Đề Tài Thử Nghiệm Và So Sánh Các Phương Pháp Đo Hoạt Tính Kháng Vi Sinh Vật Của Vi Khuẩn Lên Men Lactic Để Chọn Chủng Tiềm Năng Probiotic - Luận Văn, Đồ Án, Luan Van, Do An

Dé dn tt aghigp kha 05- Khoa MT & CNSHL GVHD: TS. Nguyéa Hodi Hung Chuong 1: Mé Dau 1.1. Dat van dé Hign nay cOng nghé sinh hoc Lk m@t inh vye dang phat trién va c6 nhiéu tidm ning Idn, Viet Nam cing da ting bude two diéu kign dé phat ign cong nghé sinh hoe, dae biét 1A nhiing tng dung trong néng nghiép va cd trong cong nghiép. Probiotic mét thanh qua khoa hoc, mét thanh qua cia céng nghé sinh hoc. NG dang duige ting dung rng rai vao ddi sing con ngudi bai vi tinh hop ly va higu qué ma né thé hign, Higu qua tée dung cia probiotic khong chi don thudn a lam thife an ngon hon ma c6 rit nhiéu téc dung, nhw: tiéu hod thife an va im bét sy r6i loan tiéu hod; dy manh sy téng hgp vitamin B va mét sé enzyme tiéu hod; cdi thién sy dung nap lactose; cdi thién chite nding mién dich; ngan chin nhing ché loét trong hé théng tiéu hod; ngan chan chting viém; gidm cholesterol; gidm t} 1é chét non; lim gidm 6 higng vi khudn gay hai; tang trong nhanh. Trén quan diém vé an todn sinh hoc, an toan thiét thyc thi probiotic dang. chiém thé thugng phong so vdi mét sO phutong cach khdc. Vi tinh higu qua cla probiotic ((inh tri bénh) 1a sy diéu hod of nhién khOng lam (én div khéng sinh, tn du téc hai trong sinh vat chi. Ma véi sit khat khe ciia con ngwi thi diéu nay 1a s6 mot. Nhu da biét ude day va cd hién nay nhiéu ndng dan sit dung cht khéng sinh trong chin nudi nhv 1a bién php (i wu nhat bdi nhitng Igi ich ma né mang lai nut: + Tang ning sudt sinh trwdng va sinh sén 6 gia sie, gia cm SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 1a tt aghi@p kha 0S- Khoa MT & CNSH. GVHD: TS. Nguyéa Hodi Hung * Tang higu qua sit dung thife an, 1am cho vat nudi thich ting nhanh chéng véi sit thay déi bat hung vé co cfu vA ching loai nguyén ligu trong khdu phan an. . Nang cao chat lvong sin phém (gidm ty 1é thit mG, tang ty 1é thit nac, lim cho thit tré nén mém hon va khéng nhiém mam bénh). . Phdng cdc bénh man tinh va ngan chan x4y ra nhitng dich bénh do vi tring. © Ting bigu qua kinh 1 trong chin nuéi. Tuy mhién, thé gidi d& nhanh chéng nhan ra nhiing téc dong xdu do vie Jam nay mang lai. Sit dyng khang sinh liéu thap trong chan nudi (sit dung khéng. diing cich trong digu tri, phdng benh va ding trong thie &n chan nudi nh chat kich thfch sinh trudng) di din dén mot h4u qua rat nghiém trong 1a 1am ting hign tugng khéng khdng sinh ca c&c loai vi khudn gay bénh trén nguii va vat nuéi. C6 § kin cho ring, vige sit dung khéng sinh lidu thép trong chin nu6i di bién vat nuéi thanh noi dé mét sé loai vi khudn “ hoc” cach v6 hiéu hod tac dung cila cdc loai khding sinh. Hau qua cba sv khing khéng sinh 3 vi khudn vé kinh t& rat 1én. Tuy nhién, nhing thiét hai vé kinh t€ khéng phai 1a chinh yéu ma van dé dang lo ngai la khéng chi vat nudi ma ngay ca loai ngudi dang ding true hiém hog xAy ra ede thdm dich do nhiing lodi vi khudn khang thude gay ra ma khong thé kiém soat dug. Nhu vay nghién cttu phat trién va ting dung probiotic vao cugc sing 1 mot cong vige can duge quan tam va dfu wf nhigu hon nia, C6 nh vay mdi tiép tue hoan thin probiotic dem lai hiéu qué cao hon, chat lugng cude sng ngdy duge cao hdn, an toan hon dép ting nhu cdu ngay cng cao va khdt khe cba chting ta. C6 thé néi day 1a suf tée dGng than hitu cia con ngudi vao ty nhién nén di mo ra mOt chién htge phat trién bén vizng va an toan, SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 2Dé 4n tt aghigp kha 0S- Khoa MT & CNSH. GVHD: TS. Nguyéa Hodi Hung Khoa hoc céng nghé luén phat trién nhim dé d4p ting lai nhu céu ngay cAng cao cila con ngw0i, Trén phutong trinh tang tin nay, con ngudi ddi héi khde khe hon vé chat htgng cia moi loai sin phdm dac biét 1a su an toan vé stfc khoé cia chinh ban than ho. Ma chinh nhdng nhu edu nay 1 kfch thich t inte tiép thtic ddy khoa hoc phat trién. “ Probiotic” 1A mét phan cia sy phat trién dy. é c6 thé c6 mot ché phim probiotic o6 dy dit nhitng hoat tinh cin thigt, khau chon loc chting vi khudn dé 1am probiotic 14 cyc ki quan trong. Bai vingay tai khau nay sé quyét dinh vai trd va téc dung cia ché phdm lén d6i trong cin quan tam. Tuy nhién, trong pham vi nhé hep ctla nghién cu nay, (6i chi thye hién dé tai 6 bude kiém tra hoat tinh khéng vi sinh vat vi thdi gian thyc hién dé ti chi trong 12 tudn kh6ng cho phép t6i thye hign ho’n chinh tft c& cde tiéu chi tuyén chon Probiotic. Chinh vi thé tdi da chon dé tai “THU NGHIEM VA SO. sA iH CAC PHUONG PHAP BO HOAT TINH KHANG VI SINH VAT CUA VI KHUAN LEN MEN LACTIC DE CHON CHUNG TIEM NANG PROBIOTIC”. 1.2. Muc tiéu nghién cfu Thit nghiém va so sinh ede phuteng phép do hoat tinh khéng vi sinh vat eda vi khudn Ién men lactic, Chon loc vi khudn lén men lactic 6 hoat tinh probiotic. 1.3. Doi tugng nghién citu Vi théi gian han hep, dé tai chi tap trung 6 nhimg adi tugng sa - Vikhudn lén men lactic 6 nguén géc t¥ thyc phém Jén men ( cd mudi, dita mudi, nem, sifa Ién men) va ¢6 ngudn gic tit ce ché phdm duge. Vi sinh vat chi thi Escherichia coli. SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 3Dé dn tt aghigp kha 05- Khoa MT & CNSHL GVHD: TS. Nguyéa Hodi Hung 1.4, Phusng phap nghién citu 1.4.1. Phung phap ludn Truéc khi bat tay vao thy hién dé tai nay, tdi da tham khdo khé nhiéu cdc nghién ctfu ty trvéc tdi nay vé probiotic cing nhu cdc phudng phép tuyén chon n6. Nhan thay cé khé nhiéu phvong phap dude sit dung dé thyc hién viée chon loc nay, t0i da xem xét va chon ra nhifng phudng phdp dién hinh nhat cho dé tai cla minh, T6i xin dé xuat so dd tién hanh nghién ctu nhy sau: Téng hap bién tap ti Liu Phan tich cfc nghién edu lién quan Trao d6i ¥ kign vai gido vien hung din Tigh hanh thit nghiém cc phuting phap Ja cde phung phép Chon phung phap hanh chon lec probiotic Bua ra két qua nghién efu Hinh 1.1; So dé nghién ctu SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 4Dé én tt aghigp Khéa 05- Khoa MT & CNSH GVHD: TS. Nguyen Hodi Huss 1.4.2. Phuong phap xi ly sé ligu St dung phan mém Excel vé 46 thi biéu dién. Sit dung phan mém Statgraphies xit ly s6 liu th6, tinh gid tri trung binh, 46 1éch chudn, vé dé thi tang quan. 1.5. Y nghia khoa hoc va thu tién Tim hiéu vé céc phutong phép dénh gid khd nang khdng vi sinh vat chi thi ca ede vi khudn len men lactic. Tao tién dé cho cde nghién cifu lién quan sau nay tai phdng thi nghiém. G6p phén chon loc dvge ching vi khudn lén men lactic c6 hoat tinh probiotic. SVTH: Dé Qué Mi Huong MSSV: 105111016 ‘Trang sDé dn tt aghigp kha 05- Khoa MT & CNSHL GVHD: TS. Nguyéa Hodi Hung Chung 2: Téng Quan Tai Ligu 2.1. Téng quan vé Probiotics 2.1.1. Gidi thigu so luge vé probiotic 21.1.1. Gidi thigu chung Viée sit dung cdc vi khudn lactic nhu thie an bd sung da xudt hién ey lau diu tir khi con ngudi biét dén sifa lén men. Viet nghién citu duge bi Metchnikoff lam viée 4 vién Pasteur Paris. Ong ta cho ring vi sinh vat trong rudt-c6 anh hung xau tdi sife khée vat nudi va nhing anh hudng xu nay c6 étvé thé duge cai thién béi viée sit dyn sia chua. Ong da trich din ic quan néng din Bungari sit dyng so lugng In sa chua va of tuéi tho rit cao. Ong phan lap duge hé sinh vat tif sita chua 6ng goi li “Bulgarian bacillus” va sit dung ching trong céc thi nghiém. Nhiing sinh vat nay ditge xéc dinh va duge bist dén 1a Lactobacillus bulgaricus vA ngay nay dugc goi la L. delbrueckii subsp bulgaricus 14 mt trong sé sinh vat dude sitdung dé len men sifa va sin xudt yoghurt, Sau khi Metchnikoff mat vao nam 1916, hoat dng nghién cxfu nay chuyén vé USA. Buge bidt 3 thai diém dé nguai ta da dé xudt vie sit dung céc L. acidophilus va nhidu thit nghiém di duigc thy hign véi sinh vat nay. [34] Thugt ngi probiotic von c6 nhigu dinh nghia khde nhau, né duge sit dyng lan ddu én nam 1965 (Lilly & Stillwell ) dé m6 td mét ch&t duge to bai mot protozoan dé kich thich sif ting tng cba mGt sinh vat khée. Bén nam 1974, Parker da sit dung dé chi cde chat bé sung thife an dong vat: IA cde sinh vt va chat c6 tée dng tich cue 1én dong vat bing céch can bing vi sinh vt rude Fuller (1989) da dv ra dinh nghia rt gan vdi hign nay 1A “ mgt bé sung vi sinh vat sng qua thite an 6 téc dng tich eye lén k¥ chi bing céch cdi thién can bing vi sinh v4t duéng rude”. [33] SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 6Dé 4n tt aghigp kh6a 0S- Khoa MT & GVHD: TS, (guyén Hodi Huwng Bang 2.1: Mot s6'san phém sita 1én men o6 chtta duing cdc vi khudin probiotic (T. Mattila-Sandholm, M. Saarela, Probiotic functional foods) Dangsin Ténsan . Vi Khu4in probiotic bé ae . 2" | Cong ty oan Noi sin xudt phéim phém sung (107-10* LAB/ml) France, Belgium, Spain, Switzerland, Yoghurt Lcl Nestle L. johnsonii LC-1 Portugal, Italy, Germany, UK Yoghurt Gefilus Valio L. rhamnosus GG Finland Netherlands, Yoghurt Vitit Mona L. rhamnosus GG remenan’s Ireland Yoghurt Vifit Sudmilch L, rhamnosus GG Germany Yoghurt Waterfor " Yo-Plus . L. acidophilus Ireland drink d Foods Yoghurt | Bio-Pot_| Onken Biogarde cultures Europe Yoghurt LAT Bauer L. acidophilus Germany Nertherlands, Fermented ~. Yakult | Yakult | L. casei Shirota strain UK, milk drink Germany ‘Cultures yoghurt: Gaio MD- E. faecit Denmark style ° Foods * faecium “ product Dairygol Yoghurt SNO me L. acidophilus Actimel Cholester ‘Yoghurt 1 Danone L, acidophilus Belgium o Control Fermented ‘Actimel D L BE ctimel_| Danone casei rope milk drink p SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 7D8 in tt aghigp KGa 05- Khoa MT & GVHD: 5. Newer Hodé Huse Yoghurt | Yoplaic | W8terter L. acidophil Ireland f p dteede . acidophilus Fermented Bifidus, L. reuterii, L Bra-Mjolk | Arla ifidus, L, reutertt Sweden milk drink acidophilus Fermented Nutri L Netherland Trahan FYOS | Nutri casei jetherlands Symbalan L. reuterii, L. casei, L. Yoghurt | >" Tonilait werth TL. casel Switzerland ce acidophilus Yoghurt Shape | Stlvel L. acidophilus Ireland, UK 2.1.1.2. Higu qua sit dung probiotic Ba cé rt nhiéu ché phim probiotic danh cho ngudi hay cho vat nudi dge ding ki bao ho sng ché. Hau hét cfc sin phdm nay chita Lactobacillus spp. hode Streptococcus spp., mot sO chita Bifidobacteria spp., Saccharomyces boulardii, hay Bacillus subtilis, Anh hudng cba cée ché phim probiotic ¢6 thé 18 trye tip hode gidn tip thong qua sy diéu chinh hé vi sinh vat duong rude. Nhimng ché phém nay c6 nhiing higu qua sit dung duge biét t6i nhut sau: [2], [3], [5], [25]. 13: > > v lactose vVvvvy v > 2], [33] C6 khd nang khéng ung thit va chdng cée yéu t6 d6t bien Kim ham vi sinh vat gay bénh dung tiéu héa Ci thién vide sit dung lactose & nhiing ngudi khéng dung nap Lam gidm Cholesterol trong huyét thanh, Kich thich hé théng mién dich Gidm nhiém tring duang nigu Tang trong (5%) 6 gia cam Gidm bénh nhiém tring 6 gia cim Gidm tiéu chay 6 déng vat non Gidm téc dung phy ciia chat khang sinh SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 8Dé dn tt aghigp kha 05- Khoa MT & CNSHL GVHD: TS. Nguyéa Hodi Hung Higu qua lam sang cia mét vai ching probiotic dutgc trinh bay trong Bang dui day: Bang 2.2: Tac dung lam sang cia m6t s@ chting probiotic [8], [15] Chiing Tac dung lm sing trén ngudi Lactibacillus rhamnosus GG (ATCC 53103) Lactobacillus johnsonii (acidophilus) LJ-1 (Lal) Bifidobacterium lactis Bb-12 Lactobacillus reuteri (BioGaia Biologics) Gidm hoat tinh enzyme phan, gidm tiéu chay do khéng sinh 4 tré em, diéu tri va dy phong rotavirus va tiéu chdy céip 4 tré em, didu tri tigu chdy tdi phat do Clostridium difficile, kich thich mién dich, giém nhe tigu ching viém da khong dién hinh 6 tré em Can bang hé vi sinh vat dudng rust, ting cudng mign dich, hé trg didu tri Helicobacter pylori Dy phdng tiéu chay du lich, diéu tr tigu chdy do virus, ké cd rotavirus, edn bing hé vi sinh v§t ditdng rudt, cai thién tinh trang téo bén, kich thich hé mién dich, gidm nhe trigu chitng. viém da khéng dién hinh 6 tré em Riit ngdn thai gian bi tiéu chay do rotavirus 6 tré em, diéu tr tiéu chdy cap 6 tré em, an toan va dung nap tt 8 bénh nhén tudng thanh HIV dung tinh Lactobacillus casei Shirota Can bing hé vi sinh vat dudng rudt, gidm hoat tinh enzyme phan, c6 tée dOng tich eve SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 9Dé 4n tt aghigp kha 0S- Khoa MT & CNSH. GVHD: TS. Nguyéa Hodi Hung d6i v6i ung thu mat bang quang va ung thu cd. ti cung, khong dnh hung téi hé mién dich cila ngui khée manh Lactobacillus plantarum DSM9843 (299v) Saccharomyces boulardii Cn bing hé bi sinh vat dung rudt, ting ham hugng acid béo mach ngn trong phan Dy phéng tiéu chdy do khdng sinh, diéu tri viém rudt két do Clostridium difficile, dv phdng tiéu chay 4 bénh nhan sit dung dinh dang qua dng Ching trong sifa_chua (Streptococcus thermophilus hay L. delbrueckii subsp bulgaricus Khéng cé tée dung trén tiéu chay do rotavirus, khdng o6 higu ting ting cng mién dich khi bi tiéu chay do rotavirus, khéng c6 tdc dung lén hoat tinh enzyme phan Vao cudi nam nhing nam 1940 6 hai nghién cxtu phat trién vé hé vi sinh vat dung rudt nay. Dau tién, thay ring thuée khang sinh bd sung trong thitc an di thiic day tang trfng ciia vat nudi, Mong mudn khém phé eo ché nay da Anh hung tdi viée ting cu’ng nghién ctu vé thanh phan cila hé vi sinh vat dudng mudt nay va c&ch thife ma né tée dong lén vat ch. Thif hai, cang ngay cng c6 nhiéu vat nudi bj bénh, cung cap cho nhifng thi nghiém dé khdm pha hé sv trong dung rust bdi nhing vat chi cé sn. Va cudi cing cho thay ring L. acidophilus Khéng 1a vi khudn Jactobacillus duy nhét c6 trong rudt non ma cé nhiéu sinh vat khédc cdn duge nghién ctu dé sit sau dé cho théy cé khodng 10" vi dung lim probiotics. Nhiing nghién cifu tiép sinh v4t thuge khodng 400 loai khéc nhau tn tai d trong mudt (Moore & Holdemann 1974), chinh vi vay vige nghién ettu vé nhitng sinh vat e6 thé sit dung kim probiotic ngay cdng duge mé rong. [30] SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 10Dé dn tt aghigp kha 05- Khoa MT & CNSHL GVHD: TS. Nguyéa Hodi Hung Sau nhiéu nghién ctfu, ngudi ta da téng két lai dutgc rt nhiéu sinh vat cé thé sit dung lm probiotic, Diu nay sé duge tinh bay & muc tigp theo. 2.1.1.3. Cac thanh phiin cia Probiotics Bang 2.3: Nhiing vi sinh vat dutge xem nhit la probiotic (Holzapfel et al. 2001) [5] Lactobacillus | Bifidobacteriu | Other lactic acid ‘Non-lactic acid ” Bacteria bacteria L acidophilus | B. adolescentis | Enterococcus faecalis | Bacillus cereus var. Toyoi Lamylovorus | B.animalis | Emerococeus faecium | Escherichia coli Nissle 1917 1 casei B. bifidum | Lactococcus lactis Propionihacterium freudenreicht Leripatus | B. breve Leuconostoc mesenteroides. | Saccharomyces cerevisiae Ldelbrucckii | B. infantis | Pediveaceus acidolacici | Saccheromyces boulardi subsp Bulgaricus | B. lactis Streptococcus thermophilus L.gallinarun | B. Longum | Sporolactobacilts inulinus L gasseri L.johnsonit L paracasei 1 plantarum L reuteri 1. rhamnosus 2.1.1.4. Tiéu chi chon loc chiing probiotic [2], [3], [33], [341 Cée sinh vat ditge ha chon lam probiotic phai dap ting dugc cdc tiéu chudn ahi: > Tiéu chudn vé an toan > Tiéu chudn vé dic diém va chtfc nang Dé c6 nhigu nghién cttu, nhigu théng tin v8 nhiag tiéu chudn danh cho probiotic, ta c6 thé téng két lai nhu sau: > Khia canh an toan eda probiotic bao gém nhiing diém cy thé sau: * — C6 dinh danh chinh xéc SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 11Dé dn tt aghigp kha 05- Khoa MT & CNSHL GVHD: TS. Nguyéa Hodi Hung «© Nhitng ching sit dung cho ngwdi tét nhat 18 c6 nguén géc tiv ngudi © — Buge phan Iép tiy dutdng tidu héa ca ngudi khée manh © Dufge ching minh 1a khong c6 kha nang gay bénh © KhOng lién quan ti bénh tat © Khong gay khit lién hgp mudi mat © Dac diém di truyén én dinh * — Khéng mang cdc gen dé khdng khang sinh cé thé truayén dye Tinh an toan ca cée chiing probiotic 1a diéu duige quan tam hing dau. C6 mét sé phuong thife gitip tién hanh dénh gid tinh an toan cia probiotic nhu: nghién cifu trén cdc dac tinh cia chiing probiotic, nghién cifu vé dude déng hoc cia ching probiotic, nghién citu céc tic dng qua Iai gitta probiotic va vat chi. Céc probiotic thung thudc nhém vi sinh vat GRAS (Generally Regarded As Safe) Bang 2.4: Vi sinh vat probiotics va tinh an toan cia ehiing [2], [15] Vi sinh vat Kha nding lay nhiém Khong gay bénh, doi khi gay nhiém tring co hoi ¢ Lactobacillus cde bénh nhin suy gidm mién dich (AIDS) Lactococcus Khéng gay benh Gay bénh co Gi, 06 S. thermophilus diige sit dung Streptococcus trong céc san phém sita Gay bénh co hdi, mOt vai ching e6 kha nang khéng Enterococcus khang sinh Bacillus Chi c6 Bacillus subtilis dutge sit dyng lim probiotics Phan In khOng gay bénh, mot s gay nhiém trong 3 Bifidobacterium ngudi Propionobacterium | Cs tiém nang trong vige sit dung lim probiotic SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 12Dé 4n tt aghigp kha 0S- Khoa MT & CNSH. GVHD: TS. Nguyéa Hodi Hung Phan ldn khOng gay bénh, mét so gay nhiém tring 4 Saccheromyces - ngudi Phan Idn nhiing vi sinh vat sit dung lam probiotic cho ngudi déu phdi dat nhiing yéu cdu khdc khe nhu da néu trén, Cdn déi véi vat nudi ta o6 thé néi Ing nhitng yéu cau nay, tay thuge vao tiing loai vat nudi va tinh an toan khi sit dung cila né, >» Truéc khi m@t probiotic cé thé mang lai nhifng Igi ich trén site 2], [4], [23] © Chiing vi sinh vat phai e6 nhitng dic didm phd hgp vdi cong nghé khée con ngudi ching thudng phai cé nhitng dic diém sai € 06 thé dita vao sin xuit, d8 nudi cay, * C6 kha nding sOng va kh6ng bj bi¢n déi chi nang khi diva vo sén phém. © Khang gay cdc mdi vi khé chju cho sin phim. © Cée vi khudn sng phai di dén duge ngi tic dong cia ching. Dé tin tai duge né phai o6 dic tinh sau: C6 kha nang dung nap vdi acid (chiu pH thap 3 da day) va dich vi cia ngudi. C6 kha nang dung nap vdi mudi mat (Ia dic tinh rt quan trong dé probiotic c6 thé sing sét dude khi di qua rudt non), * C6 Khd ndng bam dinh va niém mac dufing tiéu héa vat chi. * C6 kha nang sinh cdc enzyme hode cde sén phém cudi cing ma vat chi 06 thé str dung * C6 Kha nang kich thich mién dich nhvng khéng c6 tée dong gay * C6 kha nang canh tranh véi hé vi sinh vat tf nhién, c6 hoat tinh adi khang vdi céc vi sinh vat gay bénh, dac biét la sinh vat gdy bénh dudng rust. SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 13a tt aghi@p kha 0S- Khoa MT & CNSH. GVHD: TS. Nguyéa Hodi Hung * Sdn xudt cde chét khang vi sinh vat ( vi du ohwt bacteriocin, hydrogen peroxide, acid hitu cd). © C6 Kha nang chéng d6t bién va cdc yéu t6 gay ung du. Sau day 18 mét sO chife ndng quan trong cba nhitng ching dutge sit dung 1m probiotic “© Kha ning b4m dinh [1], [2], [10] Kha nang bam vio bé mat va sau dé 1a phat trién trong dudng tiéu héa ngudi duge xem 1 didu kién tién quyét quyét dinh chife nang ciia probiotic. Nhiing vi khudn c6 khd nang b4m dinh vao bé mat ruét sé tén tai lau hon va do 6 6 diéu kign dé biéu hign nhiing tc dng diéu hda mién dich hon 18 nhong chiing khong ¢6 kha nang ném dinh Kha nang bém dinh sé tao nén mot méi ttong téc gitta probiotic va bé mat niém mac rudt lA noi chita cde té bao lympho, diéu nay sé kfeh thich tinh mién dich tai chd va ton bG co thé, Do dé ngwdi ta cho ring chi cé nhting ching probiotic ¢6 kha ning bém dinh mdi tgo duge higu qua cdm ting mién dich va Jam én dinh hang rao bao vé niém mac rudt Su bam dinh cia probiotic cting tao nén kha ning canh tranh gain két vaio biéu m6 rudt, gitta nhitng vi khudn gay bénh va probiotic. Qua mét sé thir nghiém, déu cho thay Lactobacillus acidophilus edn sOng hay da chét do nhigt déu 06 kha nang tie ché su bam dinh cila cde vi khudn gay bénh, Trong thit nghiém vé kha ning bam dinh cla probiotic nguti ta thiong kiém tra trén nhitng ddng t€ bao ung thy trye tring nhu HT-29 va Caco-2. Hai dang té bao nay duge bigt héa thanh t€ bao rude, dutge sit dung nh Lk mot md hinh cho biéu mé ru6t non. Nhitng thif nghiém nay cho ta biét duge su khdc biét vé kha ning bam dinh cia nhiing chting probiotic khéc nhau. SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang M4a tt aghi@p kha 0S- Khoa MT & CNSH. GVHD: TS. Nguyéa Hodi Hung Ngoai sit dung hai dong té bao trén, ngudi ta cdn o6 thé nghién cifu bing ky thugt sinh thiét, bing c&ch sinh thi€t mot m&u m6 sau mot thdi gian stt dung probiotic. Ky thugt nay dugc xem Ia cho két qua chinh xde nhdt vé khd nang bdm dinh ctia probiotic. Tuy nhién van con gdp nhiéu han ché trong van dé vé dgo dite cia viée lay mé, ddi héi ngudi thy hién c6 chuyén mon cao, va 6 thé mic sai s6. “> Kha nang diéu hda mién dich [1], [2] Nhiing cai thién hé mi&n dich bdi probiotic e6 thé duge trinh bay theo 3 céch sau: 1, Tang cung hoat dng cila dai thye bao, nang cao kha nang thufe bao cba sv hay hat carbon 2. Tang khd nang sin xudt khdng thé thing 1a loai IgG va IgM va interferon (nhan t6 khéng virus khéng dic hiéu) 3. Tang cutng kha nang dinh vi khang thé ten bé mat rudt, thuding 1a IgA > Kha nang chéng lai cdc yéu to gay bénh [2], [5] Dé o6 thé tée dng lén hé sinh théi vi khudn duding rugt dh diéu kh4é quan trong dé 18 probiotic phdi c6 kha nang chdng Iai cde vi khudn gay bénh bing c4ch tiét ra e4e khang sinh hay 18 nhitng cht canh tranh, Vi khudn probiotic tao ra cde chat da dang ma tfe ché ca vi khudn Gram dyong va Gram 4m. Nhing hgp chat nay e6 thé Am gidm khong chi nhitng sinh vat mang mdm bénh c6 thé sGng dutge ma cdn Anh hudng dén sif tra déi chat cba vikhudn va su tao ra ede doe 6. Qua nghién cifu cho thay cde khéng sinh va chat canh tranh thing thay 6 c4e chiing probiotic 18: © Bacteriocin * Hydrogen peroxide SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 15Dé dn tt aghigp kha 05- Khoa MT & CNSHL GVHD: TS. Nguyéa Hodi Hung * Acid hitu co (acid lactic, acid acetic..) © Diacetyl Co ché tc déng: canh tranh véi cdc nguén bénh dé ngn chan sv bém dinh yao dung rut, canh tranh dinh dung can thiét cho su sng sét cia mdm bénh, tac dOng khéng doc t6. + Kha nang chdng dot bin va cdc yéu td gay ung thu [2], [5], [18] Trong nhiéu nam qua, ¢6 nhiing nghién cfu cho thay vi sinh vat trong thy pham hay trong hé sinh thi rudt cé kha nang chdng dét bién va chdng lai cdc yéu t6 gay ung th, Co ché ny duge nghién cifu va két lugn nhu sau: Nh@ sv gn két va phan hily cdc chat gay ung thy San xudt cdc hgp chat khéng ung thu Diéu hda nhitng enzyme tién chat gay ung thy rudt, nh cdc enzyme phan chat tién sinh ung tht (nitroreductase, B-glucuronidase ) e6 kha nang chuyén thanh chat gay ung thi trong trye trang Ue ché khéi u bang mét co ché dap ting mién dich Nhtng van dé van cdn gidi han trong mé hinh in vitro hay in vivo, vige mé rong ra trén ngudi dé dy phong ung thy con dang la van dé cn nhiéu tranh 2.1.2. Qui trinh chon loc cdc ching Probiotic Qué trinh chon Ioc ching probiotic x@y ding theo hinh 2.2 va 2.3 due tinh bay sau day: SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 16GVHD: TS. Nguyéa Hodi Hung Tuyén chon va xéc dinh chiing dua trén kiéu hinh va kiéu gen - Tén chi, loai, ky higu ching - Dang ky trong bio ting gidng quée tf nao? Xéc dinh chife nang Danh gid 46 an toan - Invitro - In vitro va trén dong vat = Trén dong vat - Trén ngui: pha 1 Pha 2: Thit nghiém ma kép ngdu nhién gém nhém thi nghiém va nhém ‘Tt nhat nén thi nghiém 46i ching udng thude vo (DBPC) aé DBPC doe lap lan 2 dé xée dinh tinh cOng hiéu ca ching khang dinh két qua hoge sin phém Pha 3: Kiém nghiém mic dé hiéu qua trén ngwdi So sénh higu qua diéu tri 1 bénh die trig bing PROBIOTIC probiotic véi phvong phép diéu tri thong thudng. Dan nhan Tén chi, loai, ky hiéu ching S6 lvgng ti thiéu vi khudn séng Diéu kién bdo quan thich hop ‘Théng tin lién hé véi khdch hang Hinh 2.1: So dé huéng din etia FAO va WHO trong tuyén chon Probiobie SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 17Dé dn tt aghigp kha 05- Khoa MT & CNSHL GVHD: TS. Nguyéa Hodi Hung Phan lap cée dong vi khudn oO Sang loc in vivo Sang loc in quy m6 ahs vitro Khong dat Dat Kigm tra kh ning gay béah Tang tnféng nhanh > Nang cao kha nang sinh sin > Nang cao sife dé khang bénh tat > Nang cao nang sudt va chat htgng sita > Nang cao sin xudt iting Probiotic khéng phai 1a mét thyc thé don lé, nhitng ché phdm probiotic khde nhau chita dufng hé vi sinh vat khéc nhau thi e6 te d6ng khéc nhau. Ngay ca nhitng ching khéc nhau cia ciing mét loai c6 thé c6 hoat déng trao déi chat khée nhau sé anh hudng dén két qua khi ching duge sit dung nhu probiotic. Két qua tiéu cy c6 thé duge gidi thich 6 nhiing vat nudi cé site dé khang yéu hay digu kign vé sinh do giai doan tang trfng ciia vat nudi, liéu Ivong sit dung, chudng trai. Véi nhiing diéu ngage nhién mang lai, cling nh nhiing két qua cha nhw mong muén cba probiotic, nhung thye t€ 18 nhitng két qua dat duge rat 6 ¥ nghia, bing céch sit dung probiotic hgp ly, theo diing ede didu kiGn va sit dung diing phung phép sé mang lai nhiéu Igi ich cho viée bd sung nguén dinh dudng trong chan nudi. SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 26Dé dn tt aghigp kha 05- Khoa MT & CNSHL GVHD: TS. Nguyéa Hodi Hung 21.4.1. Trong chain nudi gia cm [4] Trong qué trinh cai tién di truyén, nang suat cua ga thit da duge cai thién dang ké. Khi ma diéu nay 1a tét cho nganh chan nuéi gia cdm, viéc ting mat dé nudi va hic d6 th thach thifc bénh tt gia tang gay cho gia cim d& nhiém cde Joai nguén bénh khéc nhau, dic biét 18 vi khudn dudng ruét nhw E.coli, Salmonelta spp, Clostridium perfringens va Campylobacter spp. Su nhay cim vdi mdm bénh din dén vige sit dung céc chat kich thich sinh trudng chéng vi khudn — cde cht von cin bin duige sit dung dé tang site khos cho dutdng rugt va kidm sot ede kich thich phu lam sang, Voi ¥ thite ngay cing ting cla xa hoi vé sy khang thuse cia vi khudn, thi vige sit dung khéng sinh trong céc liu thude chita bénh hoge phdng bénh cho gia chm da duge han ché mét céch nghiém ng@t th4m chf bi loai bs han 4 rat nhiéu quée gia Ty lau da cé nhiéu quan tam dén viée tim ra mét loai chat dé thay the khang sinh trong chan nudi gia cdm, Vi khudn sOng trong Ong tiéu héa eta gia cdm c6 anh hung stu se dén mét vai qué tinh sinh If eva vat chi (gia cdm). Voi suy nghi a5, diéu quan trong 1a phai higu c6 ché cila he vi khudn dung rugt gia cdm nhim fim ra chat thay thé thuée khéng sinh. Trong béi cdnh binh thudng, thi trong ditdng rudt c6 mét su can bling tinh t€ gitta ede vi khudn c6 Igi va gay bénh, N6 bj Anh hudng bdi ede ntong tée vA quan hé eGng sinh va canh tranh, Cong déng vi khudn d6 khong chi bdo vé bd méy tiéu hod ma cdn ting khd nding san xudt trong ddng vat chi. Sit dung Probiotic va Prebiotic 1a hai phutong ph4p d& duge nghién ctu va c6 tiém nang gidm bét nguén dich bénh déi véi chan nuéi gia céim va déng thoi nang cao nang sudt ctia chting. Cac chat nay mdi dugc dé nghi ding dé hé tra bdo vé nhiém bénh cila thit va cdi tin phan ting mién dich cho g& ( Theo SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 27Dé dn tt aghigp kha 05- Khoa MT & CNSHL GVHD: TS. Nguyéa Hodi Hung Huang va c6ng téc vién - 2004). Thytc phdm Probiotics va Prebiotics vao thite in khOng cdn 1A nhiing phusng phép méi, ma thie t€, ching da duge sit dung trong hang thé kj nut 1A mot thanh phn tf nhién cia thife an hoge nhu thite an 1én men, thé du nhu sifa chua (yoghurt), Bénh dufgng rudt c6 Anh hudng dén nganh chan nudi gia cdm. Ching Lm gidm nang suat, tang ty 1é chét va cing 14 ngudn nhiém tiém nang cho cdc sin phdm gia cdm, va gay nén mat an toan thyc phém cho con ngudi. Viée sit dung céc thude khang sinh trong thite in gia cdm 44 duge gidm bét do vin d8 khang khang thuée cia vi khudn. Céc chat thay thé cho khang sinh trong dinh duéng gia cim can duge danh gid nghiém tic trén thyc dia. Probiotics va Prebiotics 14 ce “thi sinh” thay thé kha quan. 21.4.2. Trong chan nudi gia sic [4] Gén day, nhiéu béo edo nghién cfu chttng minh higu qua ro ring cba probiotic trén heo, bao gém: - Lactobacillus spp va Bifidobacteria spp lam ting trong Iwong va gidm ti Ie chét non, Lactobacillus casei cai thign ting trudng cila heo con va gidm bénh tiéu chay, t4e dung eda né higu qua hon so vi viGe ding khéng sinh liéu chp. - Enteracide, mét probiotic chita Lactobacillus acidophilus va Streptococcus ‘faecium thém vio thite &n cho heo con cai sita kich thich sif ting tnfdng va hoat dng cia hé thong tiéu héa. Sut thém Streptococcus faecium vao khéu phén an cho heo con lam tang trong hfgng va ting hiéu qua thite an. - Hn hgp Lactobacillus spp. va Streptococcus spp. ting sit sinh trung va chife nang min dich 4 heo con. - B6tté bao vi khudn tiéu héa tit Brevibacterium lactofermentum gidm sit tac d9ng va sy nguy hiém cia bénh tiéu chay 6 heo con. SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 28Dé dn tt aghigp kha 05- Khoa MT & CNSHL GVHD: TS. Nguyéa Hodi Hung - Heo con an Bacillus coagulans 6 ti 16 chét gidm va cdi thién viée tang trong htgng, sit chuyén héa thife &n tt hon heo con khdng c6 an bé sung cing nhtf so vdi heo ding khdng sinh liéu thap. ~ Bacillus licheniformis cdi thién trong htgng, chuyén héa thite an va gidm bénh tiéu chy, 118 chét non. - Biomate 2B plus (B.licheniformis va B. subtilis) ting higu qua thite an va tng tnfng cia heo con hon diing khéng sinh. - Heo con &n probiotic Bacillus toyoi ho&c hin hgp Saccharomyces cerevisae, Lactobacillus acidophilus va Streptococcus faecium lim tang trong higng dang é so vdi viée ding khang sinh. -Heo con an thie an bé sung nfm men (Saccharomyces cerevisae) c6 khuynh hung tiéu thu nhiéu thifc dn va ting trong hon. ~ Enterococcus faecium 18C23 ngin chin sit b4m dinh cia E.coli tao d6c 6 dung ruét vao lép mang nhay ruét non cia heo. Ngoai viéc tri bénh & heo con, cdn cé mét s& nghién cifu cho thay céc lactobacilli cing e6 kha nang ngan chan sf phét ign cia ede vi khudn gay bénh dudng rudt 4 trau bd. Theo Trovatelli va Matteuzzi (1976) do qué tinh bién Adi cia tau bd ( do mat 46 qué ding, sy so hai, thi€u thife An, sv di chuyén qué mite) va trong nguén thite &n thiGu ede vi khudn ¢6 Igi nén site dé khdng cla tau bd bi suy gidm, Bo dé, ngudi ta thudng cung ep thite in dang én men dé tg méi trudng méi trong rudt vat audi nhim mién nhiém véi vi sinh vat gay bénh, ddng thoi gitip ting trong va ting kha nding chuy€n héa thife an Fastrack, mgt sin phdm ding cho dng vgt nhai lai, chita Lactobacillus acidophilus va Streptococcus faecium, tao ra acid lactic, nim men gitp bé sung vitamin B va nhifng enzyme tiéu héa. G bé, Fastrack cdi thién tang trong, gidm SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 29Dé dn tt aghigp kha 05- Khoa MT & CNSHL GVHD: TS. Nguyéa Hodi Hung bénh tiéu chay va nhitng xéo tron tiéu héa khéc; ting san wong sifa va sy them ain d bd; t&ng Iugng thife dn d cifu va dé. 2.2. Vikhuén lén men lactic 2.2.1, Dae diém vi khudn [1], [8], [22], [29], (371 Dau tién xin gidi thigu vi tri cia vi khuan lactic trong hé théng phan loai: Vi khudn lactic thuéc lanh gidi vi khudn, nganh Firmicutes, Cing véi nganh Actinobacteria, ching qo thinh nhém céc vi khudn Gram duong. Tuy nhién so sénh vé di 1g base G+C thi nganh Firmicutes 6 d 1é hap wi voi a Actinobacteria c6 ti1¢ G + C cao, Cée chi (ging) chi y€u cla vi khudn lactic Lactococcus, Streptococcus, Leuconostoc va Lactobacillus, Ngoai ra chiing cdn 6 c&e chi khée nhut Carnobacterium, Aerococcus, Enterococcus, Vagococcus, Oenococcus, Pediococcus, — Tetragenococcus, va Weissella. Ngodi ra Bifidobacterium tude kia duige phan loai thude chi Lactobacillus (Lactobacillus bifidum) nay tich ra thanh chi Bifidobacterium, Chiing c6 nhiéu dic diém riéng bigt myc dd c6 wing dyng lm probiotics ging chi Lactobacillus va mot sé Enterococcus, Lactococcus. Cée vi khudn lactic hing duge wing dung lim probiotics 46 1a Lactobacillus acidophilus, L. plantarum, L. casei, L. casei rhamnosus, L. delbrueckii bulgaricus, L. fermentum, L. reuteri, Lactococcus lactis lactis, Lactococcus lactis cremoris, Bifidobacterium bifidum, B. infantis, B. adolecentis, B. longum, B. breve, Enterococcus faecalis, Enterococcus faecium. SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 30Dé én tt aghigp Khéa 05- Khoa MT & CNSH GVHD: TS. Nguyen Hodi Huss Domain Bacteria Finwictes Gowmol%-+C) Hinh 2.7: Vikhudn lén men lactic trong h¢ théng phan loai SVTH: Dé Qué Mi Huong MSSV: 105111016 ‘Trang 31Dé én tt aghigp Khéa 05- Khoa MT & CNSH GVHD: TS. Nguyen Hodi Huss Bang 2.6: Mét s6 dic diém ctia cdc chi vi khudn lactic [37] Vi pham vi 46 n cdn han ché nén chi thyc hién nghién citu trong pham vi cée vi Khudn thuge chi Lactobacillus. Sau day 1a dit diém chung cia chi Lactobacillus: Té bao hinh que, thung cé nhiéu dang: dai, manh va ngdn (dang tre cau Khudn coccobacilli). Kich thude t€ bao 0.5 - 1.2 x 1.0 - 10.0 um. Trong qué tinh sinh iudng, bao thydng (go thinh chudi 6 phase log. KhOng di dng, di dng khi c6 sy hign dién cla tién mao. Khéng tao bao tt, d dang gram dyong khi bdo con non, va gram Am khi € bao gid. Hinh dang khudn lac trén thach: dang Idi, mép rn, mau tng duc, thudng 6 dudng kinh 2-5 mm, ft tao s&c t6, 6 thé tao s&c t6 vang, cam hay mau gi sat va mau dé gach, Ki khi tdy nghi d0i khi hi€u khf. Phat wién manh wén moi mudng thach, ki Khi c6 5-10% CO>, catalase, cytochrome va benzidine 4m tinh, San phém ca qué tinh chuyén héa carbohydrate hon 50% 1a lactate, cdn Igi IA acetate, formate, succinate, CO> , ethanol. Khdng tao acid dé bay hoi cé s@ nguyén ti carbon hon hai. Kha nang khit nitrate kém va tao pH du@i 6.0. khong héa léng gelatin, Khong phan hy casein nhung vai ching cé thé to mgt Iutgng nhd dam hda tan, Khong tgo indole va HS. SVTH: Dé Qué Mi Huong MSSV: 105111016 ‘Trang 32Dé dn tt aghigp kha 05- Khoa MT & CNSHL GVHD: TS. Nguyéa Hodi Hung Nhu cu dinh du@ng phifc tap: amino acid, peptide, cdc din xudt acid nucleic, vitamin, mudi, acid béo, ester va mot sO nguén carbonhydrate va dic ining theo loai, Nhiét dé phat trién 5-53°C, nhiét dé t6i wu 30-40°C C6 thé phat trién tét 6 pH khodng 5 va pH téi wu [a 5.5-5.8 Duge tim thay trong cdc sin ph4m sifa, hat, sin phdm thit, nvéc gidi khat, bia, ntgu, muée ép trai cay, hoa qua, difa chua, trong nufdc thai, trong hé tiéu hoa ngudi va nhigu lodi déng vét. La nbtng sinh vit it gay bénk, of the dung tt vai dung tiéu héa. 2.2.2. Qué trinh Ién men lactic [1], [37] Vikhudn lén men lactic déng hinh Lén men acid lactic déng hinh ligng acid lactic tao nén chiém trén 80% va dude biéu didn tm tt bing phyong tinh: CsHi205 > 2CHs-CHOH-COOH Su hinh thinh nén acid lactic trdi qua hang hoat cde giai doan trung gian vai sy tham gia cila cde enzyme ting ting. Giai doan dau xay ra qué trinh hoat héa hexose duge phan c&t dé hinh thanh triosephotphate, Chat nay duge chuyén thanh acid pryruvie rdi thanh acid lactic Vikhudn lén men lactic di hink Trong lén men lactic di hinh, tao sin phém da dang ngoai acid lactic cdn 6 hang loat sin phém phy khc, cdc sin phdm phy va acid lactic duge sinh ra v6i s6 Iugng phan tit gam nh nhau. Cy thé 1a: acid lactic 40%, acid succinic va ic 10% va cdc cht khi cdn lai 20%, nugu etylic 20%, acid a Sy da dang cia sin phém tao thanh khi lén men lactic di hinh vi vi khudn. thude nhém nay c6 nhiéu he enzyme nén qué trinh chuyén héa dung phife ap hon d vi khudn lactic déng hinh SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 33,Dé én wt nghigp kha 0S- Khoa MT & CNSH GVHD: TS. Nguyba Hodi Huse > — Khi ndng 49 cia acid lactic dat 2-3% sé tie ché hoat dong cia edie vi sinh khdc, ké cd E.Coli. A awcose 30° cuneor eracmyae simon ary 2 spnceonoranae 2 2pmpmaycom Pinion pega Dales espn Gewalsngs oP det ohoupate ‘poashegyenat Presteoyrowe nan" Hinh 2.8: Qué trinh lén men lactic cia LAB. (A): Lén men lactic déng hinh. (B): Lén men lactic dj hinh [37] SVTH: Dé Qué Mi Huong MSSV: 105111016 ‘Trang 34Dé dn tt aghigp kha 05- Khoa MT & CNSHL GVHD: TS. Nguyéa Hodi Hung Cée enzyme trong qué trinh: 1. Glucokinase; 2. Fructose-1,6-diposphate aldolase; 3. Glyceradehyde-3-phosphate dehydrogenase; 4. Pyruvate kinase; 5. Lactate dehydrogenase; 6. Glucose-6-phosphate dehydrogenase; 7. 6- phosphogluconate dehydrogenase; 8. Phophoketolase; 9. Acetaldhyde dehydrogenase; 10. Alcohol dehydrogenase. 2.2.3, Kha nang téng hgp enzyme Céc vi khudn lactic c6 kha ning téng hgp mét sé lugng 1én © enzyme ngoai bao kich thich hé thng tiéu héa nhu: enzyme amylase, protease, lipase, glycolase va lactic dehydrogenase. Proteolysis (kha nang phan gidi protein): vi sinh vat téng hdp protease giip phan hijy protein thanh nhitng hgp chat ddn gian cé thé tiéu héa dutgc. Hoat tinh nay cta Lactobacilli trong dung ruét gitip phan hiy protein va vat chi cé thé tiéu thu dé dang. Lipolysis: Vi sinh vat téng hop lipase gitp phan hiby cée chat béo phife tap thanh nhiing hop chét don gidn. Diéu nay 6 thé hitu ich trong vige (ao ra cde khdu phan an dinh dung va hgp ly cho tré em, ngudi gid vA ngudi dang dong bénh. Nhiéu thi nghi da ching minh ring Lactobacilli c6 thé phan hiy Cholesterol. Bién du@ng lactose: Vi khudn sinh acid lactic e6 enzyme f-galactosidase, glycolase va lactic dehydrogenase es thé sdn sinh acid lactic tt lactose. Acid lactic e6 nhitng Idi feh nh sau * Chita tri bénh khéng dung nap lactose do thiéu cde enzyme bién dung lactose. © Tang cuing kha nang tiéu h6a protein trong sifa bing viéc im déng ty protein trong sita. © Tang curing vige sit dung Calci, Phospho, Sat. SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 35,a tt aghigp kha 0S- Khoa MT & GVHD: TS. Nguyéa Hodi Hung © Kich thich su bai tét. © Kich thich su ti¢u héa trong da day. * Bao tén nguén nang htgng trong qué trinh hé hap. 2.2.4. Kha nang téng hgp vitamin va cac chat trao ddi cé Idi cho su tang trudng Vi khudn lén men lactic ding lam probiotic déng vai trd néi bat trong rudt bing viéc téng hgp vitamin nhu: vitamin B, acid folic, biotin (vitamin H), vitamin K. 2.2.5. Kha ning san sinh cdc chat khéng khudn 2.2.51. Bacteriocin (12), [13], [21], [38], [43] Bacteriocin 1a nhitng hgp chat 6 ban cht 1a protein do vi khudn sinh tng hgp va cé kha nang te ché sy phat trién cia cc gidng vi khudn khéc cé lién hé gan vdi gidng sin xudt. Bacteriocin duge sinh tng hop bai cd vi khudn gram 4m va gram difong. Bacteriocin khéc véi khéng sinh 4 nhitng diém chit y€u sau * — Bacteriocin due téng hgp nhd ribosome © Té bao chi mién dich vdi chting + Phé khang khudn hep, vi vay thuting chi c6 kha ning teu didt nhing ching vi khudn c6 lién hé gan véi chiing san xuat, C6 rat nhigu gidng vi khudn sinh tng hgp bacteriocin, trong dé lactic acid bacteria (LAB) dufge quan tam nhiéu nhat do bacteriocin cia LAB ¢6 phé khang. Khugn rong va c6 tiém nang dutge ding lim chat bdo quan the phéim va tng dung trong duge phém. Bacteriocin duge LAB téng hgp chia thanh 4 Iép: Lép I: (Lantibiotic) 18 nhing phan tit peptide nhd (30kDa) va bén nhiét, Iép nay gém nhitng enzyme ngoai bao (hemolysin va muramidase) c6 hoat tinh sinh ly ctia bacteriocin, Bacteriocin dang nay duige thu nh§n ti’ mOt s6 ging Lactobacillus . Lép IV: I nhitng bacteriocin phite hgp, ngo&i protein cdn cé thém thanh phan lipid va carbohydrate. Hign nay vin con nhigu digu chua biét vé cau tréc va chiing nang ciia bacteriocin thudc Iép nay bi chifa cé phan tif nao thudc Iép nay duge tinh sach, SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 37Dé én tt aghigp Khéa 05- Khoa MT & CNSH GVHD: TS. Nguyen Hodi Huss lass lass Bacteriolysins (Salo msn, (Lyscstantin)| Copyright© 2005 Nowe Pubicting Group [Nature Reviews | Microbiology Hinh 2.9: eo ché khang khudn efia mét sf loai Bacteriocin [48] Giai thieh: Lép I (Nisin): Dang A Lanthibiotics gm nhiing phan tit lung tinh dai e6 thé tigu dit cée t€ bio man cdm bing céch tgo 15 trén mang sinh chat Lép Il (sakasin): Chiing mang dign duéng trong moi rung trung tinh va chiing chia mt ving ky nude vashode mgt ving lwGng cye. Ching ¢6 thé 1am thifm mang sinh chat @ bao dich, Bacteriolysins (Iysostaphin): téc dng phé hiiy vach t@ bao. SVTH: Dé Qué Mi Huong MSSV: 105111016 ‘Trang 38Dé én tt aghigp Khéa 05- Khoa MT & CNSH GVHD: TS. Nguyen Hodi Huss Bang 2.7: Mét s6 Bacteriocin va dic diém cia chting [22] Lodi Bateriocin | Phd tie ding Daediém Tactocaccus lactis | Nisin Vikhuiia gram dive | Lap I: lantibiotc, 35 kDa, 34 subsp. lactis amino acids, Lacticin 3147 | Clostridium sp Lap Tofu thinh wr 2 Listeria monocytogenes | lantibjotic, 4.2 kDa, bén v6i Staphylococeus aureus | ni, Streptococeus dysgalactiae Enterococeus faecalis Propionibacterium acne Streptococeus mutans Taciococeus tacts | Eaciococein B | Eactobacilus Lap I: Khodng 5 kDa, pha tae subsp. eremoris ong hep. Lactobacillus ‘Acidocin CHS | Vika gram dug | Lap Ti: eu thinh tr ede phan acidphilus Lactobacillus tiring I LactacinF | Lactobacillus fermentum | Lép I: 63 kDa, 37 amino Enterococcus faecalis acid, ehju due nbigt d9 121°C Lactobacillus delbrueckii trong 15 ph Lactobacillus helveticus LactasinB | Eactohacillus debrwecki? | Lap Ul: 63 kDa, chiw dso Lactobacillus helveticus | nhigt, cbf dsge wag hop Khi Lactobacillus bulgaricus | mudi edly 6 diéu kign pH 5.0- Lactococcus lactis 60 Tactobacillus Lactobin A | Lactobacillus acidophilus | Lap Ul: 4.8 kDa, 30 amino amylovorus Lactobacillus debrweckit | acid, phd tae dong hep. Lactobacillus casei | Lactocin 705 | Listeria monocytogens | Lp We edu think wt 2 Lactobacillus plantarwn | pacteriocin (m’i_ bacteriocin 34 KDA, 33 amino acid) Leuconostoc gelidwn | Levcocia A | Lactobacillus Lap I: 39 kDa, 37 amino Enterococcus faecalis | acid, 6n dink & pH th, chiv Listeria méncytogenes | duige 100°C 8 20 phit. SVTH: Dé Qué Mi Huong MSSV: 105111016 ‘Trang 39Dé én wt nghigp kha 0S- Khoa MT & CNSH GVHD: TS. Nguyba Hodi Huse helveticus Leuconostoc Mesentericin | Enterococcus faecalis | Lap I: 38 kDa, 37 amino mesenteroides ios Listeria monocytogenes | acid, chiu duge mhigt 46 60°C trong 120 phit & pH 45 Peliococeus| PediociaF | vikhudn gram dung | Lap Il: 4.3 kDa, thude enzyme acidilactici proteolytic, bn voi nigt, hoa tan chét hit ca, hoat dng & hodng pH rong, Pediosia PAI | Tisteria monocytogenes | Lp I; 46 KDA, 44 amino aid. Pediocin AcH | Vi Khudn gram am va | Lép If 46 kDa, 44 amino gram dsm. acid, phé tée dng rong, Pediococcus Pediocin A | Lactobacillus Lap If 2.7 kDa, huge enzyme pentosaceous Lactococcus proteolytic, tén tai 8 100°C Leuconostoe trong 10 phi. Pediococeus Staphylococcus Listeria Clostridun Lactobacillus sake | Lactocin S| Lactobacillus Lap 1: 37 kDa, hoat ding & Leuconostoc pHAs75 Pediococcus SakacinP | Listeria monocytogenes | Lop: 4.4 kDa, chiu big Tactobacillas Curvacin A | Listeria monocytogenes | Lap 4.3 kDa curvatus Enterococcus faecalis Tactobacilas Helveticind | Lactobacillus bulgaricus | Lap il: 37 kDa, phd tds dng Lactacoccus lactis, hep, gidim host dong sau 30 phe 8 100°C Céc chat c6 kha nang khang khudin khac Cée LAB ciing 06 kha nang tfc ché sy phat trién cita céc vi sinh vat gay bénh thong qua mét sO cdc sin phdm bién dudng khéc ngoai bacteriocin nhu: SVTH: Dé Qué Mi Huong MSSV: 105111016 ‘Trang 40Dé 4n tt aghigp kha 0S- Khoa MT & CNSH. GVHD: TS. Nguyéa Hodi Hung hydrogen peroxide, cacbon dioxide va diacetyl, acid hitu co chi yéu 1a acid lactic. Qué trinh bién dudng cia LAB 6 anh hudng dén kha ning khdng lai céc vi sinh vat c6 hai cdc dang hoat déng khéc cia chting. Duge thé hién r6 qua bang sau! Bang 2.8: Kiéu hoat ding 46i khang ctia cdc sin phdm bién duéng San phdm bién duéng Kiéu hoat dong d6i khang CO, Ue ché qué tinh decarboxylation, gidm tinh thém qua mang ( khif Carboxyl), Diacetyl Tc d6ng lén protein gdn arginine. Hydrogen peroxide Oxy héa cée protein cd ban. Lactoperoxide ‘Acid lactic Acid lactic khéng bi phan hy ma thai vao mang lam gidm pH ndi bao, N6 cting lién quan t6i qué trinh bin dung nhut: phosphoryl oxy héa. 2.3. Visinh vat chi thi 2.3.1. Gidi thigu vé vi sinh vat chi thj (indicator strains) Vi sinh vat chi thi 14 nhifng vi sinh vat gay bénh réi loan hé tiéu héa cé ngudn géc thc phém. Dé chon loc Probiotic, ngu@i ta kiém tra khd nang tie che cila cdc vi sinh vat probiotic lén sy sinh trvéng phét trién ciia vi sinh vat chi thi. Mét sé vi du vé cdc vi sinh vat chi thi nay 1a: SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 41Dé 4n tt aghigp kha 0S- Khoa MT & CNSH. GVHD: TS. Nguyéa Hodi Hung Bang 2.9: M6t s6 vi sinh vat chi thi dién hinh sit dung trong nghién cxtu chon loc probiotic Visinh vat chi thi Bacillus cereus Clostridium perfringens Listeria spp Staphylococcus aureus Ngudn nghién ctu Carherine B. Lewus, Thomas J. Montville, USA, 1991 Listeria monocytogenes Escherichia coli Salmonella N. Chauteu, I. Castellanos, A.M, Deschamps. France, 1992 Helicobacter pylori Clostridium difficile Campylobacter jejuni Escherichia coli J. Nowroozi, M. Mirzaii, M Norouzi, Iran, 2004 Escherichia coli Klebsiella pneumonia Pseudomonas aeruginosa Bacillus subtilis Staphylococcus aureus Enterococcus faecalis Pediococcus acidilaticii Lactobacillus helveticus Jin-Woo Kim, $.N, Rajagopal, USA, 2001 Samonella Enteritidis Escherichia coli Clostridium perfringens Magdalena Kizerwetter-Swida, Marian Binek, Poland, 2005 Shigella dysenteriae Escherichia coli Salmonela typhi Yersinia enterocolitica V. Padmanabha Reddy, M.D. Christopher, I. Sankara Reddy, 2006 SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 42a tt aghi@p kha 0S- Khoa MT & CNSH. GVHD: TS. Nguyéa Hodi Hung Rat nhiéu nghién ctfu chon Escherichia coli 1a vi sinh vat chi thi, dae biét Ia chi thi cho nhitng bénh lién quan t6i thye phdm hay dung tiéu héa. Chinh vi vay, dé tai nay da thic hién chon loc probiotic bing cdch kiém tra su tfc ch sinh trang ciia probiotic lén vi khudn chi thi Escherichia coli. 2.3.2. Vikhudn chi thi gay bénh du@ng rudt - Escherichia coli (4), [35] 2.3.2.1. Die diém hinh dang, nudi cy va tinh chit sinh héa Vi khudn E.coli c6 nhidu trong ty nhién, trong dudng rudt cia ngudi va gia siic, Trong dung rugt, chiing hign dign nhidu d dai trang nén cdn goi la vi khudn dai trang. Vi khudn E.coli nhiém vao dat, nude... tit phan cila dong vat. Ching tr nén gay bénh khi gép didu kién thugn Igi cho sit phat tién cia ching. Phan loai khoa hoc: Vue (Domain): Bacteria Neganh (Phylum) Proteobacteria Lép (Class): Gamma Proteobacteria BO (Ordo): Enterobacteriales Ho (Familia): Enterobacteriaceae Chi (Genus): Escherichia Loai (Species): E. coli Hinh dang: Vi khudn thudc loai tryc khudn gram 4m, di déng bing tiém mao quanh t€ bao, khéng tio bao ti, loai e6 de hfc thi e6 bao nang, loai khong 6 déc luc khéng c6 bao nang. Kich th wéc trung binh (0,5p x 1-3) hai dau tron. Mét sé dong cé khudn mao (pili). Dac diém nudi ey va sinh héa: La loai hiéu kh{ hay hiéu khi thy nghi. Nhiét d6 thich hgp 37°C nhung c6 thé moc trén 40°C, pH 7,4. SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 43,Dé én tt aghigp Khéa 05- Khoa MT & CNSH GVHD: TS. Nguyen Hodi Huss - Trén méi truding thach dinh du@ng TSA tao khém ton wét (dang S) mau tréng duc, Bé lau khém ed nén kho nhan (dang R). Kich thse khém 2- 3mm. - Trén thach méu: C6 ching dung huyét 4, e6 chiing khong dung huyét 4, - Trén méi tring chin dodn chuyén biét EMB (Eozin Methyl Blue) tao khém tim 4nh kim. - Trén mdi trvng Rapid’ E.coli tao khudn lac mau tim. - Trén mdi tring Endo, SS tao khém héng dé. - Trén céc méi trvdng dung: Lén men lactose sinh hoi, glucose, galactose, Lén men khdng déu saccarose va khOng lén men dextrin, glycogen. - Cée phan tng sinh héa: Indol duong tinh, Methyl Red (phan tng MR) difong tinh, Voges-Proskauer (phan tng VP) 4m tinh va Citrat am tinh, HS Am tinh, , Lysine decarboxylase dung tinh, E. coli 15717 E.coli SVTH: Dé Qué Mi Huong MSSV: 105111016 ‘Trang 44Dé én tt aghigp Khéa 05- Khoa MT & CNSH GVHD: TS. Nguyen Hodi Huss Hinh 2.10: Gidi thigu vé hinh théi Escherichia coli (441, [45], [46] 2.3.2.2. Dic diém khéng nguyén va doc t Gém 4 loai khang nguyén: O, K, H, F va ngi dge t6 gay tiéu chay, ngoai gc t6 gay tan huyét va phi thing. Déc t6 ciia E.coli: Loai E.coli c6 gidp mo (khéng nguyén K) gay ng6 46 manh hon loai khéng giép mo. Kh4ng nguyén K cé 13 loai KA, KB, KL. Vi du cOng thife khang nguyén cia m6t E.coli 18: OxsK HF NGi dGc 6 dung rudt: Gm 2 loai chiu nhigt va khong chju nbigt. Cd hai loai nay déu gay tiéu chdy. Logi chiu nhigt ST (Thermostable): gdm céc loai STa, STb. Logi khdng chiu nhiét LT (Thermolabiles): gdm céc loai LT1,LT2. Nhing dong E.coli san sinh déc t# (ETEC) gém nhiéu type huyét thanh khdc nhau nhving thing gp nhat 1A céc type OgHis, OsHo, OraHis, Ors 2.3.2.3. M@t sé bénh dién hinh do E.coli gay ra cho gia sic va gia cam [49] Bénh gay cho ga > Nguyén nbn: Do vi khudn E.coli gay ra. > Phuong thie lay truyén Lay qua tring do co thé me bi nhiém bénh, Lay qua dung h6 hap hode da, ni¢m mac. SVTH: Dé Qué Mi Huong MSSV: 105111016 ‘Trang 45Dé én tt aghigp Khéa 05- Khoa MT & CNSH GVHD: TS. Nguyen Hodi Huss Lay qua vé tring do nhiém ban tif phan hodc méi trudng ciia chuéng trai bj nhiém trang. Lay qua thite dn, nuféc u6ng bi nhiém tring. > Trigu chtfng va bénh tich: GA con méi nd: Rn viém, u6t, c6 mau xanh, Bung sting to, long db khdng tiéu. Tiéu chay. GA con ti 1-5 tuan tudi: ga s6t cao, udng nhiéu mutdc, khé thd, bé an, suing mat, viém két mac mat. Viém mang bao tim, viém mang bung, viém mang quanh gan 1m cho bao tim duc, mang bung ¢6 dich viém, quanh gan thy@ng phi mét lép Fibrin mau trang duc. Viém tii khi. Viém phdi Ga dé: gidm tf 1¢ dé, gd dn kém, gay Sm dan, mde sO con c6 du higu viém khép. M6 khém cho thay: Sng dn ting bi viém, léch va gan thydng suing (0 va sung huyét Gan sung to sung huyét Viém phéi inh 2.11: Anh hung do E.coli gay rad ga [49] SVTH: Dé Qué Mi Huong MSSV: 105111016 ‘Trang 46a tt aghigp kha 0S- Khoa MT & GVHD: TS. Nguyéa Hodi Hung Bénh gay cho heo: ‘> Bénh nhiém trang huyét do E.coli ¢ heo con > Nguyén nhdn : thing xay rad cde din khong am 4p, vé sinh kém, thidu hodc ft sta ddu, nude udng khng (6t, sita me kém lam gidm hofe mat nhu déng rugt, c6 thé do thiéu mau, thiéu vitamin (A, PP, BS..). Vi khudn E.coli sé xam nh4p va aban lén trong rugt, vao mau va gay nhiém tring méu, > Trigu ching: Heo bi nhiém bénh trong vong 12h sau khi sanh va c6 thé chét trong vong 48 gid’ véi cdc biéu hin sau: Heo bénh Iwi van dong, ditng riéng ra khdi dan, d rf, dudi rl xuéng hong. Déi khi 6i mifa, run ry va c6 thé chét sau khi hén mé, co giat (ty Ié chét c6 thé 80-90%). > Bénh tich: Viém mang ngoai va van tim, sung huyét than, 14 léch, c6 thé viém da va khép. ‘© Bénh tiéu chy phan tring ¢ heo con: Bénh thing xay ra 6 giai doan heo con sd sinh d&n giai doan cai. > Nguyén nhan: Bénh xy ra 4 cde dan im khOng di &m, vé sinh chuéng va thife an nuée udng kém, thiéu hode ft sifa ddu, sita me kém, thi€u méu, thiéu vitamin, Bénh thuing két hop nhiét 46 qué thép, mvfa lanh, &m wat, stress. > Triéu ching: Heo tiéu chay phan mau vang trang, tréng xdm, vé sau cé mau vang xanh, mdi héi. Heo mat nutéc, gdy stit nhanh, bu kém, di lai khong viing va nén ra sifa dong khong tiéu. Heo con bénh yéu di rt nhanh néu kh6ng didu tri kiép thdi thi heo yéu dan, léng xd va chét (ty 18 chét 06 thé 1én d&n 80- 90%). > Benh tich: Co thé mat nude, 6m, phan dinh bét vio hau mn, Mach méu rugt va hach rudt sung huyét cap tinh. ft thy viém da day rudt xudt huyét, da day chifa sifa khong tie SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 47Dé dn tt aghigp kha 05- Khoa MT & CNSHL GVHD: TS. Nguyéa Hodi Hung Heo con bi tiéu chdy phan wing Heo con yéu dan réi chét Hinh 2.12: Anh hvdng cila bénh tiéu chy phan tring lén heo con [49] ‘© Bénh pha thiing trén heo cai sifa: Bénh thudng xdy ra trén heo cai sifa hode sau cai sifa 1-3 tudn tuéi. Bénh thing xy ra trén nhifng con 1én nhat dan sau ly qua nhiing con khée. > Neuyén nhan: Do chuéng trai vé sinh khéng tét, 4m t E.coli c6 sn trong od thé két hdp véi stress khi téch me thi sé nhan ln nhanh trong ruét. Thay déi thite An dét ngét, heo con khéng cdn dutge bi do dé sé an qua nhiéu thife an din dén khong tiéu héa hét thite an. > Su sinh bénh: vi khudn E.coli gay bénh phat trién trong niém mac rugt lam san sinh dge t phé hily mao mach d&n dén phi thing khép co thé. > Trigu ching ~ Bénh thudng xy ra d6t ng6t d giai doan vai ngay dén mét tudn sau cai sta va trén heo lén tri eda bay. - Lic méi nhiém bénh heo e6 dau higu kém An, kém linh hoat. - Thé qué cp heo chét d6t ngét vA tntéc khi chét cé triéu chitng phd - 6 thé ep tinh, bénh didn bién 2-3 ngay. Ngdy dau heo bé dn, sang ngay thit 2 hode ngay thif 3 c6 triéu ching pha. SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 48Dé én tt aghigp Khéa 05- Khoa MT & CNSH GVHD: TS. Nguyen Hodi Huss - Triéu ching pha thing xuat hién chi yéu 4 ving dau nhu: mi mat, ving hau, g6c tai, d6i khi suing cd mat. ~ Phi. nao, nao bi chén ép bdi dich thodt ra tit mach mau nén gay nhiin nado dan dén triéu chifng than kinh nhu: co giat kiéu boi chéo, di xiéu veo, hay dim dau vao tung, di lai khéng dinh huéng. ~ Do thiiy thing é thanh quan nén hay kéu khan gidng ti€ng chim. ~ Nhiét d6 khong tang, sung huyét ¢ niém mac va xanh tim @ tai, mom, chép dudi..Heo r&t khé thd trudc khi chét > Ben tich: ~ Ving mé lién két dui da bi thiy thing. - Hach ving ben, hach rudt bi théy thing, xoang bung chita dich pha, phd thiing 4 mang trong rust. ~ Thiy thing mi mat, 16 tai, é quanh tim, thanh quan. Pha thing 6 két trang va chita Xuat huyét ruét non, phd mang treo nhiéu dich thiy thing rut va sting hach mang treo rudt Hinh 2.13: Anh hufdng eta bénh phd thing trén heo cai sita [49] SVTH: Dé Qué Mi Huong MSSV: 105111016 ‘Trang 49a tt aghigp kha 0S- Khoa MT & GVHD: TS. Nguyéa Hodi Hung Chusong Vat Liéu & Phuong Phap Nghién Citu 3.1. Vat liéu 3.1.1. Dia diém thyc hién dé an Dé An duge thyc hién tai hé thong phdng thi nghiém Khoa M6i Tntng va Cong Nghé Sinh Hoc. Tring Dai Hoc Ky Thudt Cong Nghé Thanh Pho Hé Chi Minh. Hé théng phdng gém cé: Phong 15: phdng thi nghiém héa Phong 14: phng thi nghiém vi sinh Phong 9 : phdng thi nghiém thye vat 3.1.2. Gidng vi sinh vat Vi khudn lén men lactic do sinh vién Nguyén Thi Bich Thy cung e@p ti két qua cia dé An tét nghiép khda 05 - lép OSDSH - Khoa M@i Trung va Cong Nghé Sinh Hoc ~ Trung DH Ky Thugt Cong Nghé TPHCM: “Phan Igp cdc vi Khugn lactic 6 nguén gée thye phém va duge phdm mang hoat tinh probiotic” Vi khudn E.coli MIS duge cung ed tit Dai Hoc Y duge TP HCM. Bang 3.1: Céc ching dugc kiém tra hoat tinh probiotic San phim Tén san phim Ki hiéu c4c ching phan lap Sita chua Sita dé chua ty nhién 1 Sla, Sib Sita dé chua tf nhién 2 82 Sita dé chua ty nhién 3 $3 Sita dé chua wy nhién 4 s4 Kefir lén men ti PTN 1 85 Kefir Ién men tit PIN 2 86 SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang $0Dé én tt aghigp Khéa 05- Khoa MT & CNSH GVHD: TS. Nguyen Hodi Huss Nem Nem I NI Nem 3 NB Nem 4 Na Nem 5 NS Dita mudi Diva mudi 2 D2 Dua mudi 3 D3 Ca mudi chua | Ca mudi cl Ché phaém Biolactyl Tla,T1b, Tle duge Biosubtyl DL T2e L-Bio-M T3e Lactomin plus Tac Probio TI Ybio T8b = Thanh phan vi sinh trong 1 géi ché phém gém: + L-Bio: Lactobacillus acidophilus + Biolactyl: Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus bungaricus, Streptococcus lactic. + L-Bio_M: Lactobacillus acidophilus, Bifidobacterium longum, Streptococcus faecalis. + Lactomin plus: Lactobacillus acidophilus, Bifidobacterium longum, Streptococcus faecalis, + Antibio Granules: Lactobacillus acidophilus: + Biosubtyl DL: Lactobacillus acidophilus, Bacillus subtillis. + Probio: Lactobacillus acidophilus. + Ybio: Lactobacillus acidophilus. SVTH: Dé Qué Mi Huong MSSV: 105111016 ‘Trang 51a tt aghigp kha 0S- Khoa MT & GVHD: TS. Nguyéa Hodi Hung 3.1.3. Héa chat 3.1.3.1. Moi truding M6i tring Pepton Water: tng sinh khéi vi khudn E.coli MGi trvdng TSA (Tripton soya agar): gitt ging vi khudn E.coli M6i tvdng BHI (Brain Heart Infusion ): mdi trudng phat ign cila E.coli sit dung trong thi nghiém thit hoat tinh khang khudn cla LAB BHI Broth 1000m1 Beef heart infusion v.00 250.02 Calf brain in fusion .........0. soesswssanees oo 200.08 Proteose peptone 10.0 5.08 NaCl. NajHPO,.12H,0 258 GMCO8E woes 2.08 pH 7.4 + 0.2, 25°C BHI thach, bé sung 2% agar M6i truding MRS (DeMan, Rogosa, Sharpe) Broth: méi tring ting sinh LAB MRS Broth 1000ml Glucose. Peptone. Beef extract... Yeast extract... CHsCOONA.... K,HPO,.3HL0...... Mg.$04.7H,0 MnSO,.4H,0 .. se 0.037863 SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang $2a tt aghigp kha 0S- Khoa MT & GVHD: TS. Nguyéa Hodi Hung Triamonium citrate 4.08 Tween 80. Iml Méi trudng MRS thach: m6i truding trong thf nghiém thit hoat tinh khéng khudn ctia LAB: MRS léng bé sung 2% agar. 3.1.3.2. Héa chat Cén 70°, 96° Cée héa chat pha méi tring dinh dvang 3.1.4. Dung cu va thiét bi 3.1.4.1. Dungcu Ong nghiém cé ndp Ong nghiém khéng n&p Dia petri Ce 50ml, 100ml, 250ml, 1000m! Erlen 250ml, 500m! Pitpet Iml, 2ml, Sml, 10ml Pipet man 101-100 yl, 20 pl-200 pl, 100 p-1000 jul Dau tip 100 jl, 1000 pl Ong ly tam eppendorf Ong duc 5 Giy tham Que c&y, que gép, que tim béng, que trang Thuée do em, mm Bila thily tinh Gig dé Sng nghiém, 16 nhuta Bong thm nude Béng khéng thém nude SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang $3,a tt aghi@p kha 0S- Khoa MT & CNSH. GVHD: TS. Nguyéa Hodi Hung Bao nilon hp, day thun, gidy g6i 3.1.4.2. Thiét bi ‘Ta cay vi sinh (Brlad France) Ta i (Memmert Germany) Ti sty (Memmert Germany) Ti lanh Toshiba Autolave (Huxky Dai Loan) May do quang (Hach) May ly tam (Tuttligen Germany) MAy do pH (Hach-Germany) Cén phan tich (Orbital Germany) Bép ti (Billy — England) May nuéc ct (Branstead USA) 3.2. Phuong phap nghién cfu 3.2.1. Chun bi giéng vi sinh vat > Chudn bi gidng vi khudn Ién men lactic: © Gidng vi khudn lén men lactic duge cung ep ti Sng nghiém thach nghiém MRS agar, duc bao quan trong td lanh 4°C. © Chuan bi moi trdng: Pha méi trdng MRS Iéng Phéi vao méi Sng nghiém 10m] MRS léng Day nip hodc lm nuit bong, cho vao bich hap Dem hap khif tring bling Autoclave 120°C, 15 phuit, 1 atm © Nudi edly LAB: Moi thao t4e duige thufc hiGn sau khi da s4t tring dung cu, ti edly, vao théo te dudi ngon ta dén én. SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang $4a tt aghigp kha 0S- Khoa MT & GVHD: TS. Nguyéa Hodi Hung Lay sinh khdi, cy chuyén vao ng nghiém chita MRS Iéng. Quin parafin quanh migng Sng nghiém dé nudi edy ki Khi. Dem 6 37°C tit 16-24h. > Chudn bi gidng E.coli: © GiGng LAB dugc cung cdp tir Sng nghiém thach nghiém MRS agar, dude bdo quan trong ti lanh 4°C. © Chudn bi méi trvgng Pha méi trvdng Peptone Water Phéi vao mdi Sng nghiém 10m] Peptone Water. Day nip hodc 1am nuit bong, cho vao bich hap Dem hap khit tring bling Autoclave 120°C, 15 phuit, 1 atm © Nudi ey E.coli ting sinh kh6i: Moi thao téc dufge thuc hign sau khi da s4t tring dung cu, ti edy, vao théo téc duéi ngon lita dén cén. Lay sinh khéi, cy chuyén vao éng nghiém chifa Peptone Water. Bem ti hiéu khi 24h, 37°C Sit dung E.coli sau khi nudi cy qua dém pha loang 107 ( tong dudng 10° tb/ml) dé kiém tra hoat tinh khdng khudn ciia céc vi khuan lactic, 3.2.2. Chudn bi méi trudng test > Méi trvdng BHI agar (2%), MOi truting MRS agar (2%): Pha méi trudng thém agar 2% trén mdi lit moi tring. Dun sOi m6i tring cho agar tan déu, cho yao binh Erlen. Lam ntit b6ng, bit lai bang nilon hodc gid'y bac. Dia petri, rita sach, sy kh6, g6i gidy, bd ich. Dem hap khit tring bling Autoclave, 120°C, 15 phiit, I atm. Dé dia khi méi trtdng 6 60-70°C, mdi dia dé khodng 15m SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang $5a tt aghi@p kha 0S- Khoa MT & CNSH. GVHD: TS. Nguyéa Hodi Hung Dé agar déng, bd vao ti d qua dém dé kiém tra va loai bé dia bi nhiém. Bao quan trong td lanh 4°C. Truc khi sit dung bé vao sy 60°C, 15p > Méi tring BHI léng (0.7%) Pha méi truding thém agar 0.7 % trén mGi lit moi trudng. Dun sdi méi trvdng cho agar tan déu, Phdi vao Tml/ éng nghiém. Lam nit bong hogec day n4p ng nghiém. Dem hap khit tring bling Autoclave, 120°C, 15 phiit, I atm. Dé ngudi, bao quan 6 ti lanh 4°C. > MOi trvdng peptone water Pha méi trdng Phdi &ml/Sng nghiém Lam nit bong hogec day n4p ng nghiém. Dem hap khit tring bling Autoclave, 120°C, 15 phiit, I atm. Dé ngudi, bdo quan 6 ti lanh 4°C. 3.2.3. BS tri thi nghiém 3.2.3.1. Thi nghiém kiém tra kha ning déi khang bing phwong phap Spot on lawn Nguyén tdc: dya vao sy di khdng tru tiép gitta vi khudn lén men lactic va vi khudn chi thi. Tao diéu kién méi tring thich hop cho ca hai vi khudn phat trién. Chuan bi: Dich nudi cay E.coli qua 21h. Pha loang 10° dude néng 46 sit dung 1a 10° té bao/ml Dich nudi cy LAB trong MRS léng 18- 24h, 4 37°C Dia méi trvdng MRS agar, sy khé 4 60°C. SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang $6a tt aghi@p kha 0S- Khoa MT & CNSH. GVHD: TS. Nguyéa Hodi Hung Méi tntting BHI (0.7% agar) gid 6 nhiét do 50°C ‘Thuée do vong khang (em, mm) Céch thy hién nh sau: Dé dia mt MRS (2% agar) E.coli nuéi cay tang sinh qua 21h Nhé 101] LAB (nudi cay ki khi 24h, | Huit Im! dich nudi cay vao 7m! BHI 37°C) lén mat thach @ (1) (0.7% agar). (2) Dé hén hgp VK chi thi + BHI agar 2) lén bé mat thach MRS U hidu khi 24-48h, 37°C Kiém tra sy tao vong khang, Bo dung kinh vong khéng D6i chitng: thay vi giot dich chifa LAB, giot MRS léng. Lam déi ching khéng cé su d6i khang. 3.2.3.2. Thi nghiém kiém tra kha nding d0i khang bing phwong phap Agar spot test (khuéch tan trén bé mat thach) Nevyén tide: Dya trén sy d6i khang trye tiép cia vi khudn 1én men lactic véi vi khudn chi thi ngay tai vi tri nhé vi khudn Jén men lactic. Chun bi: Dich nuéi cfy E.coli qua 27h. Pha lodng 10° dutéc néng dé sit dung 1a 10° té bao/ml Dich nuéi cay LAB trong MRS Idng 18- 24h, 6 37°C Dia méi trvdng BHI agar, sy khé 4 60°C. Cfch thye hign: SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang $7a tt aghi@p kha 0S- Khoa MT & CNSH. GVHD: TS. Nguyéa Hodi Hung ‘Tri 0.1 ml dich chia E.coli méi rudng BHI agar (2%) Nhé 10u1! dich nudi cy LAB lén bé mat dia U hiéu khi, 37°C, 18-24h Kiém tra si tgo vong khéng. Do dutdng kinh vong khéng Gi chiing: nhé giot méi truding MRS lng lén dia, dénh dau vi tri, So sanh vdi céc vi tri giot chifa LAB. 3.2.3.3. Thi nghiém xéc dinh kha nang d6i khéng bing phuong phap disc diffusion assay (khuéch tin qua ving gidy loc) Chudn bi: Gigy loc duting kinh 8mm ‘Méi trvng Peptone water 8ml mdi Sng nghiém Dich nuGi cy E.coli (21h, 37°C) . Pha 1 2 10” dude néng dé sit dung 1a 10° té bao/ml Dich nui cay LAB trong MRS Iéng 18- 24h, 6 37°C Céch thy hign: Dé dia mt BHI agar (2%) Ly tém dich nuéi cy LAB Trdi 0.1 ml dich chtta E.coli Cho 2011 dich ly tam Dat gidy thm U higu khi 24h, 37°C Kiém tra sy tao vong khéng. Do dung kinh vong khéng SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang $8a tt aghigp kha 0S- Khoa MT & GVHD: TS. Nguyéa Hodi Hung LAB sau khi nuéi cfy trong MRS léng qua dém 18-24h, lay 1,2ml dich nudi edly cho vao Sng effendorf, dem ly tam 14000prm trong 10 phiit, Dat nim miéng gidy thm trén bé mat agar. Di chitng: mi€ng 8 trung tam chi thm vao MRS broth Gidly thdm phai dugc khit trng tntéc khi sit dung. Dang kep khif tring qua ngon lita dén cén dé gdp gidy thdm. 3.2.3.4. Thi nghiém kiém tra kha nang déi khang bang phuong phap Agar well diffusion assay (khuéch tan qua giéng thach) Chuan bi: Dich nudi cy E.coli (21h, 37°C) . Pha loang 107, 10° duge néng d6 sit dung 1A 10°-10° té bao/ml. Khao sat hai néng d6 nay Dich nuéi cy LAB trong MRS Iéng 18- 24h, 6 37°C. Dem ly tam 14000 vong/ phiit, trong 10 phiit. Loai bd sinh khéi. Ong déng duc 18 dung kinh 8mm. Khao sat dé diy méi trang 1a Smm ting ting vdi 25m] mdi trvdng/dia, 3mm tong ting voi 1Sml mdi mudngldia Céch thyfe hign: ‘Tri 0.1 ml dich chifa E.coli méi trudng BHI agar (2%) Duc Id wén thach dung kinh 8mm. Lay 0.1ml dich nudi cy LAB (nudi edy ki khi 24h, 37°C) vao 16. U higu khi, 37°C, 18-24h Kiém tra sy tao vong khang. Do dung kinh vong khéng SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang $9a tt aghi@p kha 0S- Khoa MT & CNSH. GVHD: TS. Nguyéa Hodi Hung Qué tinh duc 16: Ma ndp dia petri, ding Sng ddng dung kinh 8mm, khit trdng trén ngon lita dén cn, dé ngudi, duc 518 trén thach. Lay que gdp, khut trng, dé ngudi, gdp thach ra khdi tao ging thach. Ho khif trong ndp petri, day nap petri Di chitng: giéng 6 trung tam dia, cho 0.1ml MRS broth dé d6i ching. 3.2.3.4 Thi nghiém xéc dinh kha nang déi khang bing phwong phdép Turbidometric assay (do 46 duc) Nguyén téc ca phuong phép do d6 dye: Bling may quang phé do mat 46 t€ bao vi sinh vat c6 trong dich nuGi cy 6 bude s6ng 600nm, ‘Thi nghiém duge tién hanh véi 2 nghiém thife: Nghiém thite 1: xe dinh hoat tinh dya trén si ite ché vi khudn chi thi E.coli bling cdc chat duge sinh ra trong qué trinh trao déi chat ciia cdc ching LAB. Nghiém thite 2: xéc dinh hoat tinh trén sy tic ché vi khudn chi thi E.coli bling cdc chat duge sinh ra trong qué trinh phat trién cia céc chiing LAB nhung a loai bé y€u 16 acid lactic bing c4ch trung hda bing dung dich NaOH IN. Chudn bi: Dich nui cay E.coli (21h, 37°C) . Pha loang 10° duc néng 46. sit dung 1a 10° té bao/ml Dich nudi cy LAB trong MRS Iéng 18- 24h, 5 37°C. Nghiém thife 1: Dem ly tam dich nudi cy LAB 14000 vong/ phtt, trong 10 phuit. Loai bé sinh khéi. Neghiém thite 2: Trung hda dich nudi edy vé pH 6 bing NaOH IN, tin hanh thanh tring 80°C trong 10 phiit. Bem ly tam 14000 vong/ phiit, trong 10 phiit, Loai bé sinh khéi. oD «100 ODde Cong thife tinh nhu sau: %OD=. Véi: OD: 18 gid tri do OD cba Sng c6 chita hgp chat khang khudn, SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 60Dé 4n tt aghigp kha 0S- Khoa MT & CNSH. GVHD: TS. Nguyéa Hodi Hung ODde: 1a gié tri do OD ciia Sng déi chting khéng c6 chita hop chat khang khudn, Céch thy hign: Dich nudi cy E.coli nudi ey tang sinh qua 24h, pha loang 107 Im] dich ly tam LAB Lay Iml Cho vao ml BHI+Iml MRS Broth Cho vao 8ml BHI U hig khi 24h, 37°C U higu khi 24h, 37°C Do OD Do OD So sénh d6 due, két Luan C6 thé l4p theo bang sau: [ Nehiem Thite 1 Nghiém Thite 2 Gng | Imi dich nudi cy E.coli qua 21h, | Iml dich nudi cAy E.coli qua 21h, néng d6 10° té bao/ml. néng d6 10° té bao/ml. + + Iml dich ly tam méi trang nudi | Iml dich ly tm da trung hda acid cdy LAB sau 18-24h, bang NaOH IN. ' ' 8ml méi tnng peptone water. 8ml mdi trung peptone water. SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 61Dé én tt aghigp Khéa 05- Khoa MT & CNSH GVHD: TS. Nguyen Hodi Huss Gng | Iml dich nudi cy E.coli qua 21h, ndng d6 10°té bao/ml. asi f+ chiing | Iml MRS Iéng + 8ml mdi truBng peptone water. SVTH: Dé Qué Mi Huong MSSV: 105111016 ‘Trang 62a tt aghigp kha 0S- Khoa MT & GVHD: TS. Nguyéa Hodi Hung Chusong Két Qua va Bién Luan 4.1. Kiém tra kha nang déi khang bing phuong php Spot on lawn Phuong phép nay c6 dang giGng chiée bénh Sandwich vi 2 mat kep 18 mdi trun MRS agar va BHI agar, kha nang khéng vi sinh vat chi thi dya én sy tip xvc tte tiép gitta ching véi vi khudn lactic, Theo nghién cttu cba Carherine B, Lewus va Thomas J. Montville (1991) vong khdng khudn thé hign rat r rang v6i khé nang lp lai cao va nhanh chéng. Cho dén gan day, Bilge H. Cadirei va Sumru Citak (2005) nhan dinh ring phutong phép nay dung nhwt cho két qué tt nhdt. Tuy nhign, hoat tinh khéng vi sinh vt 6 day 18 hoat tinh tng hgp ciia tat cd cdc sn phdm ctia qué trinh trao déi chét ciia vi khudn lactic nhv: acid lactic, acid acetic, diacetyl, bacteriocin..Vi vay, 4é x4c dinh hoat tinh riéng cia ting chat khdng vi sinh vat cila vi khudn lactic edn e6 cée phuang phap bd sung. Khi thue hign phwtong php nay mOt sé khé khan thudng gap 18 = Bé thue hién kiém tra, ta phai trai mét 1ép méng moi tng MRS, r6i nhé moe lugng nhé vi khudn LAB lén trén bé mat agar. Sau d6, nhé tiép 1 giot méi trdng BHI (0,7% agar) lén trén vi khudn lactic dé cé dinh chting trén bé mat thach, Néu lugng méi tung BHT nhé Ién qué Ién sé rita tr6i vi khudn lactic. - BE tdi mét lp méng méi trudng BHI 0,7 % agar chita vi khudn chi thj £. coli lén trén bé mat MRS agar da nhé vi khudn lactic, ta can héa ling mdi trvding BHI 4 nhiét 49 50°C. G nhiét d6 nay, E.coli dé bi tn thygng va BHI dé hda ln vi MRS agar. Navde lai, n€u ha nhigt 46 thap hon, moi trudng dé Ong, khOng tao dutge Iép méng aéu trén MRS agar nhtt mong muGn, - BHT agar tit Sng nghiém dutge chuyén sang dia petri bing cdch rét tnge tiép trén ngon lita dén cén, nguy co bi nhiém vi khudn hay bao tf tit khong Khi rt cao. SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 63Dé 4n tt aghigp kh6a 0S- Khoa MT & GVHD: TS. Nguyéa Hodi Hung - MRS agar sau khi 46 dia sy khé 15 phiit dé bé mat dia khd, khéng bi dong nvéc tranh bi nhiém do nude. Qua thit nghiém phuong php nay, vi nhiing khé khan gap phdi cing nh diéu kién tai phdng thi nghiém khéng thudn loi dé thc hién, phuong phap nay bi tam ngung dé tien hAnh phuong phép khéc. 4.2. Kiém tra kha nang d6i khang bing phuong phap Agar spot test (khuéch tan trén bé mat thach) Twong Of nhu phiong phép én, day 1a phuong phap kiém tra kha nang khdng vi sinh vat chi thi dya én sy tiép xiéc trve tip gitta chiing véi vi khudn lactic, Tuy nhién, & day méi tung BHI duge trai truée én dia Petri va effy gidng vi sinh vat chi thi, Ngdy hom sau vi khudn lactic nudi tude trong mdi tring MRS mdi duge nhé len mat thach sé to vong khang khudn xung quanh giot vi khudn lactic. Twong nf nhu phudng phép thif nhét, phutong phap nay cong nhim dénh gid khd nang khéng vi sinh vat cba tt cd cde san phim cba qué trinh trao déi cht nhung quy tinh thit nghiém don gidn hon. Tuy nhién van gap kh6 khan Ia - Khilugng vi khudn lactic qué it sé khéng di we ché vi sinh vat chi thi - Lugng vi khudn lactic qué I6n sé tran trén mat thach hda Min véi E.coli nén khong nhén dutge két qué khéng. Nhw vay 46i véi phuong phép 1 va 2 néng 46 vi khudn lactic trong dung dich MRS va ndng 46 vi sinh vat chi thi cin dude t6i vu héa dé dat két qua tt nehi¢m mong mudn, SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 64Dé én tt aghigp Khéa 05- Khoa MT & CNSH GVHD: TS. Nguyen Hodi Huss cl N3 Dich nhé LAB bj tran én bé mat thach Lugng nhé LAB qué it T7b T8b Lutgng nhé LAB qué it Dich nhé LAB bi tran trén bé mat thach Hinh 4.1: Thir nghiém khong thinh céng phuong phap Agar spot test Nav vay, tai phong thi nghiém tng Bai Hoc Ky Thugt Cong Nghe, phwong php nay bi tam ngung aé tign hanh phudng phép khée. SVTH: Dé Qué Mi Huong MSSV: 105111016 ‘Trang 65Dé dn tt aghigp kha 05- Khoa MT & CNSHL GVHD: TS. Nguyéa Hodi Hung 4.3. Kiém tra kha ning déi khang bing phwong ph4p Dise diffusion assay (khuéch tan qua vong gidty loc) Khéc vdi hai phuong phap tén, trong phuong phdp nay vi khudn lactic khdng d6i khéng trgc ti¢p vi sinh vat chi thi ma chi c6 sn phém trao déi chat cia chiing khuéch tén qua méi tru@ng thach Anh hung len tang tring cia vi sinh vat chi thj, Trong ti liu phuong phép nay thing duge tién hanh song song vi mét trong nhitng phuong php trén. Ap dung phuong phap nay ta c6 thé tach roi anh hudng cilia ting yéu te kh4ng khudn. Vi du nhv téc dung khéng khudn cia acid hitu co cé thé loai tit nhé trung héa dich nudi cay sau khi ly tém loai bé té& bao; tac dung cia H,0, dude loai tri nha xi ly dich ly am vi enzyme catalase... Bing céch 46 c6 thé d4nh gid hoat tinh kh4ng khu4n cia bacteriocin do vi khudn lactic téng hap. Khuéch tén qua ving gidy loc (disc diffusion assay) tong ty phviéng phép cia Kirby Bauer thing duge sit dung dé kiém tra tinh m4n ciia vi sinh vat d6i véi khdng sinh [9]. Vong gidy thdim dugc dat len bé mat dia Petri da céy vi sinh ‘vat chi thj réi thm dich nuéi cay vi khudn lactic li tam rdi. Tuy nhién, khi thit nghiém phvong php nay a khéng thanh céng. Két qua thé hién theo nhv hinh. 44, SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 65Dé én tt aghigp Khéa 05- Khoa MT & CNSH GVHD: TS. Nguyen Hodi Huss 17 Hinh 4.2. Thit ngi¢m kh6ng thanh cng phuong phép Disc diffusion assay Tit két qua ny e6 mOt sé nh§n xét nhtt sau: 6 hip thy cde chat khang khudn thu duge tir dich ly tam bi han ché mite 20p1, cho thy Ivgng chat duge ding dé thi hoat tinh 1a qué ft. Céc chat khéng khuén dude thm trong gidy loc c6 su khuéch tan lén mai tntting thach 1a khong cao. Ca hai nhn dinh nay déu gidi thich cho két qua khOng thanh céng SVTH: Dé Qué Mi Huong MSSV: 105111016 ‘Trang 67a tt aghi@p kha 0S- Khoa MT & CNSH. GVHD: TS. Nguyéa Hodi Hung Tuy phwong ph4p nay c6 kha nang tach riéng kiém tra dinh tinh céc chat khang khudn (acid lactic, H5O,, bacteriocin..), nhutng qua tht nghiém nhan thy néng d6 chat khdng khudn duge sinh ra 1a khéng cao, va phuong phdp nay han ché vé thé tich dufa vio gidy loc. Vi vay phutong phap khng mang lai ¥ nghia cao trong viéc tich riéng dinh tinh titng chat. Nhu vay, tai phong thi nghiém tntang Dai Hoc Ky Thuat ong Nghé, phuong phap nay bi tam ngung dé tién hanh thi nghiém phutong ph4p khéc. 4.4. Kiém tra khd nang déi khéng bing phuong phép Agar Well Diffusion Assay (khuéch tan qua giéng thach) Phuong phap Agar well diffusion assay hay cn goi la phvfong phap khuéch tan qua giéng thach, vi sinh vat chi thi duge trai m6t lp méng trén bé mat mdi trv8ng BHI agar, cho dich nudi cy LAB (cé thé Idy dich ly tam) vao giéng, ngay tai giéng chita LAB phat trign va tiét ce cht 46i khang véi vi khudn chi thi, ng&n can sy phat trién ciia vi khudn chi thi xung quanh giéng. Tuong ty nhu phuong phdp trén, khuéch tén qua giéng thach (well diffusion assay) 18 phugng ph4p danh gid Anh hudng céc sin phdm trao Gi chat ciia vi khudn lactic lén vi sinh vat chi thi ma khdng can su tiép xtic trye tiép gitta ching, cing nhu kha ndng d4nh gid riéng tc dOng cha bacteriocin sau khi loai bé téc dOng clia cdc yéu t6 khéc nhv acid hau co, HO)... Phugng phép nay cé uu diém so véi phudng phép trén la giéng thach c6 kha nang chita m6t thé tich dich ly tam Idn hon dang ké so v dich tham vao gidy loc, nén kha nang khuéch tén cae chat khéng khudn vio méi tru@ng thach cng cao hon so véi phudng phép dics diffusion assay. SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 68Dé én tt aghigp Khéa 05- Khoa MT & CNSH GVHD: TS. Nguyen Hodi Huss Dé do dugc vong khéng khudn thyc su bang phuong phdp nay, bé day méi trvng BHI trong dia Petri cdn duge chudn ha. Moi tung qué day ngan edn sy khuéch tén ctia cde chat khdng khudn trong thach. Néng d6 vi khudn chi thi cfing Anh hung lén d6 trong suét cia vong khdng khudn. Diéu nay cho thay su tufong tng giita chat khng khudn véi mat dé té bao vi khudn chi thi. Sau khi thi nghiém phuong phép vdi d6 day khéc nhau (Smm, 3mm), 46 day 3mm tfong ting véi 15 ml méi trvBng thach 1a thich hop. Twong uf ndng do vi khudn chi thi dé nhan r6 vong khang khudn 1a 10° té bao/ml. Hinh 4.3 thé hién 16 két qua thu duge. Méi tring day (5mm) va E.coli 10°t& — MGi tring thich hgp (3mm) va E.coli bao/ml 10° té bao/ml Hinh 4.3. So sdnh két qua Cl & hai dia mdi trudng véi d6 day va ndng 46 vi khudin chi thi khée nhau Vice chudn héa cho phugng php nay cdn can phai thye hién chudn héa néng 49 vi khudn 1én men lactic trong MRS léng. Khi dy chuyén vao mdi trvdng ling Iwgng sinh khdi dy khong dng déu. Theo 46 sau khi nudi cy qua 18-24h, Iugng tf bao ting sinh trong méi tvBng cing khée nhau. Khi sit dung SVTH: Dé Qué Mi Huong MSSV: 105111016 ‘Trang 69Dé én tt aghigp Khéa 05- Khoa MT & CNSH GVHD: TS. Nguyen Hodi Huss cing mét thé tich dich nuéi cay cia cdc chting dé tién hanh thi nghiém néng dé té bao /ml sé khong duge ef dinh, vi vay ma két qué c6 46 tin cay chap hon. Viée chudn héa nay due thie hién dy trén dung dung chudn vé néng dé t& bao vi khuan. Khi ign hanh do vong khdng 4 dia petri, phutong phép do rt thé sd, chi difa trén mat thudng va ding thude mm dé do, Két qua nay c6 khdng dé tin cay cao, chifa c6 49 nhay trong két qud, vi thé cdn phdi nghién citu mot phutong phap khite e6 49 nhay cao hon. Tla d= 17 mm) ‘T7b (d= 17 mm) NB (d= 16 mm) Cl (d=18mm) Minh 4.4: Vong khdng cia nhiing ching dién hinh, SVTH: Dé Qué Mi Huong MSSV: 105111016 ‘Trang 70a tt aghi@p kha 0S- Khoa MT & CNSH. GVHD: TS. Nguyéa Hodi Hung Hai mui hai ching vi khudn lactic dugc kiém tra hoat tinh khang khudn ting quat nh’ phuong phép khuéch tin qua ging thach véi dG day méi trtdng va néng d6 E.coli thich hgp 1a: d6 day 3mm va néng dé 10° té bao/ml. Dudng kinh vong khdng khudn dugc do bao gém ca dudng kinh giéng thach (Hinh 4.2 va Bang 4.1), Ta c6 céng thie tinh bé rng vanh khang khudn: Bé réng vanh khang khudn = [d vong khéng — d giéng thach]/2 (mm) Schillinger ya Lucke (1989) cho ring khi bé réng vanh khang khudn > 1 mm thi khd ning khéng ofa cde vi khuin lactic d0i véi vi sinh vat chi thj coi nit 1a manh; khi gid tri nay nim trong khodng 0,5 — 1 mm tong ting kha nding khang trung binh. Cdn theo nghién cifu cia V, Rosenfeldt Nielsen, C. N. Jacobsen, A. E. Hayford, P. L. Molller, K. F, Michaelsen, A. Perrehaard, B. Sandstrom, M. Tvede, M. Jakobsen (1999) thi bé réng vanh khang khuan duge xét trong khodng 2-Smm cho kha nding khéng (rung binh va trén Smm cho kha ning khdng manh. Gan diy, V. Padmanabha Reddy, M.D. Christopher, 1 Sankara Reddy (2006) do duge bé rong vong khéng khudn 3-Smm cla Lactobacillus acidophilus 46i v6i vi sinh vat chi thi E.coli. Theo bang 4.1, bé rong vong khang khudn do duge ctia ching (i khOng khée biét vdi cée tée gid néu trén, Bé rng vanh khéng khudn phy thuge vao néng 4 chat khéng khudn trong dich ly tim. MGt sé téc gid cdn 6 dic dich tf bao ly tam 5, 10 Hin dé ting néng d@ chat khang khudn nhiim ting bé day vanh khang khudn [36] SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 71a tt aghigp kha 0S- Khoa MT & GVHD: TS, (guyén Hodi Huwng Bang 4.1, Duong kinh vong khang khudn do duéng bang phuong phap khuéch tan qua giéng thach (well diffusion assay) Gid tri trung binh | Gid tri trang | Phan logi dutimg kink vong | bink bé rong | hogt tink SOTT Chiing . . khang khudn | vanh khdng |khang khudn (mm) Khudin (mm) 1 T8b 18.0 5.0 +H 2 cl 18.0 5.0 he 3 Tia 17.0 45 pave 4 TTb 17.0 45 He 5 D3 16.0 4.0 + 6 N3 16.0 4.0 + 7 Tle 15.0 + 8 T3e 15.0 35 + 9 86 15.0 35 + 10 Na 14.0 3.0 a in S2 14.0 3.0 + 12 s4 14.0 3.0 + 13, NI 13.0 25 + 14 T2e 13.0 4 15 NS 10.0 1.0 + 16 Sla 10.0 1.0 + 17 Sib 10.0 1.0 + 18 83 10.0 1.0 + 19 TIb 10.0 1.0 + 20 D2 10.0 1.0 + SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 72GVHD: TS. Nguyéa Hodi Hung 21 T4e 8.0 0 0 22 SS 8.0 0 0 Dé tign theo doi va dénh gié hoat tinh khing khudn cla cde ching vi khudn lactic phan Igp dutge trong phéng thi nghiém, chiing t6i 42 nghj phan logi hoat tinh khdng khudn (tam thdi d6i véi vi khudn chi thi E.coli) nh sau: Bé day vanh khdng khudn >4.0 mm tong ting hoat tinh manh (++4) Bé day vanh khang khudn = 3.0mm + 4.0 mm ngong ting hoat tinh trung binh +) Bé day vanh khdng khudn < 3.0 mm nfong tng hoat tinh y&u (+) Bé day vanh khdng khudn = 0 mm asdng ting khong c6 hoat tinh (0) Nhu vay, trong sf bn phuong phdp khuéch tin trong méi tring thach 1a spot on lawn, agar spot test, dies diffusion assay, va well diffusion assay, thi phutong ph4p cudi cing sau khi chudn héa vé d6 day moi trudng va néng d9 vi sinh vat 6 thé 4p dung dé so sdénh hoat tinh khdng khudn cia cdc ching vi khudn ngudn géc khée nhau qua dung kinh vong khéng. Tuy nhién, dé két qua c6 thé so sinh bang phutong phap thé'ng ké cdn ting sO lén lap lai. Chiing t6i da 4p dung phvong phdp nay dé so s4nh hoat tinh khéng khudn téng thé di véi E.coli cia 22 ching vi khudn lactic. Dé xdc dinh ban chat khang khudn 1a do acid lactic, H,O, hay bacteriocin, cdn tién hanh mét s6 phvong phép bé sung hu trung hda dich t€ bao ly tim, bé sung catalase...Ngoai ra, ede phuong phap khuch tén qua mi tring thach chi cé thé 4p dung dé sing loc ban dau trong quy trinh Gm kiém cdc chiing c6 hoat tinh probiotic. Bé so snh mang tinh dinh Iutgng cén fim kiém nhitng phwong phép o6 d6 tin cy cao hon, SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 73a tt aghigp kha 0S- Khoa MT & GVHD: TS. Nguyéa Hodi Hung 4.5. Thi nghiém xée dinh kha nang di khdng bing phuong phdp Turbidimetric assay (do d6 duc) TE bao vi sinh vt 18 mOt thie thé nén khi hién dién trong méi tntdng 1am cho méi tntng tr nén duc. BS duc cba huyén pho tf 1¢ thudn voi mae dd te bao. Do dé, théng qua viéc do dé duc bing méy quang phé ké 4 buéc séng nhat dinh ctia dich huyén pha té bao ta cé thé dinh tinh duge mat dé té bao 4 diéu kién cin khdo sat. Khi thye hign phyang phép do 46 duc, ching t6i 06 thay adi quy tinh so v6i tAi ligu tham khdo [16], [31]. Thay vi tiét tring dich ly tam bing phutong phép loc, ching (6i khit trang tréc eppendorf tube ding dé ly tim, sau ly tam chi thanh tring Pasteur é 80° C 10 phit 4@ trénh bign tinh bacteriocin va don gidn ha thao tac. Moi thao tac c&y truyén déu duc bdo dam diéu kién v6 tring. Phuong phép nay vin diya én nguyén tic chung cila cée phung phap phat hign cht khéng vi sinh v4t lA do kha nang tte ché ting trang cila vi khudn chi thi ctia cdc cht khdng khudn duge sinh ra ti qué trinh trao déi chat cia vi khudn lén men lactic. Khong giding nhw bén phuong phép da thyc hién 6 trén, phuong phap do 46 duc dya trén syf khuéch tan cdc chat khang khudn trong méi trvwng lng. Ti Ig phan tram OD E, coli ib vi dich ly tam té bao vi khudn LAB so véi OD E. coli i véi d6i chting (thay dich ly tam LAB bang mdi trféng MRS cing thé tich) cho ta 118 sOng s6t cia E. coli sau khi ti vdi cdc ch&t khang khudn trong dich nudi c&y té bao LAB. Nhu vay bién d9 thay adi cla c&e gid tri do duge va tinh duge rong hon so véi phwtong ph4p well diffusion assay (Hinh 4.5 va Bang 4.2). Cang trén co sé phutong phap nay o6 nhiéu wu diém so vi cée phusng phap trude, ngoai v Gc khdo sat hoat tinh khang khudn tng quét, ching (6i bude dau tim hiéu ban chat khang khudn trong dich nudi ey vi khudn LAB ly tam, SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 74Dé én tt aghigp Khéa 05- Khoa MT & CNSH GVHD: TS. Nguyen Hodi Huss Thy hién qué trinh nudi ey vi khudn 1én men lactic trong MRS léng véi didu kiGn ki khi, dich nudi cy dem phan tich ham Itong HO, sinh ra. Ket qua cho thy HO, khéng duge sinh ra trong qué tinh nui cy ki khi, nhu vay hai chat khang khudn déng quan tam cén lai la acid hifu co va bacteriocin. Dé loai bé tée dung cia acid hitu co trong hoat tinh khéng khudn, ching t6i tién hanh trung héa dich nudi cy LAB réi mdi dem ly tam ti€p 46 tién hanh quy trinh do 46 duc nhv trén, Két qua cing dufge trinh bay trong bang 4.2. Ong nghiém bén phai: Dich nudi cy | Ong nghiém bén phai: Dich nui cy Cl ly tm (Khéng trung hda) Ui v6i E, | C1 ly tam (sau khi rung hda) bi vdi E, coli coli Ong nghiém bén téi : d6i chiing (thay dich nudi cy C1 bing méi wong MRS ciing thé tich) Hinh 4.5: Kiém tra hoat tinh bing phuong phép do d6 duc SVTH: Dé Qué Mi Huong MSSV: 105111016 ‘Trang 75Dé én wt nghigp kha 0S- Khoa MT & CNSH GVHD: TS. Nguyba Hodi Huse Bang 4.2. Tile sng sét ciia E. coli sau khi i véi dich nudi cay LAB ly tam Tile song sét | Tile sdng sot Chiing vi | cila E.coli (%) | ctta E.coli (%) SOTT | khudn | khi sd vOi dich ly |khi di vdi dich by lactic |tam khong trung| tém sau khi hoa trung hoa 1 Tb 46 56.1 2 cl 4 52.7 3 Tila 3.2 62.1 4 TTb 45 57.1 3 D3 15.3 80.1 6 NB 42 63.2 7 Tle 48.2, 743 8 T3e 20.0 68.8 9 86 343 63.4 10 Na 419) 67.9 i s2 12.3 16.8 12 S4 34.0 54.3 13 NI 15.8 55.8 4 T2e 63.2 63.5 15 NS 48.1 65.6 16 Sla 45.7 58.0 17 Sib 85.1 76.0 18 83 46.5 58.1 19 Tib 92.6 80.3 SVTH: Dé Qué Mi Huong MSSV: 105111016 ‘Trang 76Dé 4n tt aghigp kha 0S- Khoa MT & CNSH. GVHD: TS. Nguyéa Hodi Hung 20 D2 57.0 57.6 21 T4c 89.5 715 22 SS 53.2 73.5 Tit bang 4.2 ta 06 thé thay ring td sOng s6t eta E. coli c&ng tha thi hoat tinh khang vi sinh vat cng cao, Nhu vy dé so sdnh tre ti¢p kha nang tte cht ting trudng E, coli cla céc sin phém trao déi chat vi khudn LAB, chiing t6i stt dung dai hygng (100% ~ t 16 % sO st eta E. coli) va bidu dién wen dé thi 4.1 Se de ch€ ting trading E. coli fl q i ti i H i H i H @ Ching vikbusa LAB 5c hen ri: King tng hoa CCotbén phi: sau khi rong he Dé thi 4.1: Tilé te ché ting trvdng E. coli ca dich nudi c&y vi khudn lactic ly tém kh6ng trung hda va sau khi trung hoa Bé thi 4.1 cho thay: TiIg We ché ving tring vi sinh vat chi thi ca dich nudi ey LAB ly tam khOng trung hda thudng Idn hon mau wong ting sau khi trung hda (19/22 trudng hgp) Bigu nay hgp ly vi trung hda nhim logi bé téc dung eda acid hitu cd SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 77Dé 4n tt aghigp kha 0S- Khoa MT & CNSH. GVHD: TS. Nguyéa Hodi Hung trong hoat tinh kh4ng khudn. Trung hgp Sib, Tlb va T4e cho két qud nguge Iai, Tuy nhign c&c ching ndy kh6ng gay chi ¥ dic biét vit Id tfe che E. coli cao nhat xdp xi 20%, thudc loai thép nbit so véi cde chiing cdn Iai. - Nhiéu ching cé thé ec ché trén 80% tang trféng cia E. coli khi kh6ng trung hda (T8b, Cl, Tla, N3, S2, N1), nhung sau khi loai bé téc dung cia acid hitu c¢ (go thinh thi kh ning tte ehé cn Iai tn dvdi 40%. Nhiéu ching hoat tinh khdng E, coli lai khong phi do acid hitu cd tao thanh ma chil yéu do yéu té khac, rat nhiéu kha nang 1a do bacteriocin (T2c, N5, Sla, D2) va hoat tinh nay cing xap xi 40%. So sénh véi kt qua phuong phap khuéch tén trong giéng thach (well diffusion assay) nhin chung ta e6 thé phan bigt nhiing ching c6 hogt tinh manh va yéu bling c& hai phitong php, vi du m6t so chiing duige dénh gid 1a ¢6 hoat tinh manh trong well diffusion assay We ché én 90% ting tntng E. coli (mau khong trung hda). Nguge lai nhiéu chiing lam gidm 46 duc E.coli trong tf nhung bé day vanh khang khudn lai nhé (T3c, $2, N1). C6 vé ring nhitng chiing ife ché ting truding vi sinh vat chi thi c&ng manh do téc dng eta acid hitu co thi bé day vanh khing khudn cdng lén. Cé thé 46 1A do kha nding khuéch tén ca bacteriocin trong méi trvdng thach kém hon acid hitu cd. Dé thi 4.2 biéu dién tvong quan gitfa ti lé sng s6t E. coli (A%) theo phudng phép do d6 duc theo bé rng vanh khang khudn (mm) vdi sé ligu xif ly va dé thi ve bing phdn mém Statgraphies. SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 78Dé én tt aghigp Khéa 05- Khoa MT & CNSH GVHD: TS. Nguyen Hodi Huss Plot of Fitied Model A% = 24.1972 + 14.6845°D mm Oomm Dé thi 4.2: Sy wong quan gitta phuong phép do d6 duc (turbidimetric assay method) vai phong phép khuéch tn qua ging thach (well diffusion assay) Phuong trinh biéu dién tong quan la: A(%) = 24.2 + 14.7 D duge thiét lap vdi hé sO tvong quan (correlation coefficient) 12 0.8366 va R2 = 66.3 %. Nint vay, vé mgt théing ké coi nhu khong c6 sy tong quan gitta két qua ca hai phuong phap khuéch tan qua giéng thach do 9 due. Tém lai, phutong phap khuéch tan qua giéng thach chi cho két qua dinh tinh lai tn nhigu thdi gian va cOng phu, cdn phufong phép do dQ duc cho két qua mang tinh dinh lugng hon vi vay c6 thé dé dang sit dung dé danh gid hoat tinh kh4ng vi sinh vat cia cdc thinh phan chat khang khudn. Ching téi dé nghi phat trién phvong phdp nay trong Phong thi nghiém trong céng viéc tuyén chon céc ching LAB lam probiotics hay sin xuat bacteriocin, SVTH: Dé Qué Mi Huong MSSV: 105111016 ‘Trang 79a tt aghigp kha 0S- Khoa MT & GVHD: TS. Nguyéa Hodi Hung Chuong 5: Két Luan & Kién Nghi 5.1. Két Luan é chon lc c&e ching c6 tiém nang probiotics, tuyén chon kha nang khang vi sinh vat déng vai trd quan trong nhat va 18 hang rio tyén chon dau tién can 4p dung. Trong bén phitong ph4p thit nghiém trén méi tring thach 1a spot on lawn, agar spot test, dics diffusion assay va well diffusion assay thi phuong phép thit nt 1a cho két qua trong phng thi nghiém cia chiing ti. Phuong ph4p nay ding dé nh gid hoat tinh tng hop cdc cht Khdng khudn do vi khudn lactic sinh ra trong qué trinh trao déi chit. Dé thyc hién cdc phvong phép nay da ti vu héa diéu kién 1a: Sit dung méi tring thach day 3mm tufong ting vai 1Sml moi trong. Sit dung E.coli néng 46 10° t& bdo/ml ding cho tat cd c&e phutong phép kiém tra sy d6i khang. Trong cdc phutong phép sit dung dé kiém tra hoat tinh khng vi sinh vt chi 6 phitdng phép do 46 duc (turbidometric assay method) cho két qua dinh higng. Ap dung phong phép nay c6 thé danh gid anh hung riéng ré cla céc chat khdng vi sinh vat chi thi, Bang phyfong phép well diffusion assay chting t6i xac dinh ditge nam ching cé vong khéng khudn tét nhat 1a: C1, T8b, Tla, T7b, N3. Trong 46 Cl va N3 c6 nguén g6e tir thyc phiim I cd mudi va nem. Cdn Tb, Ta, T7b e6 ngudn ge tr dude phém, la céc ché phdm Biolactyl, Probio va Ybio. Bing phuong phép do d6 duc ching (6i x4e dinh duge nam ching 06 kha ning (ao chat khang khudn, ife ché sy ting sinh cia vi khudn chi thi E.coli cho két qua tt nhét la : Tla, C1, N3, T7b, TSb, trong tng vi thi nghiém kiém tra vong khang khudn, SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 80Dé dn tt aghigp kha 05- Khoa MT & CNSHL GVHD: TS. Nguyéa Hodi Hung 5.2. Kiénng Kién nghj tai phong thf nghiém Dai Hoc Ky Thuat Cong Nghé nén trién Khai phvong ph4p turbidometric assay trong nghién citu chon loc chiing tiém nang probiotic. Dé thyc hién hoan chinh phvéng phap nay cn phai diéu chinh, t6i vu h6a mét sé digu kién nhy sau: © Vigc chudn hoa nay dude thy hién diya trén dung du@ng chudn v8 néng 46 1 bao vi khudn, kiém soat va 6 dinh néng d6 té bao vi khudn én men lactic sit dung trong phutong phép + Kiém soat va of dinh néng a9 tf bao E.coli sit dung trong physng phap ch ly vA dinh Igng bacteriocin va xit If dich ly tam bang proteinase K dé xée dinh cée hop chat c6 hoat tinh khéng khudn e6 ban chat 1a protein hay khong. * Tigp we tin hanh cdc kiém tra hoat tinh Probiotic cba vi khudn lactic véi ede tiéu chi vé: kha ning bam dinh canh tranh véi vi khudn chi thi, kha ning chiu dude pH hoe dung hoa véi acid, kh ning chiu duge ndng 46 mudi mat cao, kiém tra khd nding déi khdng tnyc tiép véi vi khudn chi thi trong didu kign in vivo. SVTH: Dé Qué Mi Huong MSSV: 105111016 Trang 81

Đề Tài Thử Nghiệm Và So Sánh Các Phương Pháp Đo Hoạt Tính Kháng Vi Sinh Vật Của Vi Khuẩn Lên Men Lactic Để Chọn Chủng Tiềm Năng Probiotic - Luận Văn, Đồ Án, Luan Van, Do An

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Đề Tài Thử Nghiệm Và So Sánh Các Phương Pháp Đo Hoạt Tính Kháng Vi Sinh Vật Của Vi Khuẩn Lên Men Lactic Để Chọn Chủng Tiềm Năng Probiotic - Luận Văn, Đồ Án, Luan Van, Do An

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