinds of antibiotics (BMD, colistin, chlotetracylin ..), acidifiers, CuSO4, ZnSO4..etc. It was concluded that these strains could be considered as useful microorganisms for the probiotic production.

Key words: Microorganism, probiotics, lactic bacteria, yeast, compatibility.

T VN T thi c i con ngi bit s dng cc Vi sinh vt (VSV) ch bin v bo qun thc phm, nhng tng to ra nhng sn phm VSV sng hu ch b sung vo thc n hoc nc ung nhm ci thin sc kho v nng cao nng sut ca vt nui ch c quan tm nhiu trong vi thp k tr li y. Nm 1974 Paker a ra khi nim probiotic ch nhng VSV v nhng cht lm cn bng h VSV trong ng rut ca ngi v vt nui, k t c rt nhiu nhng pht hin mi v vai tr, tc dng ca cc VSV sng nh: tng cng sc kho ca h tiu ho thng qua ci thin cn bng h vi sinh vt rut (Fuller, 1989), tng p ng min dch, phng chng ung th ng tiu ho (FAO/WHO, 2001), gim colesterol mu (Pereira v Gibson, 2002)... Cc (VSV) probiotic nht thit phi l cc VSV sng hu ch, chng c th l cc vi khun (VK) (thng l cc VK khun lactic, mt s chng Bacillus) v nm men (ch yu l cc chng thuc loi Saccharomyces cerevisiae v Saccharomyces bouladii). Trong t nhin, cc VSV hu ch rt phong ph, a dng nhng chng ch c th l cc VSV probiotic khi chng tho mn c mt s iu kin: (i) c sc sng cao; (ii) tn ti c trong mi trng ng tiu ho (pH thp, mui mt, c kh nng bm dnh vo nim mc ng tiu ho), (iii) pht trin v cnh tranh c vi cc vi sinh vt c hi trong ng rut, (iv) c kh nng sn sinh cc bacteriocin v hoc enzyme tiu ha; (v) c kh nng lm tng p ng min dch. V nhng c im ny m vic phn lp, tuyn chn cc vi sinh vt probiotic tr nn rt kh khn, phc tp. Hu nh rt him c nhng chng VSV hi cc iu kin trn. Bi vy,

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c cht lng cao, sn phm probiotic thng gm nhiu loi VSV thuc cc loi khc nhau (Multispecies Probiotic) (Timmerman v cs, 2006). Nghin cu ny c tin hnh nhm phn lp, nh gi, tuyn chn cc chng vi sinh vt c cc c tnh probiotic phc v cho vic sn xut cc ch phm Probiotic dng trong chn nui.

VT LIU V PHNG PHP NGHIN CU Vt liu nghin cu Cc vt liu phc v cho vic phn lp tuyn chn cc vi sinh vt probiotic: Phn, cht cha ng tiu ho (rut non, manh trng) ca ln v gia cm (g, ngan, vt). Mt s sn phm probiotic thng mi (Lacto-Sac ca hng Alltech-Canada; EM ca Nht; E-Lac ca Hn qucvv).

Cc vi sinh vt kim nh (Salmonella enteritidis, E. coli v Shigella flexneri); cc vi sinh vt probiotic c phn lp, tuyn chn v nh gi c tnh probiotic1 c bo qun ti bo tng ging chun vi sinh vt thuc Vin Vi Sinh Vt v Cng ngh sinh hc-i hc Quc gia H ni.

Cc ha cht: dng tch ADN v phn gii trnh t: EDTA, Tris- SDS, chlorofom-isoamyl alcohol, dNTP, Taq polymeraza, Ethidium Bromit, kit QIAgen...vv.

Cc vt liu khc: Gi , ng gluco, K2HPO4, MgSO4, natri glutamat, pepton, cao nm men; dextrin, tinh bt sn, bt sa whey.

Phng php nghin cu Phn lp, tuyn chn cc chng vi sinh vt hu ch Mu c pha long nng t 10-3-10-5 v cy gt trn mi trng MRS (i vi vi khun Lactic) v YM (i vi nm men). Sau khi cy cc mu c gi trong t m nhit 37oC cho vi khun (VK) v 30oC cho nm men trong 48 gi. Sau 48 gi trn mi trng MRS chn cc khun lc to vng phn gii CaCO3; trn mi trng (Yeast Malt- YM) chn cc khun lc nm men trn u, trn nhn soi knh c hnh trng v ny chi.

nh gi c im hnh thi, phn loi v nh danh cc chng VSV hu ch Hnh thi t bo c quan st sau khi nui cy t 24 n 48 gi trn cc mi trng MRS (vi VK Lactic) v YM (vi nm men). Hot tnh catalaza v sinh kh CO2 t cc ngun ng khc nhau c nng 1% (raffinoza = 2%) c xc nh bng phng php ca Kozaki v cs, (1992).

Ngoi ra, cc chng nm men cn c th phn ng DBB v hot tnh Ureaza. Vic phn loi cc chng vi khun c thc hin da trn c s kho phn loi ca Bergeys (Holt v cs, 2000) (theo cc ch tiu ch yu nh: hnh thi t bo, khun lc, nhum Gram, phn ng catalaza v ln men mt s loi ng) v phn tch, gii trnh t 16S rARN theo phng php c gii thiu bi Lane (1991). Xc nh trnh t rADN 16s ca cc chng vi khun lactic theo phng php ca Saito v Miura (1963) v trnh t rADN ITS1, ITS4 ca cc chng nm men theo phng php ca Manitis v cs (1982).

nh gi mt s c tnh probiotic ca cc chng VSV hu ch nh gi kh nng chu nhit: Cc chng vi sinh vt c nui cy trn mi trng MRS

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dch th (VK Lactic) v mi trng YM (nm men) (pH =7.0), trong cc my lc n nhit (200 vng/pht) vi cc gii nhit khc nhau 30; 37; 45; 55oC.

nh gi kh nng sinh trng trong mi trng c pH khc nhau Tng t nh trn, cc chng vi khun v nm men cng c nui cy trn mi trng dch th trong m acetat c pH khc nhau (2,2; 3,0; 4,0; 5,0; 6,0; 7,0), trn my lc n nhit (200 vng/ pht ti 37oC cho vi khun Lactic v 30oC cho nm men).

nh gi kh nng sinh trng trong mi trng c nng mui mt khc nhau Cc chng nghin cu cng c nui cy trn mi trng dch th nh trn c nng mui mt (0,2; 0,5; 1;1,5; 2; 3%). Nui cy lc (200 vng/pht ti 37oC i vi vi khun v 30oC i vi nm men). m mt t bo sau 48h nui cy gt trn a Petri c cha mi trng thch hp. Sau 48 gi, tc sinh trng ca cc chng vi khun c nh gi thng qua s lng VSV tnh bng cfu/ml v kh nng sn sinh axit lactic. Kh nng sn sinh axit lactic ca cc VK Lactic c nh gi theo phng php Therner v cs (1894). Kh nng khng cc vi khun kim nh (Salmonella enteritidis, E. coli, Shigella flexneri): c tin hnh theo phng php o vng khng khun.Tnh i khng ca cc vi sinh vt: c th bng k thut cy vch trn a thch.

nh gi kh nng bm dnh vo nim mc ng tiu ha Chun b vi sinh vt v vt liu bm dnh: Vi sinh vt (vi khun v nm men) c nui cy thun khit trong mi trng thch hp cho tng chng. Chun b cc mu rut g ti: Ra cc on rut non (t g broiler mnh khe) 3 ln vi m PBS sao cho tt c cc vi sinh vt khng cn trn b mt nim mc rut. Ra 1 ln vi mi trng (MRS cho vi khun v YM cho nm men). Tin hnh th bm dnh: Ph dch t bo chun b trn ln trn b mt nim mc rut. 90 pht trong t m (37oC). Ra mu rut ln 1 bng nc mui sinh l1%. Ra mu rut 3 ln bng m PBS. Thu ly dch ra ca c 3 ln v trn u. m s lng VSV trong dch ra xc nh kh nng bm dnh ca cc VSV nghin cu. nh gi tnh tng thch ca cc chng vi sinh vt vi cc thnh phn c hot tnh trong khu phn n cho ln v gia cm: Cc chng nghin cu c nui cy trn mi trng dch th tng ng (MRS cho VK Lactic; thch thng cho VK Bacillus v YM cho nm men) nhng c b sung cc thnh phn c hot tnh thng c trong cc khu phn n cho ln v gia cm gm mt s loi khng sinh: Bacitracin Methylene Disalicylate (BMD) (50 ppm); Saigon Nox (100 ppm), Colistine 98% (100 ppm), chlotetracyclin (CTC) 15% (100 ppm); mt s loi khong CuSO4 (250 ppm Cu); ZnSO4 (100 ppm Zn) v hn hp axit hu c (gm axit lactic axit, axit formic, axit citric...) vi liu 200mg/lt.. Nui cy lc (200 vng/pht) trong t m (37oC). m mt t bo trn mi trng a thch sau 48h.

KT QU V THO LUN Phn lp, tuyn chn, phn loi v nh danh cc chng vi sinh vt hu ch T cc ngun khc nhau (cht cha ng rut ca ln, g v cc mu sn phm probiotic thng mi), 64 chng vi sinh vt c phn lp, trong c 27 chng vi khun lactic (42,2%) v 39 chng nm men (57,8%) (Bng 1).

Trong t cht cha ng rut ca ln v gia cm phn lp c 25 chng vi khun lactic, 32 chng nm men. T mt s ch phm probiotic hin ang lu hnh trn th trng, phn lp c 2 chng vi khun lactic v 7 chng nm men.

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Bng 1: Kt qu phn lp cc vi sinh vt t cc ngun khc nhau

Vi sinh vt Ngun phn lp VK Lactic Nm men

Cht cha rut non v rut gi ca ln. 10 4 Cht cha rut non, rut gi ca g 15 28 Bn (4) mu sn phm probiotic thng mi 2 7

Cng 27 39 Tng s 64

Kt qu phn lp cc mu sn phm probiotic thng mi cho thy, tt c cc sn phm ny u l cc sn phm probiotic a chng. lm c s cho vic tuyn chn, cc chng VSV trn tip tc c thun khit trn mi trng a thch v chn c 4 chng (2 chng VK lactic v 2 chng nm men) nh gi cc c tnh hnh thi, sinh l, sinh ha quan trng lm c s cho vic phn loi. Kt qu c trnh by Bng 2 v 3.

Bng 2: c im hnh thi, sinh l, sinh ho ca 2 chng vi khun lactic

K hiu chng TT c im NC1 NC2

1 Hnh dng t bo Que ngn Que ngn 2 Nhum Gram Gram + Gram + 3 Phn ng catalaza - - Kh nng ng ho ngun cacbohydrat

4 Riboza - + 5 Xyloza - + 6 Arabinoza + + 7 Rhamnoza + + 8 Trehaloza + ++ 9 Lactoza +++ ++ 10 Mannitol ++ ++ 11 Sucroza +++ +++ 12 Cellobioza +++ +++ 13 Raffinoza ++ ++ 14 Galactoza + ++ 15 Tinh bt + - 16 Gluconat ++ +++ 17 Fructoza + ++ 18 Thnh t bo c meso-DAP + +

Hai chng vi khun Lactic c la chn (NC1 v NC2) u l nhng VK hnh que ngn, Gram dng v phm ng katalaza m tnh. y l nhng c im c trng ca cc VK Lactic. Tuy nhin, kh nng ng ho cc ngun carbohydrate ca 2 chng ny c nhng khc bit, Chng NC2 c kh nng ng ha hu ht cc ngun carbohydrate nghin cu tr tinh bt, trong chng NC1 c kh nng ng ha tinh bt mc thp, nhng li khng c kh nng ng ha riboza v xyloza. C 2 chng u c kh nng ng ho tt 2 ngun carbohydrate c bn l Sucroza v Cellobioza, hai chng nm men u c hnh trng, elip, nhng SC ny chi

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1,2,3 pha, trong khi SB ch ny chi 1 pha. C 2 chng u c hnh thi khun lc nh nhau (trng sa, trn nhn bng v c vng ng tm) kt qu Bng 3.

Nhn chung, c im ng ha v ln men cc ngun carbohydrate ca 2 chng nm men tng t nhau, c hai u c phn ng m tnh vi DBB v thu phn ur. Cn c vo kho phn loi ca Bergeys v cc d liu cc bng 2 v 3 th chng NC1 thuc nhm Lactobacillus fermentum, chng NC2 thuc nhm Lactobacillus casei, chng SC thuc Saccharomyces cerevisiae v chng SB thuc nhm Saccharomyces nhng cha r loi.

Bng 3: Mt s c tnh hnh thi v sinh l sinh ho ca hai chng nm men SC v SB

STT c im Chng nm men SC SB 1 Hnh dng t

bo Hnh trng, elp,

ny chi 1,2,3 pha Hnh trng, elp, ny chi 1 pha

Trng sa trn nhn bng li c vng ng tm

Trng sa trn nhn bng li c vng ng tm

2

Hnh thi khun lc Kh/nng ng

ho ngun cacbohydrat

KN ln men ngun

cacbohydrat

KN ng ho ngun

Cacbohydrat

KN ln men ngun

cacbohydrat 3 Sorboza - - - - 4 Riboza - - 5 Xyloza - - - - 6 Arabinoza - - - - 7 Rhamnoza - - - - 8 Trehaloza + + + + 9 Lactoza - - - - 10 Sucroza + + + + 11 Cellobioza - - - - 12 Raffinoza + + + + 13 Galactoza + + + + 14 Tinh bt - - - - 15 Melibioza - - _ _ 16 Glucoza + + + + 17 Sinh axit - - 18 Phn ng DBB - - 19 Phn ng thu

phn ur - -

Tuy nhin, gi tn chnh xc, cc chng trn c gii trnh t ARN v ADN 16S. Cc kt qu nh danh c trnh by nh sau: Trnh t rADN 16S ca chng NC1 >NC1 GCCAACGAGTGGCGGACGGGTGAGTAACACGTAGGTAACCTGCCCAGAAGCGGGGGACAACATTTGGAAACAGATGCTAATACCGCATAACAGCGTTGTTCGCATGAACAACGCTTAAAAGATGGCTTCTCGCTATCACTTCTGGATGGACCTGCGGTGCATTAGCTTGTTGGTGGGGTAACGGCCTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACAATGGGACTGAGACACGGCCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTAAAGAAGAACACGTATGAGAGTAACTGTTCATACGTTGACGGTATTTAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGT

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GGCAAGCGTTATCCGGATTTATTGGGCGTAAAGAGAGTGCAGGCGGTTTTCTAAGTCTGATGTGAAGCCTTCGGCTTAACCGGAGAAGTGCATCGGAAACTGGATAACTTGAGTGCAGAAGAGGGTAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAAATATATGGAAGAACACCAGTGGCGAAGGCGGCTACCTGGTCTGCAACTGACGCTGAGACTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCGAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTGCGCCAACCCTAGAGATAGGGCGTTTCCTTCGGGAACGCAATGACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAGATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCGAACTCGCGAGGGCAAGCAAATCTCTTAAAACCGTTCTCAGTTCGGACTGCAGGCTGCAACTCGCCTGCACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCCCACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAGCCAGCCGCTAAGGTGGGACAGATGATTAGGGTGAAGTCGTAACAAGGTAGCCGTAGGAGAACCTGCGGCTGGATCACCTCCTTT Trnh t rADN 16S ca chng NC1 tng ng vi trnh t rADN 16S ca Lactobacillus fermentum 99,7 % (1446/1450). Trnh t rADN 16S ca chng NC2 >NC2 ACGCTGGCGGCGTGCCTAATACATGCAAGGCGAACGAGTTCTCGTTGATGATCGGTGCTTGCACCGAGATTCAACATGGAACGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAGATCCAAGAACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTGTCGGGTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGTTGTCGGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAAGCGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTTTTGATCACCTGAGAGATCAGGTTTCCCCTTCGGGGGCAAAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATGACTAGTTGCCAGCATTTAGTTGGGCACTCTAGTAAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAGACCGCGAGGTCAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCGAAGTCGGTGGCGTAACCCTTTTAGGGAGCGAGCCGTCTAAGGTGGGACAAATGATTAGGGG

Trnh t rADN 16S ca chng NC2 tng ng vi trnh t rADN 16S ca Lactobacillus casei 99,7 % ( 1483/1486 ). Trnh t ADN 26S ca chng SC. >SC-I1 TTAAAGAAATTTAATAATTTTGAAAATGGATTTTTTTGTTTTGGCAAGAGCATGAGAGCTTTTACTGGGCAAGAAGACAAGAGATGGAGAGTCCAGCCGGGCCTGCGCTTAAGTGCGCGGTCTTGCTAGGCTTGTAAGTTTCTTTCTTGCTATTCCAAACGGTGAGAGATTTCTGTGCTTTTGTTATAGGACAATTAAAACCGTTTCAATACAACACACTGTGGAGTTTTCATATCTTTGCAACTTTTTCTTTGGGCATTCGAGCAATCGGGGCCCAGAGGTAACAAACACAAACAATTTTATTTATTCATTAAATTTTTGTCAAAAACAAGAATTTTCGTAACTGGAAATTTTAAAATATTAAAAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAACGCAGCGAAATGCGATACGTAATGTGAATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCCTTGGTATTCCAGGGGGCATGCCTGTTTGAGCGTCATTTCCTTCTCAAACATTCTGTTTGGTAGTGAGTGATACTCTTTGGAGTTAACTTGAAATTGCTGGCCTTTTCATTGGATGTTTTTTTTCCAAAGAGAGGTTTCTCTGCGTGCTTGAGGTATAATGCAAGTACGGTCGTTTTAGGTTTTACCAACTGCGGCTAATCTTTTTTATACTGAGCGTATTGGAACGTTATCGATAAGAAGAGAGCGTCT

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AGGCGAACAATGTTCTTAAAGTTTGACCTCAAATCAGGTAGGAGTACCCGCTGACTTAGCATATCAATAGCGAAGAAAGAAAGCATATCAATAAGCGGAGGAAAAGAAACCAACCGGGATTGCCTTAGTAACGGCGAGTGAAGCGGCAAAAGCTCAAATTTGAAATCTGGTACCTTCGGTGCCCGAGTTGTAATTTGGAGAGGGCAACTTTGGGGCCGTTCCTTGTCTATGTTCCTTGGAACAGGACGTCATAGAGGGTGAGAATCCCGTGTGGCGAGGAGTGCGGTTCTTTGTAAAGTGCCTTCGAAGAGTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCTAAAGCTAAATATTGGCG AGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAGAACTTTGAAAAGAGAGTGAAAAAGTACGTGAAATTGTTGAAAGGGAAGGGCATTTGATCAGACATGGTGTTTTGTGCCCTCTGCTCCTTGTGGGTAGGGGAATCTCGCATTTCACTGGGCCAGCATCAGTTTTGGTGGCAGGATAAATCCATAGGAATGTAGCTTGCCTCGGTAAGTATTATAGCCTGTGGGAATACTGCCAGCTGGGACTGAGGACTGCGACGTAAGTCAAGGATGCTGGCATAATGGTTATATGCCGCCGTCTTGAAACAGGACC

Trnh t gen ca chng SC tng ng 100% vi Saccharomyces cerevisiae >SB-I1 GCCGGGCCTGCGCTTAAGTGCGCGGTCTTGCTAGGCTTGTAAGTTTCTTTCTTGCTATTCCAAACGGTGAGAGATTTCTGTGCTTTTGTTATAGGACAATTAAAACCGTTTCAATACAACACACTGTGGAGTTTTCATATCTTTGCAACTTTTTCTTTGGGCATTCGAGCAATCGGGGCCCAGAGGTAACAAACACAAACAATTTTATCTATTCATTAAATTTTTGTCAAAAACAAGAATTTTCGTAACTGGAAATTTTAAAATATTAAAAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAACGCAGCGAAATGCGATACGTAATGTGAATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCCTTGGTATTCCAGGGGGCATGCCTGTTTGAGCGTCATTTCCTTCTCAAACATTCTGTTTGGTAGTGAGTGATACTCTTTGGAGTTAACTTGAAATTGCTGGCCTTTTCATTGGATGTTTTTTTTCCAAAGAGAGGTTTCTCTGCGTGCTTGAGGTATAATGCAAGTACGGTCGTTTTAGGTTTTACCAACTGCGGCTAATCTTTTTTTATACTGAGCGTATTGGAACGTTATCGATAAGAAGAGAGCGTCTAGGCGAACAATGTTCTTAAAGTTTACCTCTCAAATCAGGTAGGAGTACCCGCTGAACTTAAGCATAC Trnh t gen ca SB tng ng 100% vi Saccharomyces boulardii, sai khc 12 /707 bp vi Saccharomyces cerevisiae. Cc kt qu phn loi theo phng php c in v da vo phn tch trnh t ADN v ARN c trnh by Bng 4.

Bng 4: Kt qu phn loi v nh danh cc chng c la chn

STT K hiu Da vo phn tch trnh t 16S rARN Theo phng php c in 1 NC1 Lactobacillus fermentum Lactobacillus fermentum 2 NC2 Lactobacillus casei Lactobacillus casei 3 SC Saccharomyces cerevisiae Saccharomyces cerevisiae 4 SB Saccharomyces boulardii Saccharomyces sp.

Cc kt qu Bng 4 cho thy, c s ph hp rt cao ca vic phn loi gia phng php c in v phn loi da trn c s phn tch trnh t AND v ARN riboxom. T kt qu phn loi trn cho thy, vic phn lp v tuyn chn cc chng VSV hu ch chn thm c hai chng VK Lactic: Lactobacillus fermentum-NC1 v Lactobacillus casei-NC2. c tnh probiotic ca cc chng VSV hu ch: Cc chng vi khun Lactic thuc nhm L. fermentum v L. casei cng nh cc chng nm men thuc cc nhm S. cerevisiae v S. boulardii l nhng chng VSV lnh tnh (khng phi l nhng VSV gy bnh), thng c s dng nh ngun VSV Probiotic (Sanders v Klaenhammers, 2001).

Tuy nhin, mi mt chng thuc cc nhm ny c cc c tnh probiotic mnh yu khc nhau. Bi vy, trc khi c s dng, cc chng ny cn c nh gi cc c tnh probiotic. Kt qu nh gi c trnh by cc Bng 5; 6; 7; 8 v 9. Kt qu Bng 5 cho thy, 2 chng vi khun lactic (NC1 v NC2) pht trin tt phm vi nhit t 30-37oC. Khi nui cy nhit cao hn (45 v 55oC) sinh khi v kh nng sn sinh axit lactic gim r rt (t 5 n 8 x 107 37oC xung 2,8-5 x 106 cfu/ml 45oC v

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t 2-4 x 103 cfu/ml 55oC). Tng ng, vi s gim sinh khi VSV, hm lng axit lactic cng gim mnh. p ng v sinh trng i vi nhit nui cy ca 2 chng nm men (SC v SB) cng c xu hng tng t i vi nhm VK lactic, nhng 2 chng nm men t ra sinh trng tt hn khi nui cy nhit 30oC. Ngoi tr nhng VSV c phn lp t mi trng bn ngoi, cn hu ht cc VSV c phn lp t ng tiu ha ca vt nui u khng thch nghi vi mi trng nhit cao hn so vi nhit c th vt nui.

Bng 5: nh hng ca nhit nui cy n kh nng sinh trng v sn sinh axit lactic (g/l) ca cc chng VK Lactic v nm men.

Nhit nui cy (oC) 30oC 37oC 45oC 55oC

K hiu chng CFU/ml HA*

(g/l) CFU/ml HA

(g/l) CFU/ml HA

(g/l) CFU/ml HA

(g/l) NC1 4 x 107 2,68 8 x 107 3,08 2,8 x106 1,8 4 x 103 0,63 NC2 2,4 x107 2,34 5 x 107 2,58 5 x 106 1,1 2 x 103 0,49 SC 2 x 109 - 6 x 107 - 4 x 106 - 8 x 104 - SB 1,8 x109 - 4 x 107 - 4 x 107 - 2 x 104 -

* HA: hm lng axit lactic (g/l)

Tuy nhin, kt qu NC ca Phm Th Ngc Lan v L Thanh Bnh, (2003) cho thy, khi phn lp t phn g ti 789 chng vi khun lactic, c hai chng la chn (CH123 v CH156) c cc c tnh probiotic v c hai chng u c kh nng sinh trng khung nhit t 15-45oC. i vi cc VSV probiotic, kh nng sng trong phm vi nhit rng c ngha khng nhiu khi chng hin din trong ng tiu ha ca vt nui, nhng li rt quan trng khi chng tn ti trong iu kin bo qun v trong trng hp ch bin thc n.

Bng 6: nh hng ca pH mi trng nui cy n kh nng sinh trng v kh nng sn sinh axit lactic (g/l) ca cc chng vi khun Lactic v nm men.

Chng vi sinh vt pH mi trng NC1 NC2 SC SB

CFU/ml 1x108 1x107 8x106 1x107 2 HA(g/l) 2,48 2,12 - - CFU/ml 1,2x108 1,2x107 1x108 2,7x108 3 HA(g/l) 2,5 2,18 - - CFU/ml 1,3x108 1,3x107 4,5x108 4,6x108 4 HA(g/l) 2,56 2,2 - - CFU/ml 1,3x108 1,3x108 2,5x109 2,2x109 5 HA(g/l) 2,55 2,18 - - CFU/ml 1,1x108 1,1x108 3,0x109 4x109 6 HA(g/l) 2,45 2,08 - - CFU/ml 8,6x107 9,0x107 2,4x108 2,5x108 7 HA(g/l) 2,4 2,0 - -

HA: Hm lng axit lactic (g/l)

Mt c tnh rt quan trng khc ca cc vi sinh vt probiotic l kh nng sng trong cc mi trng c pH khc nhau. Cc kt qu nghin cu nh hng ca pH mi trng nui cy n mt v kh nng sn sinh axit lactic ca cc VSV probiotic c trnh by bng 6.

TRN QUC VIT - Phn lp , tuyn chn v nh gi .

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Cc s liu bng ny cho thy, p ng v sinh trng ca cc chng VK lactic v nm men khng thay i nhiu khi pH mi trng thay i (2; 3; 4; 5; 6 v 7). Tuy nhin, chng NC2 v 2 chng nm men (SC v SB) pht trin mnh hn mi trng c pH 5 v 6. mi trng pH 7 mt cc chng gim nhng vn duy tr c mc 107 n 108 cfu/ml.

Kt qu ny cho thy, c 2 chng VK Lactic v 2 chng nm men c la chn u c kh nng sng trong mi trng pH thay i t rt toan (pH = 2) n trung tnh. iu ny c ngha rt quan trng i vi cc VSV probiotic v mun pht huy tc dng, chng phi thch ng c vi mi trng trong ng d dy-rut ca ln v g, ni m pH mi trng thay i (t rt toan d dy n gn trung tnh rut non). Kh nng chu mui mt cng l mt c tnh quan trng ca cc VSV probiotic, cc kt qu nghin cu v kh nng ny ca cc chng NC1, NC2, SC v SB c trnh by Bng 7. Cc s liu Bng 7 cho thy, cc chng nm men (SC v SB) khng chu nh hng nhiu bi mui mt. Bng chng l khi tng dn nng mui mt t 0,2 ln 3% chng vn sinh trng v pht trin bnh thng, mt nm men vn t trn 109 cfu/ml. So vi 2 chng nm men, hai chng VK Lactic cng pht trin tt trn mi trng c mt mui mt cao (3%). Tuy nhin, mt km hn so vi nm men.

Bng 7. nh hng ca mi trng c nng mui mt khc nhau n kh nng sinh trng ca cc chng vi khun Lactic v nm men.

Nng mui mt (%) STT K hiu chng 0,2 0,5 1 1,5 2 3 1 NC1 8,6 x 107 1,1x108 1,3x108 2x108 2,2x108 1,8x108 2 NC2 1,3x107 61,3x108 1,1x108 1,3x 108 1,3x 108 9,0x107 3 SC 2,2 x 109 3,4 x 109 2,9 x109 3,4 x109 4,5 x109 2,3 x109 4 SB 2,1 x 109 2,5 x 109 3,9 x109 1,6 x109 2,8 x109 2,0 x109

Kh nng bm dnh vo nim mc ng tiu ho ca cc chng VK lactic v nm men c trnh by Bng 8.

Bng 8. Kh nng bm dnh ca cc chng VSV trn biu m rut.

TT K hiu chng Nng ban u (CFU/ ml) Kh nng bm dnh

(CFU/ gam rut) 1 NC1 6,28 x 109 1,5 x 108 2 NC2 2,4 x 109 1 x 108 3 SC 4,6 x 108 2,6 x 108 4 SB 3,8 x 109 4 x 108 5 E. coli 3 x 107 2,8 x 106

Kt qu Bng 8 cho thy, cc chng VK Lactic v nm men u bm dnh tt vo nim mc rut nhng mc khng ging nhau gia hai nhm. Cc chng nm men t ra c bm dnh cao hn (t 2,6 n 4 x 108) so vi 2 chng VK Lactic. iu ny rt d gii thch v cc chng ny u c ngun gc (c phn lp) t cht cha ng tiu ha ca vt nui.

S liu c trnh by cc Bng 6, 7 v 8 cho thy, c 2 chng VK Lactic v 2 chng nm men u c kh nng sng v pht trin trong mi trng tng t nh trong ng tiu ha ca ln v gia cm. Tuy nhin, nu ch l nhng VSV v hi, th d c sinh sng v pht trin tt trong ng tiu ha ca vt nui th cng khng c nhiu ngha nu chng khng c kh nng sn sinh cc Bacteriocin (khng cc vi khun kim nh) v c tnh probiotic khc. Cc

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s liu Bng 9 cho thy, cc chng VK Lactic NC1 v NC2 c kh nng khng c 3 loi vi khun kim nh nhng cc mc khc nhau.

Bng 9. Hot tnh khng vi khun kim nh ca chng vi khun lactic v nm men

Kh nng khng cc vi khun kim nh (D-d, mm) STT K hiu chng Salmonella entriristic E. coli Shigella flexneri

1 NC1 8 6 12 2 NC2 6 3 9 3 SC 0 0 0 4 SB 0 0 10 5 6H2 21 12 10 6 C3 16 11 10 7 7 10 7 7

Chng NC1 c sc khng vi E. Coli, Salmonella v Shigella mnh hn so vi chng NC2, c bit l kh nng khng E.Coli. Trong s 2 chng nm men, ch c chng SB c kh nng khng Shigella, chng SC khng khng bt k vi khun kim nh no. V khng c mt s c tnh probiotic nh cc VK Lactic v Bacillus, nhng cc chng nm men hu ch tc ng c li i vi vt nui qua c ch sau: (i) kch hot mt s enzyme thuc nhm disacharridase (surcrase, lactase, maltase) lm tng kh nng tiu ho ng a (Buts v cs, 1986); (ii) trung ha mt s loi c t ca vi khun (Castagliuolo v cs 1998); (iii) kt dnh mt s VK gy bnh c roi bm vo biu m rut nh cc th quan mannose v loi chng ra ngoi theo phn; v (iv) kch thch h min dch, tng sn xut IgA (Czerucka v Rauspal, 2002). Cc kt qu nghin cu v tnh tng thch.

Bng 11. Kh nng sinh trng ca cc chng VSV probiotic trong mi trng c cha mt s thnh phn c hot tnh ca khu phn.

S lng t bo (CFU/ml) Thnh phn C3 7 6H2 NC1 SB H4

i chng 7,0 x 108 8 x 1010 7,6 x 109 5,2 x 109 2,0 x 109 6 x 1010 Khng sinh

- BMD 4,3 x 103 2,5 x 106 3,9 x 109 4,8 x 109 7,5 x 108 6,2 x 108 - Saigon Nox 120 4 x 104 4,8 x 103 1,1 x 103 6,8 x 108 9,8 x 105 - Colistine 7,0 x 108 3,2 x 109 5,8 x 109 2,8 x 109 5,6 x 108 2,0 x 1010

- CTC 3,7 x 104 6 x 104 3,4 x 104 2,6 x 104 1,0 x 108 2,0 x 105 Mui khong

- CuSO4.5H2O 5,7 x 108 1 x 1010 6,8 x 109 1,8 x 109 1,0 x 109 7,2 x 109 - ZnSO4.5H2O 6,0 x 108 9,3 x 109 4 x 109 8,8 x 108 9,0 x 108 8,0 x 109 Hn hp axit hu c 5,2 x 107 1,0x1010 9,2 x 108 4 x109 1,8 x109 3,0 x 109 *Saigon Nox: Khng sinh hn hp ca Bayer gm Kitasamycin 50g/kg + Sulphamethazon 50g/kg.

Trong s cc yu t nh hng n sc sng v hot tnh ca cc VSV probiotic th s tng tc ca chng i vi cc thnh phn khu phn (ch yu l cc thnh phn c hot tnh nh mt s loi mui kim loi, cc cht axit ha v mt s loi khng sinh) c mt ngha rt quan trng (Suita Cruce v Goulet, 2001). Kt qu c trnh by Bng 11.

TRN QUC VIT - Phn lp , tuyn chn v nh gi .

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Qua Bng 11 ta thy, cc chng nm men (SB) v VK Bacillus (H4) vn sinh trng v pht trin bnh thng trong mi trng hin din mt s loi khng sinh, cc mui sulfat ca ng v km cng nh hn hp mt s loi axit hu c vi liu tng t nh liu khuyn co b sung trong khu phn cho ln v g.

Trong s cc VK Lactic, chng 6H2 v NC1 sng tt trong mi trng c BMD, Colistine, CuSO4.5H2O v ZnSO4.5H2O v hn hp axit hu c, ngoi tr Saigon Nox v CTC. Hai chng VK lactic cn li (C3 v 7) pht trin yu trn mi trng c BMD, Saigon Nox v CTC. Qua kt qu cho thy, cc chng vi khun nghin cu c tnh tng thch cao vi cc axit hu c, cc mui sulfat ca ng v km v mt s loi khng sinh c s dng ph bin trong thc n chn nui. iu ny c ngha rt quan trng trong vic hng dn s dng cc sn phm probiotic c cha cc chng trn trong chn nui ln v g. Vi sn phm probiotic m chng VSV trong c tnh tng thch cng cao vi cc thnh phn c hot tnh ca mi trng th tnh ng dng cng ln.

KT LUN V NGH Kt lun : Trn c s cc kt qu nghin cu, mt s kt lun c rt ra nh sau: Trong 64 chng c phn lp, nh gi, tuyn chn v nh danh bng phng php sinh hc phn t c 4 chng gm 2 chng nm men (S. Cerevease; S. Boulardii) v 2 chng VK Lactic (L. Casei; L. fermentum). Bn chng ny u l nhng VSV lnh tnh, c cc c tnh Probiotic c trng (bm dnh tt vo biu m rut, sinh trng v pht trin bnh thng trong iu kin tng t nh trong ng tiu ha ca vt nui, khng cc VK kim nh v c kh nng sn sinh enzyme tiu ha v axit hu c).

Cc chng VSV hu ch c tuyn chn (S. Cerevease; S. Boulardii; L. Casei; L. fermentum) u c tnh tng thch cao vi mt s thnh phn c hot tnh trong khu phn thc n nh: BMD, Chlotetracycline, Colistin, Kitasamycine, Sulfamethazon. Chlotetracycline; CuSO4.5H2O; ZnSO4. 7H2O v hn hp axit hu c. Cc chng VK Lactic c tnh tng thch yu hn. ngh : Cho c sn xut th mt s ch phm probiotic t cc chng c la chn v nh gi trn.

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*Ngi phn bin : Ths. Nguyn Giang Phc; TS. Nguyn Hu To