Chng 1

M U
Xu hng ca con ngi ngy nay l quay v s dng cc sn phm mang tnh thin nhin, khai thc nhng kinh nghim c truyn kt hp vi k thut hin i v hn ch ti a vic a ho cht vo c th. c bit l cc sn phm thc phm c b sung cc chng vi sinh vt gip con ngi tng cng sc khng, ci thin h tiu ho v gip chng li rt nhiu bnh tt pht sinh t bn trong c th. Tuy nhin cc sn phm thc phm ny khng phi l thuc, m c xp vo nhm thc phm chc nng. Chnh v nhng li ch trn v theo quan im phng bnh hn cha bnh, cc nc pht trin c xu hng s dng cc loi thc phm c li ch cho sc khe nhiu hn l s dng thuc iu tr. Sa l thc phm chnh ca tr em v ngi gi, do thi quen khng s dng sa nn enzyme lactase b mt dn, dn n bnh khng dung np lactose. S thiu ht lactase ph bin rng ri trn ton th gii. Trong c th ngi, lactase hot ng khong nm nm. Bc Chu u v nhng ngi sng pha Ty Bc ca tiu lc a n , lactase c xu hng bo tn ngi ln, l do vic s dng cc sn phm t sa nh pho mt trong sut ma ng khi ngun cung cp lng thc c th b hn ch. Chng ta c th kt lun rng vic thiu ht lactase (c gi l khng dung np lactose hoc alactasia) khng phi l mt bnh, nhng s tn lu ca lactase vo tui trng thnh l s thch ng sinh hc vi vic s dng cc sn phm t sa. Lactase l mt enzyme c tm thy mp ca rut non. N phn hy lactose thnh ng glucose v galactose. Tn s thiu ht lactase trn ton th gii khc nhau. Mc lactase trong rut v cc triu chng khng c s ha hp. Hnh nh in hnh l kh chu ngay sau khi dng sa hoc cc sn phm t sa. iu ny c th thy t vic b au bng, bun nn, cm gic y hi ngn, nhanh, chy nc mt v tiu chy[56].

Khi b tiu chy, cc tc nhn gy bnh hoc c t ca chng gy nn s tn thng nim mc thnh rut, khin men tiu ho ng lactose b mt i, v hu qu l ng lactose khng tiu ho c tch lu trong lng rut, tip tc gy nn tiu chy[56]. Ngoi ra, trinh trang km hp thu lactose do bnh ng tiu ha ti mt s quc gia l kh chnh xc, c th gy km hp thu lactose - hi chng rut ngn, kem hp thu ng galactose bm sinh. Trong thc t, trang thai bt thng hoc sai lch l vic duy tr hot ng lactase trong sut giai oan trng thnh. C 3 nguyn nhn c bn dn n s bt dung np lactose: (A) nguyn pht, (B) th pht, (C) bm sinh[57]. (A) Nguyn pht: y l nguyn nhn thng gp nht ca bt dung np lactose do thiu lactase tng i, xut hin tr em vo nhng tui khc nhau trong nhng nhm chng tc khc nhau. (B) Th pht: Do tn thng rut non sau vim d dy rut do siu vi (Rotavirus), tr b bt dung np lactose thong qua, c th hi phc sau khi vn bnh vim d dy rut c gii quyt hoc tr b tiu chy ko di dn n nim mc rut tn thng, men lactase khng sn sinh nn c th tr khng th hp thu c lactose dn n triu chng bt dung np. Khi y s khin cho tnh trng tiu chy ko di v trm trng hn l tr b suy dinh dng, v khi tr bi suy dinh dng th lng men lactase cng gim, v vy suy dinh dng v tiu chy l vng lun qun kh gii quyt. (C) Bm sinh: Nguyn nhn ny rt him gp, biu hin ngay sau sinh do ri lon nhim sc th, gy ngn cn sn xut men lactase. Trong h tiu ha ca chng ta c mt h vi sinh vt vi s lng rt ln, trong c hn 400 loi vi sinh vt khc nhau (Melissa peterson et al., 2002), chng c xp vo 2 loi: vi sinh vt gy bnh v vi sinh vt c ch. Cc loi vi sinh vt sng chung vi nhau to thnh h sinh thi n nh, cn bng v l hng ro bo v, gip c th chng li cc tc nhn gy bnh ng rut cng nh duy tr mt s hot ng chuyn ha ca c th. Vi sinh vt c ch cng nhiu th c th cng khe mnh.

 tng dung np lactose trong sa, l tng nht l tng sinh tng hp c enzyme ny ngay trong c th ngi. Tuy nhin con ng ny rt kh khn. Enzyme t ngun vi sinh vt vn l nhu cu trc mt. Do la chn ngun enzyme vi sinh l hiu qu hn tuy nhin phi chn loi enzyme c hot ng pH ti u tng t nh sa trnh hin tng ng t sa. Hin nay lactase c nghin cu kh y v sn phm lactase thng mi hin nay ch yu l sn xut t nm men. Cc loi nm men nh Kluyverromyces lactics, Kluyverromyces marxianus, Torulopsis spherical, Torulautilis sp, Saccharomyces fragilis, Candida pseudotropicalis. Ngoi ra lactase cn c sn xut t: vi khun (ch yu l Gram dng, thuc ging Lactobacillus, Lactoccoccus, Bacillus v vi khun Gram m l E.coli ), nm si (Trichoderma sp, Fusarium sp, Asperigillus sp) ti : Gp phn xy dng b su tp vi sinh vt sinh tng hp lactase v ng dng - Phn lp, thu thp vi sinh vt c kh nng sinh tng hp lactase t nhiu ngun. - nh danh cc chng vi khun n cp ging v hng n nh danh cp loi. - Kim tra hot tnh enzyme lactase ca nhng chng vi sinh vt phn lp c. - Bo qun ging vi khun c hot tnh lactase.

Chng 2
TNG QUAN
3

2.1. Enzyme lactase 2.1.1. Lactose Lactose (-D-galactopyranosyl (1 4)--D-glucopyranose) l ng i c ng vt c v, ngoi tr mt vi loi, nng trong khong 2 10%. Hm lng lactose trung bnh sa b khong 4,8%, khong 4,4 5,2% (theo Ganzle, 2008). Trong qu trnh sn xut phomai, phn ln lactose trong sa c trong dch whey. Khc vi trc y, whey khng c xem nh ph phm. Ngy nay, whey c s dng hiu qu hn. Whey c th c sy kh sn xut cc loi bt whey khc nhau hoc phn on bng k thut lc mng sn xut sn phm whey protein v phn i qua mng giu lactose (theo Fox 2009; Lifran, 2009). Lactose l mt loi ng c nhiu trong sa. c th hp thu c th ng lactose cn phi c phn tch thnh ng glucose v galactose trc. Qu trnh ny xy ra phn trn ca rut non bi men lactase. V th, bt dung np ng lactose tht ra l trong c th chng ta thiu men lactase chuyn lactose thnh ra hai loi ng nh trnh by trn. Nu thiu men lactase rut non th ng lactose khng c phn tch v s khng c hp thu vo mu m b tn ng rut, dn n nhng ri lon tiu ha, gi l bt dung np ng lactose. y l nguyn nhn lm cho ta khng ung c sa. Nhiu ngi nhm ln gia bt dung np ng lactose v d ng sa. Bn cht ca d ng sa l do h min dch ca c th phn ng vi protein c trong sa dn n nhng ri lon. Lactase c m ha bi mt locus gen trn nhim sc th s 2, th hin c quyn trong rut ng vt c v nh, v mc rt thp trong rut kt ca qu trnh pht trin thai nhi. Con ngi c sinh ra vi mc lactase cao. Sau khi cai sa gen sinh tng hp lactase trng thi bt hot v vy lactase trong rut non gim. Vic gim lactase ng ngha vi vic gy ra cc triu chng ph bin nh khng dung np hypolactasia hoc khng dung np lactose. Bt dung np ng lactose khng lin quan n h min dch, ch v c th chng ta thiu lactase tiu ha lactose v th lactose s b tng ra ngoi vi dng tiu chy. ngi, vn kh khn trong tiu ha lactose tng theo la tui, tui thanh thiu nin chim khong 70% s

thanh thiu nin ca th gii (Paige 2005). Enzym thng mi c xu hng pht trin mnh, galactosidase cho php sn xut sn phm cha hm lng lactose thp (Gekas v Lopez-Leiva 1985; Pivarnik, 1995; Nakayama v Amach 1999; Rehman 2009). Khi , lactose t ha tan v t ngt, s thy phn lactose lm gim cc tinh th lactose sn phm giu lactose trong qu trnh bo qun v lm tng ngt (Gekas v Lopez-Leiva 1985; Pivarnik, 1995; Nakayama v Amachi 1999; Rehman 2009). 2.1.2. Lactase v chc nng Enzyme thy phn lactose c tn lactase thuc nhm -galactosidase (E.C.3.2.1.23) vit tt l LCT. Enzyme ny c hot tnh ch yu l thy phn lin kt 1-4 glucosidic ca phn t ng i lactose to thnh 2 phn t ng n c ngt cao hn ng lactose. Do vy, enzyme lactase ch yu c s dng trong cng nghip cc sn phm t sa gim hm lng lactose, mt thnh phn cacbonhydrat chnh trong sa m phn ln ngi trng thnh c kh nng dung np rt km v c bit mt s t ngi ln k c tr s sinh c d ng mnh vi ng lactose. Hn na, phn t lactose d dng b kt tinh dng ht ct nhit lnh nn gy kh khn cho sn xut mt s sn phm sa lnh nh kem, v vy ngi ta s dng enzyme ny gim hm lng lactose ng thi tng ngt ca sn phm nh cc ng n to thnh. Enzyme lactase trong mt s trng hp cn c hot tnh transgalactosyl ha, trc tin enzyme ph v lin kt 1-4 glucosidic to thnh galactosyl v sau chuyn cho phn t mt hp cht cha nhm hydroxyl. Do vy ngi ta s dng hot tnh ny tng hp galactooligosaccharide, c hot tnh dc hc. Lactase c th c tm thy nhu ng rut non ngi v ng vt, trong mt s thc vt (da hu, tri m) v vi sinh vt (vi khun, nm men, nm si). Ty theo ngun gc lactase s c kch thc phn t khc nhau, cc chui peptide (tiu phn theo cu trc bc 4) di ngn v s lng cng rt khc nhau. Mc d vy nhng chui peptide chnh mang hot tnh lactase thng thng c trng lng phn t khong 120kDa v cha tm xc tc v tm ny c cu trc tng

t gia cc loi (c bo tn qua tin ha) v trong l vai tr khng th thay th ca cc tay Glu trong phn ng xc tc axit base hoc tn cng vo nhn. .

Hnh 1. Enzyme lactase rut non ngi[52] Hot tnh phn hy lactose c xc nh bng 2 cch, hoc l thng qua s mt i ca c cht l lactose hoc l thng qua s to thnh ca sn phm c trng cho ph v lin kt

glycosidic, thng thng dng c cht c hiu ONPG (o-nitrophenyl--D-glucoside) v xc nh lng ONP (o-nitrophenyl) to thnh. Cc lactase khc nhau c di hot ng pH t axit yu n trung tnh t khong 4.0 6.5 ty thuc vo ngun gc. Lactase ca nm si c pH hot ng axit hn, cn lactase ca cc vi khun v nm men a pH gn trung tnh hn. Bng 1. Mt s c trng ca chui peptide ng vai tr xc tc trong enzyme lactase mt s loi vi sinh in hnh Ngun gc enzyme Trng lng phn t Chiu di Nguyn t proton Nucleophile/baz K.lactic 117618 1025 Glu482 Glu551 E.coli 116351 1023 Glu461 Glu537 E.coli 118016 1031 Glu449 Glu512 A.niger 119160 1006 Glu200 Glu298

Phn ln lactase hot ng trong khong nhit 35-45 0C (a nhit trung bnh), mt s khc t 45-650 C (a nhit), dng c bit c lactase hot ng xc tc nhit lnh 4 0C. Di y l tng hp mt s c tnh ca lactase t cc ngun vi sinh vt khc nhau (Bng 2). c bit mt s enzyme i hi ion kim loi hot ha. Ngi v ng vt c kh nng dung np lactose l do trong thi k tr mi sinh, rut non c kh nng tng hp lactase tiu ha lactose trong sa m, thnh phn chnh cho tr sinh trng. n tui trng thnh cc gen ny hu nh ngng hot ng do vy c th tiu ha lactose rt km. iu ny cng ng cho ng vt. Nghin cu ch ra rng s dung np latose c th c h tr bi h vi sinh nim mc rut non, trong c vai tr ca cc vi sinh vt ln men lactose nh Lactococcus v Bifidobacterium. y chnh l c s ca vic a cc vi sinh vt ny vo mt s dng sn phm sa chua ung c men sng h tr tiu ha.

Bng 2. Mt s c trng ca enzyme lactase mt s loi vi sinh in hnh Gi tr pH ti u 3,0-4,0 5,0 6,9-7,3 7,2 6,2-7,1 3,4-4,3 6,0 6,8 6,5-7,5 Gi tr nhit ti u 55-60 50-55 37 35 40 55-57 30-55 60-65 65 42-45 55 500-600 Trng lng phn t 124 90 201 135 540 530 Hot ng cao sa khng bo Mn+2 , K+ Mn+2 , Na+ K+ , Na+ Hot ha ion. Ch Ch thch khc

Ngun

A.niger A.oryzae K.fragilus K.lactic E.coli L.thermophilus C.inaegualis B.circulans Bacillus sp L.bulgaricus S.thermophilus

2.1.3. C ch, cu trc v qu trnh sinh tng hp Lactase c th thy phn mt lot cc cht nn. Trong ng ch nht l lp enzyme galactosidase, lactase cng c glucosidase v glycosylceramidase hoat ng. Trong qu trnh trao i cht, -glycosidic trong D-lactose thy phn to thnh D-galactose, D-glucose, c th c hp thu qua thnh rut vo mu. Phn ng xc tc lactase tng th l : C 12 H 22 O 11 + H 2 O C 6 H 12 O 6 + C 6H 12 O 6 + Q

Lactase cng xc tc chuyn i phlorizin phloretin v glucose. C ch xc tc thu phn lactose D gi li cu hnh cht nn anomeric trong cc sn phm. Trong khi cc chi tit ca c ch l khng chc chn, t c phi thng qua mt phn ng chuyn i. Cc nghin cu v lactase ca E.coli xut rng: qu trnh thy phn c bt u khi mt tc nhn nucleophin glutamate tn cng enzyme t pha bn trc carbon galactosyl ca -glycosidic. V Mg ph thuc vo axit xc tc nn vic loi b cc nhm ri D-glucose c th c thun li, enzyme c gii phng t phn na -galactosyl khi tn cng nucleophin s sn xut D-galactose.

Hnh 2. C ch thy phn lactose [53]

Cc nghin cu v cht nn chng minh rng 3-OH v 2-OH trn vng galactopyranose l rt cn thit thy phn enzyme. Nhm 3'- hydroxy c tham gia ngay t ban u trong khi nhm 2 l khng cn thit, nhng cn thit trong cc bc tip theo. iu ny c chng minh bi thc t rng 2-deoxy l mt cht c ch cnh tranh hiu qu (K i= 10mM). Loi b cc nhm hydroxyl trn phn na glucopyranose khng hon ton loi b cht xc tc. Pre-pro-lactase c mt cu trc polypeptide chnh duy nht bao gm 1927 axit amin chia thnh 5 nhnh: (1) 19 amino axit chia tn hiu theo th t, (2) chui min ln khng hin din trong lactase giai on trng thnh, (3) cc phn khc lactase giai on trng thnh, (4) mt

mng tri di k nc, (5) mt on ngn a nc cacboxyl cui. Chui tn hiu c phn tch trong li ni cht, pro-LHP t chiu di 215kDa c chuyn sang b my Golgi, ni c nhiu glycoxyl ha v protein thy phn. Cc prodomain c hin th hot ng nh mt chaperone intramolecular trong ER, ngn nga s phn tch trypsin v cho php LPH p dng cc cu trc 3-D cn thit vn chuyn n b my Golgi.

Hnh 3. Qu trnh sn sinh lactase ngi [53] 2.1.4. H vi sinh vt sinh tng hp lactase Lactase c sinh tng hp bi mt s loi nm men (Kluyverromyces lactis, Kluyveromyces marxianus, Torulautilis sp, Torulopsis spherica, Sacchromyces fragilis, Candida pseudotropicalis). Ngoi ra, cn c cc vi khun Gram dng, thuc ging Lactobacillus, Lactococcus, Bacillus, nhm vi khun Lactobacillus acidophilus, chng Escherichia coli Nissle 1917. Bn cnh th nm si cng c quan tm nhiu (Trichoderma sp, Aspergillus sp, Fusarium sp) nhng loi Aspergillus sp c nghin cu nhiu hn c. Tuy nhin qu trnh tng hp lactase c quy m nghin cu v quy m sn xut ch yu l t nm men Kluyveromyces v vi khun lactic. Cc enzyme lactase c sinh tng hp bao gm c ngoi bo v ni bo, loi chu nhit v khng chu nhit, c bit loi lactase chu lnh ng dng trong lm kem. S dng lactase (c nh hoc t do) ch yu sn xut sa ti v sa bt khng cha (hoc cha rt t) lactose hoc sinh tng hp cc hp cht galacto-ooligosaccharides.

10

Bng 3. H vi sinh vt sinh tng hp lactase Nm men Kluyverromyces lactic Kluyveromyces maxianu Kluyveromyces sp Torulopsis spherical Torulopsis pseudotropicalis Torulautilis sp Saccharomyces fragilis Candida pseudotropicalis Zygosaccharomyces lactic Nhng t bo nim mc phn trn ca rut non (l nhng t bo sn xut ra men lactase) b tn thng. Hin tng ny hay gp tr em v thng xy ra sau khi b tiu chy, vim d dy rut hoc c th do ha tr liu. Khi tr b tiu chy, cc tc nhn gy bnh hoc c t ca chng gy tn thng nim mc thnh rut, khin men tiu ho ng lactose b mt i, v hu qu l ng lactose khng tiu ho c tch lu trong lng rut, tip tc gy nn tiu chy v bnh ngy cng trm trng hn. Thng k cho thy 50% -70% tr tiu chy nng nhp vin c biu hin bt dung np lactose. Lactose l ng ch yu c trong sa m v cc sa cng thc. Lactose l ngun cung cp ng glucose cho s hot ng ca no v c th, lm phn mm, to s vt tri ca cc Bifidus v Lactobacillus l nhng vi khun c li gip cho s pht trin h min dch v tiu ho trong c th tr. Men lactase mng rut l men tiu ho bin ng lactose tr thnh ng glucose. Khi tr b tiu chy cp, mng rut b tn thng bi vi khun,virt hoc c t ca chng lm thiu men lactase. Do thiu men lactasa lm ng lactose khng tiu ho c trong rut gy bt dung np lactose. ng lactose khng c tiu ho s ng li trong rut, Vi khun Lactobacillus acidophilus Lactobacillus sp Lactococcus lactic Bacillus subtilis Bacillus curculans Streptococcus salivarius Streptococcus thermophilus E.coli Nm si Aspergillus sp Aspergillus niger Aspergillus oryzea Aspergillus candidus Fusarium sp Fusarium moniliforme Trichoderma sp Trichoderma reesei Rut-C30

11

ht nc lm tiu chy tng thm v ko di. Trng hp ny gi l thiu lactase th pht khc vi thiu men lactase tin pht thng xut hin ngay sau khi tr sinh v rt him gp (ch 1 trong s 1000 tr). Bt dung np lactose th pht thng ko di trong 1-2 tun nht l tr tiu chy ko di. Hu qu l tr b suy dinh dng nng, km n cng thm tp qun king n lm chm hi phc nim mc rut. Bt dung np lactose c th gp trong nhng trng hp tiu chy do nhim vi khun, virt, c bit tiu chy do Rota virt, d ng sa b l nhng bnh l rt thng xy ra tr nh di 3 tui[56]. 2.2. Tng quan v vi khun lactic (LAB) 2.2.1. Khi nim Vi khun lactic (LAB) l mt nhm cc vi khun Gram dng c s thng nht v hnh thi, c im sinh l, sinh ha v s trao i cht. Chng hn nh chng l trc khun ngn hay que (rod) v cu khun (cocci) khng h hp, khng sinh bo t. Chng thng c tm thy trong cc cht b phn hy v sn phm cha lactic, axit lactic c to ra nh l sn phm ch yu ca s trao i cht v l kt thc ca qu trnh ln men carbohydrate. V vy LAB c dng trong thc phm ln men, axit ha c ch s tng trng ca tc nhn gy h hng. Mt s chng LAB c th sinh bacteriocin c ch sinh vt gy bnh (Klaenhammer, 1987). Hn na, axit lactic v cc sn phm trao i cht khc gp phn vo tng gi tr cm quan v cu trc ca thc phm. Vi khun ln men lactic c Pasteur tm ra t sa b chua v hin nay chng c cng nhn l an ton sinh hc (generally recognized as safe - GRAS), do c s dng thng xuyn trong thc phm v c ng gp trong h vi sinh vt c ch ca con ngi. Theo bn phn loi c sa i hin nay, vi khun lactic gm khong 20 ging thuc h Lactobacillaceae, chng thng c dng hnh cu (hoc oval) v hnh que, trong cc ging sau y l ch yu nht: Aerococcus, Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leucnostoc, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus v Weissella. Trc y ging Bifidobacterium cng c xp vo nhm vi khun lactic trong kha phn loi ca Bergey 1957, trong chng c xem l Lb. bifidum mc d Bifidobacterium khng ph hp

12

vi cc m t chung ca vi khun lactic m chng lin quan nhiu hn n nhm Actinomycetaceae, mt nhm vi khun Gram dng v c con ng ln men ng khc vi vi khun lactic.

Hnh 4. Cy pht sinh loi ca vi khun lactic [26] Ch : - Khong cch tin ha gn nhau - Trong nhm c ng khung c xem l nhm vi khun an ton Vic phn loi vi khun axit lactic ch khc nhau phn ln l da trn hnh thi hc, ch v con ng ln men khc nhau, tng trng nhit khc nhau, qui trnh sn xut axit lactic, kh nng pht trin nng mui cao, v chu c axit hoc kim. Phn loi theo con ng ha hc nh thnh phn axit bo v cc thnh phn ca thnh t bo, ngoi ra phng php sinh hc di truyn hin i cng c s dng trong phn loi. 2.2.2. c tnh chung Vi khun lactic l nhng vi khun Gram dng, khng sinh bo t, catalase m tnh v l vi khun vi hiu kh (aerotolerant organisms), trao i cht ch yu bng con ng ln men v

13

khng h hp do khng c cytochromes, ch tr ging Bifidobacterium l k kh bt buc. Vi khun lactic c th ln men c cc ng monosaccharid, ng disaccharid, protein tan, pepton v axit. Phn ln chng khng ln men c tinh bt v cc polisaccharid khc. ng knh ca cc dng cu khun lactic t 0,5 - 1,5m. Cc t bo hnh cu xp thnh cp hoc hnh chui c chiu di khc nhau. Kch thc t bo trc khun lactic t 1 - 8m. Trc khun ng ring r hoc kt thnh chui. Cc loi vi khun lactic c kh nng rt khc nhau khi to thnh axit lactic trong mi trng, v sc chu axit (hay bn axit) cng rt khc nhau. a s cc trc khun lactic ng hnh to thnh axit lactic cao hn (khong 23%) lin cu khun (khong 1%). Cc trc khun ny c th pht trin pH 3,84 (cu khun khng th pht trin c mi trng ny), hot lc ln men tt nht ca trc khun vng pH 5,56. Nhit sinh trng ti thch ca vi khun lactic a m l 25350C, a nhit l 40450C v a lnh l thp hn 50C. Khi gia nhit khong 60800C th hu ht chng b cht sau 1030 pht. Trong t nhin, vi khun lactic thng gp trong t, trong nc, trong khng kh, nhng ch yu l thc vt v cc sn phm thc phm ln men (trn cc loi rau, qu, sa, tht,). Mc d c ch vn cha c lm sng t tuy nhin LAB c cho l c mt s hiu ng c li cho chc nng min dch. LAB c th bo v chng li cc mm bnh bng cc phng tin ca s c ch cnh tranh, y mnh s bo hiu cho t bo ch lm gim p ng vim, to p ng min dch lm gim d ng (Kerr v Martha, 2003) tng s lng t bo plasma sn xut IgA khng th chnh trong cht nhy c th, hot ha cc i thc bo cng nh tng t l T lympho (Reid et al., 2003), (Ouwehand et al., 2002), kch thch p ng min dch bm sinh (mc d cha c c ch c th) (O'Toole v Cooney, 2008). C th nh Lacticin 3147 do Lactococcus lactis sinh ra c tc dng dit khun trn nhng t bo nhy cm bi s tng tc u tin vi thnh t bo. l nguyn nhn m trn mng t bo to ra nhng knh cho K + v pht pht v c i ra khi t bo. Trong s n lc ti tch ly li nhng ion ny, nhng h thng hp thu ph thuc ATP dn ti thy phn ATP bn trong. Khi ATP

14

c yu cu cho s duy tr ca nhng chc nng quan trng ca t bo, nh gradient pH ti mng t bo, nhng chc nng t bo b ph v v t bo dn dn mt nng lng v cht. Trong bacteriocin c bn cht l cc peptid khng khun sinh ra bi vi khun chng li vi khun khc. Nh vy, loi vi khun to ra loi bacteriocin no th c kh nng khng li chnh bacteriocin . Ngoi ra khng gy ra phn ng d ng trong con ngi v cc vn v sc khe, b phn hy nhanh bi enzym protease, lipase. Chng c kh nng tiu dit cc vi khun khc do s to thnh cc knh lm thay i tnh thm ca mng t bo, nhiu loi bacteriocin cn c kh nng phn gii ADN, ARN v tn cng vo peptidoglycan lm suy yu thnh t bo. Bacteriocin ca vi khun lactic c th phn loi da trn cu trc c bn nhng n cn da trn kiu hot ng ca chng. Bacteriocin class I (i din: nisin ca Lactococcus lactis) gn vo lp lipid II, ngn s vn chuyn cc tiu n v peptidoglycan t t bo cht n vch t bo, o ngn tng hp vch t bo hoc do bm c vo lp lipid II, cc phn t nisin to l xuyn mng t bo dn n tiu bo; bacteriocin class II (i din sakacin ca Lactobacillus sake) l cc peptide lng tnh c kh nng xuyn mng t bo to knh/l trn mng. Lp III (cn gi l bacteriolysin nh lysostaphin) protein khng bn nhit, tc ng trc tp ln vch t bo ch (Riley v Chavan, 2007), (Cotter et al., 2005). iu chnh thnh phn cu to ca vi khun ng rut. nhng phn khc nhau ca h tiu ha th tn ti cc vi khun khc nhau. Khi tp trung khoang rut, chng to nn s cn bng tm thi ca h sinh thi vi sinh vt ng rut, s thay i ny c nhn thy mt vi ngy sau khi bt u tiu th thc phm c VSV cha lactase, ph thuc vo cng dng v liu lng ca ging vi khun. Gim thiu tiu chy do khng sinh: Nguyn nhn l do khng sinh lm mt cn bng h vi sinh ng rut v khi Clotridium difficle v Klebsiela oxytoca tng ln nhanh chng v gii phng c t gy bnh tiu chy v vim rut. C nhiu nghin cu cha bnh tiu chy do khng sinh, kt qu cho thy chng S. bolardii, S. boulardii, Lactobacillus rhamnosus GG, Enterococcus faecium SF68 c tc dng tt, chng lm gim ng k thi gian phc hi khi mc

15

bnh v s dng L. acidophilus tr bnh tiu chy do s dng thuc khng sinh erythromycin th mang li hiu qu (D'Souza, 2002), (Cremonini, 2002), (Mcfarland, 2006). Tnh hiu qu ca phng chng tiu chy ph thuc vo chng VSV c s dng v liu lng, c th gim n 50% mc ca bnh. Cc loi thc phm b sung LAB, c dng iu tr v phng nga bnh tiu chy cp, v gim mc nghim trng v thi gian nhim trng rotavirus (virus gy tiu chy cp tr em v tiu ngi ln) (Reid et al., 2003), (Ouwehand et al., 2002). Nhng li ch ca chng LAB l ngn nga nhim trng th pht, mt bin chng thng gp khi dng khng sinh. Thuc khng sinh c cho l lm cho h thng min dch vo trng thi "tt" trong khi LAB li gip h tr h min dch v nhiu hn na c th nhanh chng phn ng vi ly nhim mi. 2.2.3. c im hnh thi mt s chng LAB Ty thuc vo hnh dng t bo m ngi ta chia vi khun lactic thnh dng hnh cu v hnh que. Kch thc ca chng thay i ty tng loi.

2.2.3.1. Ging Lactobacillus Phn loi khoa hc: Bng 4. Phn loi khoa hc ging Lactobacillus Gii : Vi khun

Nghnh : Firmicutes Lp B H : Bacilli : Lactobacillales : Lactobacillaceae Lactobacillus

16

Ging :

Chi Lactobacillus hin bao gm hn 125 loi nh : L. acidophilus, L. brevis, L. casei, L. fermentum, L. plantarum, L. bulgaricus

Hnh 5. Lactobacillus sp[51] im ( c : Beijerinck, 1901)

T bo c dng hnh que thng di v thon, khng di ng, Gram dng, him khi to sc t (mu g st v mu gch). glucose, cc aldehydic hexoses tng t, cacrbohydrates l ng n v cc ru a chc c chuyn ha bng con ng ln men lactic ng hnh v c d hnh to ra axit lactic, axit acetic, ru v CO2. Khng c kh nng chuyn ha nitrate, tr loi L. plantarum khi iu kin nht nh. Mt s loi c th pht trin mi trng ngho cht dinh dng, v chu c nhit cao. K kh khng bt buc, khng to ra catalase, khng ha lng gelatin, khng phn hy casein nhng vi chng c th to mt lng nh m ha tan. Khng to indole v H2S. T bo cn non cho kt qu Gram dng v tr thnh Gram m khi gi. Khng c kh nng di ng.

17

c tm thy trong cc sn phm ln men t ng vt v thc vt, c bit l trong cc sn phm sa. Cc loi thuc ging Lactobacillus ln men ng hnh: Lactobacillus acidophilus, L. bulgaricus, L. plantarum, L. casei Cc loi thuc ging Lactobacillus ln men lactic khng ng hnh: Lactobacillus brevis, L. pentoaceticus, L. lycopersici, L. fermenti 2.2.3.2. Ging Streptococcus Phn loi khoa hc: Bng 5. Phn loi khoa hc Ging Streptococcus (Rosenbach, 1884) Gii : Vi khun Nghnh: Firmicutes Lp : Bacilli B : Lactobacillales H : Streptococcaceae Ging : Streptococcus Gm cc loi: S. agalactiae, S. anginosus, S. bovis, S. canis, S. equi, S. iniae, S. mitis, S. mutans, S. oralis, S. parasanguinis, S. peroris, S. pneumoniae, S. pyogenes, S. ratti, S. salivarius, S. salivarius ssp. thermophilus, S. sanguinis

18

Hnh 6. Streptococcus [51] c im: C dng hnh trn hoc hnh trng (ovan), t khi ko di thnh dng que. Sau khi phn chia theo mt phng chng thng xp ring bit, cp i hoc chui ngn, l loi vi khun khng nha bo, Gram dng, khng di ng, v thng khng to sc t, v t bo ca mt s loi c th nhn thy trong iu kin nht nh, ng knh t bo 0.5-1m. Trong mi trng lng, sinh khi c th c dng nhm c dng ht v ni trn mt nc hoc l dng mn gy c mi trng. Trn mi trng agar, khun lc thng nh hn 1mm, trong cng 1 loi c th chuyn t dng nhm sang dng mn v to chy nhy, b mt khun lc li. Sau khi ln men ng, sinh ra rt t hoc khng sinh CO2 v c th to ra mt lng ng k ethanol, axit acetic, axit formickhi ln men glucose trong mi trng c tnh kim (Gunsalus v Niven, 1942). Khng c kh nng chuyn nitrate thnh nitrite, k kh khng bt buc, khng sng c trong mi trng vi 10% mui mt, nhng chng phn hy protein ch c trong nhm enterococcus. Tt c cc chng trong Streptococcus u c nhu cu dinh dng rt phc tp, mt s loi c nhu cu v vitamin B v cc amino axit trong khi s khc c nhu cu v cc axit bo khng no v gia tng lng CO2. V vy nhu cu v dinh dng cng gip ch trong vic xc nh mt loi. (Dubos, 1948). S phn loi cc loi thuc Streptococcus c ngh bi (Sherman, 1937). Theo , chng c chia ra thnh 4 nhm: nhm sinh m (pyogenic), viridans, Enterococcus v nhm lactic. S phn chia ny da trn cc c im sinh l c trng tng ng vi mi nhm, c bit l khong nhit pht trin. Tuy nhin vi cc loi mi c cng nhn lm cho c im mi nhm tr nn t khc bit nhau v chng cng lc mang nhiu c im ca hn 1 nhm. V d: Streptococcus tiberis Diernhofer thuc nhm viridans, nhng n li mang nhng c im m ta

19

c th t n vo nhm Enterococcus. Trong khi Streptococcus acidominimus Ayers v Mudge cng thuc nhm viridans nhng cng c th xem nh thuc nhm pyogenic. Cc loi thuc ging Streptococcus ln men lactic ng hnh: Steptococcus cremoris, Streptococcus lactis, Streptococcus thermophillus, Streptococcus salivarius subsp. thermophilus Cc loi thuc ging Streptococcus ln men lactic khng ng hnh: Streptobacterium brassicae fermentati 2.2.3.3. Ging Leuconostoc Phn loi khoa hc: Bng 6. Phn loi khoa hc ging Leuconostoc (Van Tieghem, 1878) emend. (Hucker v Pederson, 1930). Gii : Vi khun Ngnh : Firmicutes Lp : Bacilli B H : Lactobacillales : Leuconostocaceae

Ging : Leuconostoc Gm cc loi nh: L. carnosum, L. citreum, L. durionis, L. fallax, L. ficulneum, L. fructosum, L. garlicum, L. gasicomitatum, L. gelidum, L. inhae, L. kimchii, L. lactis

20

Hnh 7. Leuconostoc [49] c im: ( Van Tieghem, 1878), (Orla-Jensen, 1919), (Hucker v Pederson, 1930). Leuconostoc c t bo thng c dng hnh cu hay oval n l, cp i hay dng chui ngn. Trong mi trng nht nh nh nc tri cy hoc rau qu c tnh axit, t bo c th ko di thnh dng nhn hay dng que. Mt s loi to cht nhn c trng khi sng trong mi trng cha sucrose. i vi mi trng bnh thng, s tng trng c kch thch nh vic b sung cao nm men, nc chit c chua hoc rau c. Leuconostoc l mt loi ky kh khng bt buc. Khi ln men to ra mt lng hn ch axit lactic. L-lactic axit lun c to ra v i khi cng xut hin D-lactic axit. Axit acetic v ru cng c to thnh v khong lng glucose ln men c chuyn thnh CO2. Khng lm ng t sa, chuyn ha fructose thnh mannitol, c tm thy trong ln men sa v rau qu, v d nh : Leuconostoc mesenteraides (Cienkowski) Van Tieghem. 2.2.3.4. Ging Pediococus Phn loi khoa hc: Bng 7. Phn loi khoa hc ging Pediococcus (Claussen, 1903) Gii: Ngnh: Lp: B: H: Ging: Vi khun Firmicutes Bacilli Lactobacillales Lactobacillaceae Pediococcus

Gm cc loi nh: P. acidilactici, P. cellicola, P. claussenii, P. damnosus, P. ethanolidurans, P. inopinatus, P. parvulus, P. pentosaceus, P. stilesii. c im: ( Balcke, 1884)

21

Pediococcus l nhng t cu khun hoc song cu khun, c khi n l hoc thm ch chui ngn, Gram dng, khng di ng, catalase m. Khong nhit ti u l 25-32oC, chng l vi khun k kh khng bt buc, c th pht trin trn mi trng rn vi s c mt ca khng kh. Qu trnh trao i cht ch yu l qu trnh ln men ng hnh, khng c kh nng chuyn ha nitrate thnh nitrite, kh nng phn hy protein km v vy chng c nhu cu rt cao v thnh phn dinh dng t mi trng. Loi in hnh l Pediococcus cerevisiae Balcke.

Hnh 8. Pediococcus sp [50] 2.2.4. c im sinh l sinh ha 2.2.4.1. Nhu cu dinh dng ca vi khun lactic Cc loi vi khun lactic khc nhau th c nhu cu dinh dng khc nhau. Chng khng ch c nhu cu v cc ngun c cht cha cc nguyn t c bn nh cacbon, nit, photphat v lu hunh m cn c nhu cu v mt s cht cn thit khc nh vitamin, mui v c a. Nhu cu dinh dng cacbon Vi khun lactic c th s dng nhiu loi hydrat cacbon t cc monosaccarit (glucoza, fructoza, manoza), cc disaccarit (saccaroza, lactoza, maltoza) cho n cc polysaccarit (tinh bt, dextrin).

22

Chng s dng ngun cacbon ny cung cp nng lng, xy dng cu trc t bo v lm c cht cho qu trnh ln men tng hp cc axit hu c. b. Nhu cu dinh dng nit Phn ln vi khun lactic khng t tng hp c cc hp cht cha nit. V vy m bo cho s sinh trng v pht trin chng phi s dng cc ngun nit c sn trong mi trng. Cc ngun nit vi khun lactic c th s dng nh: cao tht, cao nm men, trypton, dch thy phn casein t sa, pepton,Hin nay cao nm men l ngun nit c s dng nhiu nht v c hiu qu nht. tuy nhin quy m cng nghip khng th s dng ngun nit ny v rt tn km. c. Nhu cu v vitamin Vitamin ng vai tr l cc coenzyme trong qu trnh trao i cht ca t bo, nn rt cn thit cho hot ng sng. Tuy nhin, a s cc loi vi khun lactic khng c kh nng sinh tng hp vitamin. V vy cn b sung vo mi trng cc loi vitamin. Cc cht cha vitamin thng s dng nh nc chit t khoai ty, ng, c rt hay dch t phn nm men d. Nhu cu cc hp cht hu c khc Ngoi cc axit amin v vitamin, vi khun lactic cn cn cc hp cht hu c khc cho s pht trin nh cc baz nit hay cc axit hu c. Mt s axit hu c c nh hng thun li n tc sinh trng ca vi khun lactic nh axit xitric, axit oleic. Nn hin nay ngi ta s dng cc mui citrat, dn xut ca axit oleic lm thnh phn mi trng nui cy, phn lp v bo qu cc chng vi khun lactic. Tng t nh hai axit hu c trn, axit axetic cng c nhng tc ng quan trng n s sinh trng ca t bo. Nn ngi ta thng s dng axit axetic di dng cc mui axetat lm cht m cho mi trng khi nui cy vi khun lactic. e. Nhu cu cc mui v c khc

23

 m bo cho sinh trng v pht trin y , vi khun lactic rt cn cc mui v c. Nhm cung cp cc nguyn t khong nh ng, st, natri, kali, photpho, lu hunh, magie c bit l mangan, v mangan gip ngn nga qu trnh t phn v n nh cu trc t bo. f. Nhu cu dinh dng oxi Vi khun lactic va c kh nng sng c trong mi trng c oxy v va sng c trong mi trng khng c oxy. + Trong iu kin hiu kh sinh khi vi khun s pht trin nhanh hn so vi iu kin k kh, trong iu kin ny t mt phn t glucose s b oxy ha hon ton thnh CO 2 v H2O v tng hp cc enzyme, t mt phn t glucose to ra 36 hoc 38 ATP. + Trong iu kin k kh t mt phn t glucose ch to ra 2 ATP do lng c cht b phn hy rt nhanh v tng hp mt s cht khng khun. 2.2.4.2. Qu trnh trao i cht Mt tnh nng cn thit ca LAB trong qu trnh trao i cht l kh nng ln men carbohydrate, cc ATP to ra c s dng cho cc mc ch tng hp sinh hc khc v sn phm cui cng ch yu l acid lactic (t 50% carbon ca ng). LAB c kh nng ln men cc loi ng hexcose (glucose, mannose, galactose, fructose), disaccharide (lactose, saccharose); pentose (arabinose, xylose, ribose) v cc hp cht lin quan. Chng ch s dng c cc loi ng dng ng phn D. Tuy nhin, LAB c th thch ng vi nhiu iu kin khc nhau lm thay i cch thc trao i cht v dn n cc sn phm cui cng to ra cng khc nhau. Da vo kh nng ln men lactic t glucose, ngi ta chia vi khun lactic lm hai nhm: ln men lactic ng hnh v ln men lactic d hnh. (Owen R. Fennema et al. 2004). a. Ln men lactic ng hnh

24

Trong ln men ng, LAB s dng 2 cch thc ch yu l vn chuyn ng t do (transport) v phosphoryl ha ng (phosphorylation) bi glucokinase (cn 1 ATP). Mt s loi s dng phosphoenolpyruvate: sugar phosphotransferase system (PTS), mi phosphoenolpyruvate l mt phn t cho phosphate, trong trng hp ny i hi mt lin kt phosphate c nng lng cao hot ha phn t ng. Con ng glycolysis hay con ng EMB (Embden-Meyerhof-Parnas pathway) c s dng bi hu ht cc LAB (ngoi tr leuconostocs, nhm III Lactobacilli, Oenococci v Weissellas) to ra fructose-1,6-diphosphate (FDP) v nh FDP aldolase tip tc chuyn thnh dihydroxyacetonephosphate (DHAP) v glyceraldehyde-3-phosphate (GAP) i vi nhng cht c mc phosphoryl ha 2 v tr, sau to thnh pyruvate. Trong iu kin c nhiu ng v hn ch oxy, pyruvate b kh thnh axit lactic bi lactate dehydrogenase (nLDH) v NAD+, do NADH c oxy ha trc , khi th oxy ha kh c cn bng, sn phm cui cng c to ra ch yu l axit lactic v qu trnh ny c gi l ln men lactic ng hnh. b. Ln men lactic d hnh Ngoi ra cn c mt s con ng ln men khc nh: con ng pentose phosphate, con ng pentose phosphoketolase pathway, con ng hexose monophosphate, con ng 6phosphogluconate v c gi chung l 6-phosphogluconate/phosphoketolase (6-PG/PK). c im ca con ng ny l s kh hydro ngay t bc u to 6-phosphogluconate. Theo sau l s tch carbon to pentose-5-phosphate v tip tc chuyn ha thnh glyceraldehyde-3-phosphate (GAP) v acetyl phosphate.GAP c to thnh tng t nh trong con ng glycolysis v kt qu l to ra axit lactic. Trong iu kin khng c mt ca cc cht nhn in t, acetyl phosphate s b kh to thnh ethanol thng qua CoA v acetaldehyde. Khi qu trnh ny to ra mt lng ng k cc sn phm khc nh CO2, ethanol... th n c gi l ln men lactic d hnh. Thng thng, LAB ln men ng hnh ch yu ln men bng con ng glycolysis v ngc li LAB ln men d hnh s dng con ng 6-PG/PK. Tuy nhin cn ch nhn xt trn khng phi dng cho tt c cc trng hp.

25

(Owen R. Fennema et al. 2004).

26

Hnh 9. Con ng ln men glucose

27

(A) Ln men ng hnh (con ng glycolysis,, EMB) (B) Ln men d hnh (con ng 6-phosphogluconate/phosphoketolase) Cc enzyme tham gia vo qu trnh: 1. Glucokinase; 2. Fructose-1,6-diphosphate aldolase; 3. Glyceraldehyde-3-phosphate dehydrogenase; 4. Pyruvate kinase; 5. Lactate dehydrogenase; 6. Glucose-6-phosphate dehygrogenase; 7. 6-phosphogluconate dehydrogenase; 8. Phosphoketolase; 9. Acetaldehyde dehydrogenase; 10. Alcohol dehydrogenase. 2.2.5. Sn phm qu trnh ln men v kh nng sinh cc cht khng khun vi khun lactic Cc vi khun lactic c kh nng tng hp mt s lng ln cc enzyme ngoi bo kch thc h thng tiu ha nh: tng hp enzyme amylase, protease, lipase, glycolase v lactic dehydrogenase. Khi s dng chng c tc dng h tr tiu ha, phn gii protein v lipid, gip gim cholesterol, ci thin kh nng dung np lactose LAB ng vai tr ni bt trong rut bng vic tng hp vitamin: vitamin B, axit folic, biotin (vitamin H), vitamin K. Vi khun sinh axit lactic to ra mt s cc sn phm bin dng c kh nng c ch s pht trin ca cc vi sinh vt gy bnh nh: H2O2, CO2, diacetyl v cc bacteriocin(Bng 8). Vi khun lactic c xem l c kh nng c ch v cnh tranh cc vi khun gy hi cng nh cc mm bnh bn trong c th (in vivo) bng cch sinh ra cc cht c tnh khng khun nh nu trn. C rt nhiu nghin cu tp trung vo bacteriocin v ph khng khun ca bacteriocin, kt qu cho thy kh nng khng khun ca chng vn cn rt hn ch, phn ln chng ch c th khng li cc vi khun Gram dng nh khng mt s loi c lin quan mt thit (cc chng Lactobacillus khc) hoc khng cc vi khun sinh bo t Gram dng nh Bacillus v Clostridium (Holzapfel et al.,1995). Tuy nhin vn c mt s nghin cu khc cho thy LAB c th khng li mt s vi khun Gram m nh: Salmonella, Escherichia coli v Helicobacter licobacter (Skytta et al., 1992; Helander et al., 1997; Niku-Paavola et al., 1999) c th l nh vo vic sn sinh ra cc cht c

28

trng lng phn t thp (nh H2O2, axit lactic, cc hp cht d bay hi) v cc hp cht th cp trong qu trnh trao i cht lm cho ph khng khun ca chng c m rng hn i vi vi khun Gram m. Chng L. acidophilus LA1 c kh nng khng li c vi khun Gram dng v Gram m nh Staphylococcus aureus, L. monocytogens, Salmonella typhimurium, Shigella flexneri, Klebsiella pneumoniae, Pseudomonas aeruginosa v Enterobacter cloacae v Helicobacter pylori (Bernet-Camard et al., 1997), (Michetti et al., 1999). Mc d LAB khng c kh nng khng li nhiu loi vi khun Gram m nhng trong thc t th ngi ta quan tm nhiu n kh nng khng vi khun Gram m ca LAB hn v cc vi khun Gram m thng l nhng vi khun c hi v gy bnh cho ngi nh E. coli gy tiu chy, Salmonella gy bnh thng hn v Helicobacter gy vim lot d dy Bng 8. Sn phm bin dng v kiu hot ng i khng Sn phm bin dng CO2 Kiu hot ng i khng c ch qu trnh decarboxylation, gim tnh thm ca mng. Diacetyl Hydrogen peroxide Lactoperoxide Axit lactic Axit lactic khng b phn hy m thm vo mng lm gim pH ni bo. N cng lin quan n qu trnh bin dng nh: oxidative phphotylation. Bacteriocin Tc ng ln mng , tng hp enzyme v tng hp protein. Tc ng ln protein gn arginine. Oxy ha cc protein c bn.

2.3. Tng quan v phng php phn lp v tuyn chn vi khun lactic 2.3.1. Nhu cu tm kim cc ngun mi

29

Trong nhng nm gn y, kh nng khng li thuc khng sinh ca vi khun kh ph bin trong s iu tr bnh ca con ngi v ng vt. V th s tip tc tm ra nhng cht khng vi khun mi ang tr nn quan trng trong lnh vc thuc. hn ch s dng qu nhiu thuc khng sinh ha hc trong thc phm v thc n chn nui, mt s la chn hp l l ng dng ca mt s protein ca vi khun nh thuc khng sinh. Trong s , vic sn xut lactase t vi khun lactic thu ht nhiu s ch do LAB c ph khng khun rng hn so vi cc ging khc v c tim nng c dng lm cht bo qun thc phm v ng dng trong dc phm. 2.3.2. Phng php tm kim cc ngun mi Tuy hu ht cc sn phm v lactase trn th trng u ging nhau v thnh phn vi sinh, ch khc nhau v nh sn xut, m mi chng li c ph tc dng khc nhau. V vy vic phn lp tuyn chn ra nhng chng, ging mi s gp phn lm phong ph thm ngun lactase, lm tng kh nng phng v tr bnh cho ngi v ng vt. Vic la chn v phn lp cc chng vi khun sn xut lactase l mt quy trnh la chn cn thn. Vi khun lactic c vai tr quan trng trong cng nghip sa, mui chua rau qu, lm yaourt cng nh trong nng nghip v dc phm. V mt c tnh khc ca vi khun lactic tr nn c xem trng l chng c kh nng to ra bacteriocin (cht khng khun) nh lactacin, brevicin, lacticin, helveticin, sakacin, plantacin,... c tc dng c ch mt s vi sinh vt gy bnh, ngn chn s pht trin ca cc ngun bnh trong thc phm (Batt, 1999; Dubernet et al., 2002). V cc chng vi khun lactic thng c tr trong ng rut ca ngi v ng vt vi mt lng sn c s gip kch thch tiu ho thc n v tiu dit mt s vi khun gy bnh ng rut khc nn, ng thi v chng c ngun gc t thc phm nn a s l m bo v mt an ton cho con ngi v vt nui. Vi khun lactic c phn lp trn mi trng chuyn bit MRS (DeMan, Rogosa, Sharpe.1960) t nhiu ngun khc nhau: - T cc sn phm thc phm ln men nh: kefir, sa chua, da chua, nem chua, kim chi. - T phn ca cc gia cm: th, g, heo v t m g

30

- T sa m, phn tr s sinh. Mt chng vi khun sau khi c tuyn chn, to dng thun th vic quan trng k tip l phi xc nh chng thuc ging no, loi no. V vy, cc phng php nh danh c p dng. Cng vic tuyn chn ngoi vic tm ra chng m bo nhng thuc tnh c bit ca probiotics, m bo s n nh v thun khit cao v t l chon chng c hot lc cao nht theo tng mc ch s dng, tng i tng cho ph hp. Kho st tnh an ton ca chng (i vi con ngi, cho ng vt v mi trng) trc khi mang i s dng. 2.3.3. Cc phng php xc nh, phn loi vi sinh vt 2.3.3.1. nh danh vi sinh vt theo phng php c in. Ban u vi khun lactic c dng ch cc vi khun lm chua sa v sau mt dng vi khun thun khit cng c cho l vi khun lactic (c th l Lactococcus lactis) c a ra bi J. Lister vo nm 1873. Mt bc quan trng trong phn loi vi khun lactic c hnh thnh khi c s ging nhau gia cc vi khun trong sa chua v cc vi khun to axit lactic khc c trong cc ngun khc nhau. Tuy nhin vn cn nhiu nhm ln khi chuyn kho ca Orla-Jensen xut hin (Orla-Jensen. 1919) v n c nh hng ln n h thng phn loi ca LAB, mc d c s sa i dng k nhng c s cho vic phn loi vn khng thay i, Orla-Jensen vn phn loi da trn phng php c in nh: so snh hnh thi vi khun (cocci, rod hoc dng tetrad), hnh thc ln men glucose (ng hnh, d hnh), kh nng pht trin cc nhit khc nhau (10oC v 45oC), kh nng ln men cc ngun ng khc nhau. Nm 1985 c thm nhiu ging LAB mi c m t, tuy nhin vic phn tch theo phng php c in da vo hnh thi vi khun vn gi vai tr quan trng trong phn loi LAB. Cc ging LAB cng c m t li trong Bergeys manual 1986, y c xem nh l phin bn cui cng v ch yu tip tc phn nh kt qu ca Orla-Jensen 1919. Trong c s ph hp vi cc m t chung ca cc ging LAB in hnh: Aerococcus, Lactobacillus, Leuconostoc, Pediococcus v Streptococcus. S thay i chnh trong Bergeys manual 1986 l

31

ging Streptococcus ln u tin c chia thnh 3 nhm : Enterococcus, Lactococcus v Streptococcus sensu stricto (Schleifer. 1987). Mt vi ging LAB ging vi Lactococci c ngh xp vo ging Vagococcus, cc ging Lactobacillus, Leuconostoc, Pediococcus khng c thay i nhiu, tuy nhin mt vi loi trong Lactobacills c xp vo ging Carnobacterium, v loi Pediococcus halophilus c a ln cp ging l Tetragenococcus, mt s LAB ln men d hnh trong Lactobacillus v Leuconostoc c xp vo ging Weissella, ngoi ra mt s ging mi cng c m t c lin quan nhiu n LAB nh Alloiococcus, Dolosicoccus, Dolosigranulum, Eremococcus, Facklamia, Globicatella, Helcococcus, Ignavigranum v LactosphaeraBn sa i c thc hin vo nm 1986 cng h tr nhiu trong vic phn loi vi sinh vt da trn thnh phn ha hc v di truyn. (Owen R. Fennema et al. 2004) 2.3.3.2. S phn loi LAB n cp ging Nh cp, c s chung cho vic phn loi LAB vo cc ging khc nhau ch bng phng php nh danh c in v so snh vi kha phn loi ca Bergeys manual 1986, tuy nhin vic ny ngy cng gp kh khn t kt qu tin cy khi phn loi mt s ging mi nhng n li l bc quan trng trc khi tin hnh cc phn tch su hn. LAB c phn chia theo hnh thi nh sau: dng trc khun (rod) gm Lactobacillus v Carnobacterium; dng cu khun gm tt c cc ging cn li v mt ngoi l l i vi ging Weissella c m t gm c dng trc khun v cu khun; thm vo , s phn chia t bo theo hai hng vung gc trn cng mt mt phng dn n s hnh thnh dng t cu khun (tetrad) v n cng c s dng m t mt dng khc ca cu khun, bao gm cc ging Aerococcus, Pediococcus v Tetragenococcus. Phn chia theo hnh thc ln men glucose, LAB c chia vo 2 nhm: ln men ng hnh chuyn ha glucose ch yu thnh axit lactic v ln men d hnh to axit lactic, ethanol, axit acetic v CO2. Trong thc nghim, kim tra s sinh kh CO2 phn bit gia 2 nhm: nhm

32

(Sharpe. 1979) Leuconostoc, Oenococci, Weissella v phn nhm ca Lactobacilli l ln men d hnh, tt c cc LAB khc l ln men ng hnh. S pht trin cc nhit khc nhau cng gp phn phn bit mt s ging thuc cu khun: Enterococci pht trin 10oC v 45oC, Lactococci v Vagococci pht trin c 10oC nhng khng th sng 45oC, Streptococci thng khng pht trin 10oC trong khi li c th pht trin 45oC ty vo loi. S chu mui (6,5% NaCl) cng c s dng phn bit gia Enterococci v Lactococi, Vagococci v Streptococci (Mundt. 1986). c bit kh nng chu mui (18% NaCl) c gii hn trong Tetragenococcus. Kh nng chu axit kim cng rt hu ch trong vic phn loi. Aerococci, Carnobacteria, Enterococci, Tetragenococci v Vagococcicos th pht trin pH cao nhng khng pht trin pH=9.6. Ngoi ra s hnh thnh cc dng ng phn ca axit lactic trong qu trnh len men glucose cng c s dng trong phn bit gia Leuconostocs (ch to D-lactic axit) v Lactobacilli ln men d hnh (to axit lactic c 2 dng D v L). Tuy nhin Weissella c th gy nhm ln trong vn ny. (Owen R. Fennema et al. 2004) a. Cc phng php da trn c im hnh thi: - Nhum Gram (phng php Hucker ci tin). - Kim tra kh nng di ng: nhum tin mao, phng php kim tra trn mi trng thch mm. - Nhum bo t: nhum lc Malachit (phng php Schaeffer-Fulton), nhum Carbolic Fuchsin. - Nhum v nhy (Capsule): phng php nhum m bn, phng php Congo v phng php dng Tm kt tinh (Crystal violet). - Nhum vi khun khng axit b. Cc phng php da trn iu kin nui cy v c im sinh l: - Hnh thi khun lc.

33

- Nhit sinh trng, chu axit, chu mui mt - Nhu cu oxygen. c. Cc phng php da trn cc phn ng sinh ha: Ty thuc vo ngun phn lp m ta nh hng cho cc phn ng sinh ha nh danh cho ph hp nh: kh nng ng ha cc ngun carbon, kh nng ng ha cc ngun nit, kh nng sinh sc t hunh quang, tnh mn cm i vi cht khng khun, ng ha Malonat, xt nghim ng ha Citrat, nhu cu mui v tnh chu mui, kh nng ng ha tartrat, kh nng sinh trng vi KCN, xt nghim oxidaza, xt nghim catalase, kh nng ln men/xy ha amygdalin, arabinnoza, xenlobioza, fructoza, glucoza, lactoza, maltoza, manitol, melezitoza, saccaroza, sorbitoi, phn ng Methyl - Methyl Red, phn ng V.P. (Voges-Proskauer), phn ng ONPGaza Ngoi ra cn mt s da trn phn ng sinh ha chuyn bit hn nh: + API20E KIT : da vo 20 phn ng khc nhau. Hin nay c nhiu ni vn dng k thut ny nhng nhn chung kt qu cng cn nhiu sai s do nhiu nguyn nhn: trng hp gene quyt nh phn ng sinh ha nm trong plasmid li b mt do tui t bo, lng ging cy hay thay i trong qu trnh nui cy dn n lm sai kt qu. + Phn bit bng thc khun th: Cc vi khun c mn cm vi thc khun th khc nhau. C thc khun th xm nhim lm tan t bo ngay lp tc sau thc khun th nhn ln thnh cc ht trong t bo ch, trong khi vi mt s vi khun th thc khun th xm nhim nhng li khng lm tan t bo vi khun v chng cng tn ti vi t bo vt ch. Da vo s khc bit ny m ngi ta dng cc thc khun th khc nhau phn bit cc i tng vi khun nghin cu. Tuy nhin, phng php ny cng bc l mt s nhc im kh gii quyt l cc c tnh mn cm ca vi khun vi thc khun th li thay i do iu kin ngoi cnh hoc l vi khun li c mc mn cm khc nhau i vi cc thc khun th khc nhau. Mt khc na, thc

34

khun th rt d thay i cc c tnh do cng lm thay i c ch xm nhim vo vi khun ch. + Phn bit theo Typ huyt thanh: y l phng php c dng kh lu nhng rt hiu qu v hin vn ang c s dng (v d nhm vi khun Bt). Nguyn tc l da vo nhm quyt nh khng nguyn trn t bo vi sinh vt (b mt t bo tin mao hoc protein v). u th ca phng php ny l cc khng huyt thanh c dng bit ha nhiu chi khc nhau, trong nhiu trng hp c trng cho loi. Ni chung y l phng php kh n nh nhng c hn ch l: yu cu k thut sn xut khng huyt thanh v tiu chun ha phn ng khng huyt thanh khng ng nht ti cc phng th nghim v tnh n nh gia cc ln lp li. + Phn bit bng loi hot cht khng khun (Bacteriocin): Bacteriocin bn cht l peptid khng khun sinh ra bi vi khun chng li vi khun khc. Nh vy, loi vi khun to ra loi bacteriocin no th c kh nng khng li chnh bacteriocin . Bi vy c nhiu loi vi khun c phn loi da vo kiu bacteriocin. 2.3.3.3. nh danh vi sinh vt theo phng php hin i. Vic phn loi LAB ch yu da trn hnh thi v c im sinh l, sinh ha. Tuy nhin trong thc nghim, cc c im ny khng nh danh chnh xc mt chng n cp loi c th. Ngy nay vi cng ngh xc nh trnh t DNA mt cc t ng v nhanh chng, ngi ta trc tip xc nh c trnh t ca gen m ha cho 16S rRNA v to phng tin d dng cho phn loi vi khun (Owen R. Fennema et al. 2004) Vic xc nh trnh t ca gen 16S rRNA phn tch s pht sinh loi ca LAB c th hin vo nhng nm 1990, bt u t s pht trin cc u d DNA nh danh vi khun. Vic phn loi LAB bng cc u d 16S hoc 23S RNA c pht trin v s dng i vi Lactococci, Enterococci, Lactobacilli, Carnobacteria v phn bit Vagococci vi cc LAB khc D liu v trnh t cc gen 16S rRNA ca LAB c tch ly trong sut nhiu nm qua v danh mc cc u d hin c c a ra bi Pot et al.

35

2.4. Cc nghin cu v lactase trong v ngoi nc 2.4.1. Trong nc Cc nghin cu v lactase Vit Nam c th k n: Nhm ca Nguyn Thu H v cng tc vin (2006) nghin cu s biu hin ca enzyme lactase 2 chng Lactobacilus reuteri L103 v L461. y l nghin cu hon ton c bn, cho thy 2 enzyme biu hin 2 tiu phn ( 2 chui peptide c trng lng l 35kDa v 85kDa). Cc enzyme ny c khong pH hot ng kh hp, mt enzyme t pH 3.8 4.0 ( chng L461) v mt pH 4.6 4.8 (chng L103). Kt qu nghin cu ny sau c trnh by ti Hi ngh ln th V, Trng i hc Khoa hc H Ni, 2006, v ng trong tp ch quc t. Nhm tc gi ny v sau c mt s nghin cu tinh sch enzyme s dng tng hp galactooligosaccharides mang hot tnh sinh hc. Nhm nghin cu ca tc gi Trn Vn Giang (2009) c 2 nghin cu tp trung vo nghin cu enzyme ti t hp. Mt l nghin cu biu hin -glucosidase trong E.coli BL21. Mt nghin cu khc l nhn dng v phn tch trnh t gen m ha B-glucosidase t chng Bacillus subtilis G1. 2.4.2. Ngoi nc 2.4.2.1. Nghin cu lactase t nm men c nghin cu kh y v sn phm lactase thng mi hin nay ch yu l sn xut t nm men. Cc loi nm men nh Kluyveromyces lactis, Kluyveromyces marxianus, Torulopsis spherical, Torulautilis sp, Sacchromyces fragilis, Candida pseudotropicalis. Lactase t Sacchromyces fragilis c nghin cu t rt sm, 1956, khi Davids. A [47] bit rng lactase trong nm men ny l dng ni bo v tm ra phng php ph t bo bng ha cht tng hot tnh enzyme. Nghin cu ny cng ch ra l lactase b c ch bi nng ng Glc, Suc, nhng km c ch hn vi Gal v Lac.

36

V lactase ca Candida pseudotropicalis, nghin cu ca Sonia a. de Bales v Francisco J. Castillo, 1979 [46], nui trong canh trng cha 10-12% nc chit rt sn xut phomai (whey) b sung thm khong v nc chit nm men. Hot tnh enzyme thu c l 468 U/ml v 4.35 U/g sinh khi. Hot tnh enzyme ti u pH 6.2 v nhit 470c. Hot tnh ny c th bo tn 95% sau 3 thng bo qun -200c Gn y nht (2008) [6] l nghin cu v sinh tng hp lactase t Kluyveromyces marxianus c nh trn gi th rn vi nng c cht lactose l 50g/L, b sung them ngun nit v phosphor. Sau 30h nui cy, 90% c cht b chuyn ha v thu c hot tnh lactase ni bo l 1147.7 UI/g sinh khi. 2.4.2.2. Nghin ca lactase t vi khun Ch yu l t Gram dng, thuc ging Lactobacillus, Lactococcus, Bacillus, vi khun Gram m l E.coli. Chang B.S v cng tc vin (1989) [41] tinh sch lactase t Streptococcus salivarius phn nhnh thermophilus n mc 109 ln so vi ban u, vi hiu sut l 41% v hot tnh l 592UI/mg protein 370c. Nu t enzyme trong sa th hot tnh ny bn nhit (60650c) ti 146 pht tc gp 40-80 ln khi mi trng phn ng khng phi l sa. Nm 2005, mt nghin cu n , S.K.Akolkar v cng tc vin [7] sinh tng hp lactase t Lactobacillus acidophilus, chng phn lp t sn phm len men ragi, mt mn n truyn thng. Vi sinh c nui trong canh trng lng 5L, cha nng lactose rt thp 0.75% cho thm c 1% ragi nn hiu qu v kinh t. Enzyme thu c c hot tnh 5400U/ml-1 tc l thm ch cao hn chng sn xut cng nghip l nm men Kluyveromyces lactis. Lactase trong E.coli c bit n t nm 1941 [48], khi ngi ta phi ph t bo vi khun thu dung dch lactose th . Enzyme nay c bit c gii pH trung tnh, ti pH 5.0 gn nh mt hot tnh . Nhit ti u l 360C. Gen m ha cho -galactosidase ca E.coli sau ny c tch dng, ti t hp li trong vect c hiu vi mc ch lm cng c trong k thut di truyn tuyn chon cc t bo biu hin gen thng qua sng lc khun lc xanh trong mi trng cha X-gal.

37

2.4.3.3. Nghin cu lactase t nm si in hnh l t Trichoderma sp, Aspergillus sp, Fusarium sp. c trng chung l cc enzyme ny l ngoi bo nn quy trnh tinh sch kh d dng. Phn ln l enzyme chu nhit v chu pH acid yu (3.0-5.0). Bn cnh u im trn th nhc im ln nht l hot tnh ring ca enzyme kh thp so vi vi khun v nm men. Lactase t Aspergillus sp c nghin cu nhiu hn c l cc enzyme t loi Aspergillus niger v Aspergillus oryzae. Mt vi nghin cu tiu biu sau: Park, Y.K v cng tc vin (1979) [45] tinh sch lactase t Aspergillus oryzae bng kt ta amon sulphate v sau l sc k ct thu c enzyme kh n nh pH 3.0-8.0 v hot ng tt nhit 500c. Tuy nhin enzyme ny trong mi trng b sa gim hot tnh ng k. Sanjeev Agrawal v cng tc vin (1982) [43] phn lp c 48 chng nm si c hot tnh lactase, trong c 8 chng thuc Aspergillus sp. Nghin cu cng cho thy l enzyme ngoi bo v thuc tnh enzyme thuc dang chu nhit Gn y Zhang W v cng tc vin (2005) [40] chuyn gene lactase ca Aspergillus candidus vo nm men Pichia pastoris, tinh sch enzyme v thu c enzyme c trng lng 113kDa, hot tnh l 3600U/mg, ti u pH 5.2 v nhit 600C. i vi lactase t Trichoderma sp, Francisco J. Castillo v cng s (1984) [42] nui chm Trichoderma reesei Rut-C30 trong mi trng cha lactose thu c hot tnh enzyme ngoi bo 27.3U/L. Hot tnh enzyme t c cc i 600c v pH 4.6. Loi lactase ny c gi l lactase axit v ph hp dng trong mi trng c pH tng i axit nh nc chit rt t len men phomai. Lactase cng c th tch chit t nm si Fusarium sp. Macris BJ, Markakis P (1981)[44] thu nhn enzyme ngoi bo t chng Fusarium moniliforme nui bng nc chit rt t sn xut phomai (whey) v ta bng amonium sulphate. Kt qu l enzyme c hot tnh tt nht pH 3.8 v nhit 500c. Tng t nh vi enzyme t Trichoderma sp, enzyme ny thuc dng lactase axit v thch hp thy phn lactose trong nc chit. Trong iu kin thch hp, 10 n v hot tnh enzyme c th thy phn 50% lng lactose trong vng 5h.

38

2.4.3.4. Mt s chng c thng mi ha[58]

Chng Bifidobacterium animalis subsp. lactis BB-12 Bifidobacterium breve Yakult Bifidobacterium infantis 35624 Bifidobacterium lactis HN019 (DR10) Bifidobacterium longum BB536 Escherichia coli M-17 Lactobacillus acidophilus LA-5 Lactobacillus acidophilus NCFM Lactobacillus casei DN114001 (Lactobacillus casei Immunitas(s)/Defensis) Lactobacillus casei CRL431 Actimel/DanActive ProBactrix Escherichia coli Nissle 1917 Mutaflor Bifiene Align Howaru Bifido Tn sn phm Nh sn xut Chr. Hansen Yakult Procter & Gamble Danisco Morinaga Milk Industry BioBalance Ardeypharm Chr. Hansen Danisco Danone Chr. Hansen Ci thin sc khe tiu ha, kch thch min dch, lm gim khng sinh-lin quan n tiu chy, iu chnh s cn bng men vi sinh trong rut gi, lm gim s tng cn (Burrington, Kimberlee. 2007). Tc ng n con ngi

Lactobacillus casei F19

Cultura

Arla Foods

39

Lactobacillus casei Shirota Lactobacillus paracasei St11 (or NCC2461)[55] Lactobacillus johnsonii La1 (Lactobacillus LC1)

Yakult Lactobacillus fortis

Yakult Nestl Nestl Kch thch min dch, ci thin tiu ha, lm gim khng sinh - tiu chy Dnh cho tr em

Lactococcus lactis L1A

Norrmejerier

Lactobacillus plantarum 299V

GoodBelly / ProViva/ TuZen

NextFoods Probi Ferring Phng, chng v gim thiu tiu chy tr em, BioGaia Biologics ci thin h tiu ha. Phng bnh t em v ngi ln

Lactobacillus reuteri ATTC 55730 (Lactobacillus reuteri SD2112)

Lactobacillus rhamnosus ATCC 53013 (discoveredby Gorbach & Goldin (LGG) Lactobacillus rhamnosus LB21 Lactobacillus salivarius UCC118

Vifit v mt s mt hng khc

Valio Kch thch min dch, ci thin tiu ha, lm gim khng sinh-tiu chy

Verum

Norrmejerier

Saccharomyces cerevisiae (boulardii) lyo

DiarSafe v mt s mt hng khc

Wren Laboratories v mt s hng khc

chng li khng sinh-c lin kt v tiu chy [Clostridium difficile] bnh nhim trng, iu tr tiu chy cp tnh ngi ln & tr em

40

Hn hp: Lactobacillus rhamnosus GR-1 & Lactobacillus reuteri RC-14

Bion Flore Intime Jarrow FemDophilus

Chr. Hansen

Ngn nga vim m o

Hn hp : mixture of 8 strains of Streptococcus thermophilus & four Lactobacillus spp & three Bifidobacterium spp strains Hn hp: Lactobacillus acidophilus CUL60 & Bifidobacterium bifidum CUL 20

VSL#3

Sigma-Tau Pharmaceuticals, Inc.

Hn hp: Lactobacillus acidophilus CL1285 & Lactobacillus casei

BioK Plus

Bio-K+ International

Ci thin sc khe tiu ha. Ngn nga Tiu chy C lin h Antiobic (AAD) v Clostridium (C Difficile). Kim hm In vitro nhng chng Listeria monocytogenes v L. innocua, Escherichia coli, Staphylococcus aureus, Enterococcus faecalis v Enterococcus faecium. tiu ha ng lactose v kch thch min dch

41

Lactobacillus helveticus R0052 & Lactobacillus rhamnosus R0011 Hn hp pha trn t 16 chng: Bifidobacterium bifidum Bifidobacterium breve Bifidobacterium infantis Bifidobacterium lactis Bifidobacterium longum Lactobacillus acidophilus Lactobacillus brevis Lactobacillus bulgaricus Lactobacillus casei Lactobacillus gasseri Lactobacillus paracasei Lactobacillus plantarum Lactobacillus rhamnosus Lactobacillus salivarius Lactococcus lactis Streptococcus thermophilus

A'Biotica v mt s Institut Rosell mc hng khc

Flora Source Multi-Probiotic

Nutri-Health

2.4.3.5. Sn xut ch phm lactase trn th gii Lactose hin din nng khong 4,7% (w / v) trong sa v whey (ni trn mt) cn li sau giai on ng t lm pho mt. S hin din ca n trong sa lm n khng ph hp vi phn ln dn s ngi ln trn th gii, c bit nhng khu vc khng c truyn thng v ngnh cng nghip sa. Bt dung np lactose c gii hn ch yu dn tc c ngun gc nm Bc u hay tiu lc a n v nh n nh lactase, tt c cc ng vt c v tr r rng c th tiu ha sa, nhng trong nhiu trng hp kh nng ny gim sau khi cai sa. Thi Lan, Trung Quc v ngi M en tng ng vi 97%, 90% v 73%, c bo l khng dung np lactose, trong khi ngi M trng v dn s Thy in tng ng vi 84% v 96%. Thm vo , ch c rt him mt s c nhn khng dung np lactose chuyn ha bm sinh hoc thiu lactase, c hai u c th nhn thy khi sinh. S cn thit ca sa c hm lng lactose thp c bit quan trng trong chng

42

trnh vin tr thc phm nh mt nc m nghim trng, tiu chy v thm ch t vong do n sa c cha lactose, tr em khng dung np lactose v ngi ln b suy dinh dng protein c hm lng calo thp. Trong tt c cc trng hp, thy phn lactose thnh glucose v galactose s ngn chn cc vn tiu ha (nng)[55]. Mt vn khc c trnh by bi lactose l kh nng ha tan thp dn n hnh thnh tinh th nng cao hn 11% (w / v) (4C). iu ny ngn cn vic tp trung s dng xi-r whey trong qu trnh sn xut thc phm, to nhiu kt cu phc tp v d dng b h hng vi sinh. Thm vn na l vic x l cht thi whey nh vy l rt t tin do nhu cu oxy sinh hc ca n cao. Nhng vn ny c th c khc phc bng cch thy phn lactose trong sa, sn phm c cc yu t nh ngt, ha tan, kh nng hnh thnh tp trung, an ton vi sinh v xi-r (70% (w / v))[55]. Lactose c th c thy phn bi lactase, mt b-galactosidase:

Hnh 10. Qu trnh thy phn lactose[54] Lactase thng mi c th c ch bin t sa nm men Kluyveromyces fragilis ( K. marxianus var. marxianus), vi pH ti u (pH 6,5-7,0), hoc t cc loi nm Aspergillus oryzae hoc A. niger, vi Optima pH (pH 4,5-6,0 v 3,0-4,0 hoc tng ng) ph hp vi thy phn whey hn. Cc men ny c th c mc khc nhau v s c ch sn phm ca

43

galactose. Ngoi ra, nng lactose v galactose cao, lactase cho thy kh nng truyn enzyme ng k v sn xut b-1,6 lin kt vi oligosaccharides galactosyl[55]. Lactase ang c s dng trong sn xut kem, sa c c v c hng v ngt. Khi c thm vo sa hay sa lng (2000 U kg -1 ) v khong mt ngy 5C s c khong 50% lactose c thy phn, a ra mt sn phm c ngt m khng kt tinh nu ngng t hoc ng lnh. Phng php ny cho php khng lng ph whey thay th mt s hoc tt c sa bt tch kem c s dng trong cng thc nu kem n truyn thng. N cng ci thin kh nng m rng v tnh mt ca sn phm. S lng lactase nh hn c th c thm vo sa tit trng sn xut mt sn phm lactose tng i r tin ( v d nh 20 U kg -1, 20C). Ni chung, vic s dng lactase khng t c tim nng y ca n, v enzyme hin nay tng i t tin v ch c th c s dng nhit thp[55]. Lactase t t bo ng vt, thc vt v nm men v vi khun l dng ni bo, cn t nm mc l dng ngoi bo. V tr tn ti ca enzyme trong t bo s quyt nh phng php tinh sch cng nh t d ton chi ph cho cng on ny, thng thng chim t trng ln trong qu trnh sn xut . Trong cng nghip hin nay ch yu sn xut enzyme c ngun gc t vi sinh vt do thi gian sinh trng nhanh. Lactase t vi khun c c trng l hot tnh thng thng cao hn nhiu v lng enzyme cng to ra nhiu hn so vi lactase c ngun gc t vi nm , tuy nhin phng php tinh sch v tn km hn. Cc sn phm lactase c th : - Dng lng s dng trc tip nh vo sa phn hy lactose - Dng c nh sn xut sa ngho lactase - Dng vin nn ung hc tr tiu ha lactase ti rut non i vi enzyme cho sn xut sa th ch yu l enzyme t nm men cn h tr tiu ha th enzyme t nm si c s dng nhiu hn.

44

Bng 9. Cc cng ty sn xut enzyme lactase

Chng 3
VT LIU V PHNG PHP
Sa chua Sa men sng Nem chua Phn xu

Chng c s dng Kluyveroymces hoc

Cng ty sn xut enzyme Gist brocades, H Lan Nutritional Biochemical, M Kyowa Hakko Koygo, Nht Novo A/S, an Mch Baxte Laboratories lnc, M Dairy Food Lab, M Dainylad Food Labs, M Kyowa Hakko Koygo, Nht

Sn phm Maxilact Lactaid Lactase-Y Lactozym Lactase-LP Plexazym L1

Sacchromyces lactic Kluyveromyces hoc

Sacchromyces fragilis Aspergillus niger hoc Aspergillus oryzae

Mi trng thun li cho LAB Loi b vi khun k kh bt buc Escherichia coli

Tng Pharmaseutical, Noda, Nht Seishu sinh trong MRS broth

Rhom Pharma (Weiterstadt, FRG)

Cy truyn sang MRS agar Phn lp ging thun khit

MRS b sung 0,02% NaN3 loi vi khun hiu kh bt buc

CF-Boeringer GmnH, Mannheim, c st hnh thi khun b-galactosidase Quan Tp on Northington Biochemical, M (tinh sch dung cho nghin cu)
lc

Loi b vi khun Gram (E.coli, Salmonella)

Nhum Gram Quan st hnh thi t bo

+
Loi VK sinh bo t hnh que(Bacillus spp.Clostrididium)

Hnh que Nhum bo t

Hnh cu

Loi VK sinh bo t hnh cu Sporosarcina

45

Loi VK khng axit nh Mycobacterium Loi Arthrobacter Corynebacterium Propionibacterium

Nhum khng axit

Quan st

Catalase

nh tnh axit lactic Kim tra kh nng di ng

Vi khun ln men lactic

Ln men nghnh

3.1. Phng php th nghim S th nghim:

Ln men ng Ln men d hnh

Mu

Phn lp

46

nh danh s b

Sng lc hot tnh lactase

nh danh bng Kit 3.1.1. Thu thp mu 3.1.2. Phng php phn lp vi khun lactic 3.1.2.1. Tng sinh Mc ch: Kch hot cc vi khun c trong mu pht trin li bnh thng v chng c th b suy yu trong qu trnh bo qun ca cc mu. Trong cc vi khun lactic c kh nng pht trin nhiu hn v ta s dng mi trng chn lc i vi vi khun lactic. ng thi y cng l bc u gip thu lng sinh khi vi khun phc v cho cc th nghim tip theo. Mi trng s dng MRS broth c b sung 0,02% NaN3, hp kh trng 1210C, trong 15 pht. Vi cc mu l dng dch nh: sa chua, sa chua men sng, ht 1ml cho vo ng nghim cha khong 10ml MRS broth, cn li i vi cc vi cc mu l dng rn ly khong 2g, nh ti cho vo ng nghim cha MRS broth. Qun mng PVC (mng bc thc phm) quanh np ng nghim cho kn em 370C, 24 gi. 3.1.2.2. Phn lp

47

Mc ch: Tch cc khun lc ring l khc nhau, gip chn la ra cc chng vi khun ng nht. Cc mu sau khi tng sinh em pha long ti pha long l 10-9 Gieo cy mu tch khun lc vi khun ring r: s dng mi trng phn lp l mi trng MRS agar. Tin hnh cy trang cc pha long t 10-5 10-9 kt hp vi phng php cy ria tch cc khun khc nhau, sau cy truyn cc khun lc ng nht vo cc ng thch nghing 370C, 24 gi, bo qun ng ging nhit 40C. 3.1.3. Phng php nh danh vi khun lactic 3.1.3.1. Nhum Gram Mc ch: Gip ta phn bit vi khun thnh 2 nhm ln: vi khun Gram dng (Gram-positive) bt mu tm v vi khun Gram m (Gram-negative) bt mu hng. Ngoi ra nhum Gram cho php ta quan st r hnh thi t bo hn so vi soi vi khun sng. Sau khi thu c cc chng vi khun ng nht, ta tin hnh nhum Gram v loi b cc vi khun Gram m, gip sng lc ra cc vi khun Gram dng c kh nng l vi khun lactic. 3.1.3.2. Nhum bo t Mc ch: Da trn cu trc c bit ca mng bo t: dy, chc, kh bt mu, cha nhiu lipit. Trc ht x l t bo cht bo t d bt mu bng nhit v axit. Nhum mu c t bo cht ca bo t v t bo bng thuc nhum c hot tnh mnh. Ty mu t bo cht ca t bo v nhum n bng thuc nhum khc b sung. Nh t bo cht ca bo t v t bo cht t bo bt mu phn bit. Trong bo t s c mu xanh v t bo c mu . Cc vi khun Gram dng thu c t bc trn c tip tc kim tra kh nng sinh bo t. S dng sinh khi vi khun khi gi nhum bo t. T kt qu thu c ta loi b cc chng c bo t, do LAB l vi khun Gram dng nhng khng sinh bo t. 3.1.3.3. Nhum khng axit

48

Vi khun khng axit c v bn ngoi chng c cn v axit nn n vn gi mu nhum sau khi b x l vi dung dch cn v axit, v vy n c phn loi l vi khun khng axit. Cn nhng vi khun khng mang c tnh khng axit mu nhum ban u s b ra tri v bt mu thuc nhum b xung. Trong vi khun khng axit s bt mu . Vi khun khng khng axit bt mu xanh. Mc ch: Nhum khng axit loi b nhng vi khun khng axit nh Mycobacterium. Chng khng phi l vi khun lactic m ta mong mun v chng l nhng chng c kh nng gy bnh cho ngi. Cc chng phn lp l Gram dng khng sinh bo t c tip tc tin hnh nhum khng axit. 3.1.3.4. Th nghim catalasa Mc ch: Kim tra kh nng phn hu H2O2 to ra O2 ca vi sinh vt nh sn sinh ra enzyme catalasa. Cc chng vi khun thu c sau bc nhum khng axit c tin hnh th nghim catalase. Nu thy si bt l dng tnh, khng si bt l m tnh. C th nh trc tip dung dch H2O2 ln khun lc trn thch a ghi nhn kt qu. Trong th nghim ny, ch c nhng chng vi khun cho catalase m tnh l c kh nng l vi khun lactic. 3.1.3.5. Th nghim kh nng sinh axit. Mc ch: Kim tra kh nng sinh axit ca vi khun trong qu trnh trao i cht ca chng. Trong th nghim ny cc chng cho catalase m tnh thu c t bc trn s c kim tra vi thuc th Uffelmann. Cc chng phn lp l vi khun lactic khi chng to axit lactic v lm i mu tm ca thuc th thnh mu vng nht. 3.1.3.6. Th nghim kh nng di ng Mc ch: Xc nh kh nng di ng ca vi sinh vt trong mi trng thch mm

49

V vi khun lactic khng c kh nng di ng, ch mc theo ng cy thng ng trong ng cha mi trng thch mm v khng lm c mi trng xung quanh nn trong th nghim ny ta s loi b c cc vi khun di ng khng phi l vi khun lactic, chng s mc lan ra khi ng cy v lm c mi trng xung quanh. 3.1.3.7. Th nghim kh nng ln men ng v kh nng sinh kh Mc ch: kim tra vi sinh vt c kh nng ln men cc loi ng no. Cc loi ng c s dng l: glucose, lactose, saccharose, maltose v mannitol. Kt qu t th nghim ny gip ta nh danh n cp ging cc chng vi khun lactic phn lp c da theo kha phn loi ca Bergey. * Tin hnh: - Mi trng MRS broth vi lng ng glucose ln lc thay th bng cc loi ng cn kim tra. - t vo mi ng nghim 1 ng nh (ng Durham) ln ngc u hng kh CO 2 sinh ra nu vi khun c kh nng ln men ng. Kh trng trong 15 pht 1210C.. - Cy vi khun mi hot ho vo cc ng nghim, t 37 0C, theo di hin tng sinh axit sau 1-3 ngy. Trng hp dng nt bng cn qun bng mng PVC (mng bc thc phm) bt kn ming ng nghim. 3.1.3.8. Th nghim kh nng sinh H2O2 * Mc ch : kim tra kh nng sinh, l mt cht tham gia vo hot ng khng khun ca ca vi sinh vt. * Nguyn tc : H2O2 l mt cht va c tnh kh va c tnh oxy ha. Trong mi trng axit, H2O2 c tnh oxy ha mnh, khi tc dng vi cht kh KI to ra I2, nhn bit phn ng bng cho thm h tinh bt. H2O2 + 2KI * Tin hnh : I2 + 2KOH

50

- Nui cc chng vi khun trong mi trng lng 24 gi - Ly mi mu 5ml dch nui cy, thm 1ml H2SO4 1M v 1ml KI, sau thm 3 git h tinh bt Quan st s chuyn mu v ghi kt qu. 3.1.3.9. Th nghim kh nng khng vi sinh vt bng phng php o c (turbidimetric method). Mc ch: Nhng vi sinh vt lactic l nhng sinh vt c kh nng c ch s pht trin ca vi khun gy bnh nn trong th nghim ny cn phi kim tra kh nng c ch s pht trin vi khun gy bnh ca nhng chng vi khun LAB phn lp c nhm tuyn chn nhng chng mang c tnh , trong vi khun E. coli v Salmonella c chn lm vi sinh vt ch th. Nguyn tc: Khi mt pha lng c cha nhiu phn t khng tan s to thnh mt h huyn ph v c c bi cc phn t hin din trong mi trng lng cn nh sng, lm phn tn chm nh sng ti. T bo vi sinh vt l mt thc th nn khi hin din trong mi trng cng lm cho mi trng tr nn c. c ca huyn ph t l thun vi mt t bo. Do , thng qua vic o c bng my quang ph k bc sng 610nm ca dch huyn ph t bo ta c th nh tnh c mt vi sinh vt iu kin cn kho st. Da trn kh nng khng khun ca cc sn phm trao i cht ca LAB i vi vi sinh vt ch th trong mi trng lng. Vi sinh vt ch th b c ch tng trng dn n nng t bo gim so vi i chng. ng i chng l s pht trin bnh thng ca VK ch ch E. coli. ng kim tra l ng c cha dch nui LAB ly tm, nu vi khun LAB c tnh khng khun th ng kim tra s c mt t bo thp hn ng i chng. Th nghim c tin hnh vi 2 nghim thc : + Nghim thc 1: xc nh hot tnh da trn s c ch vi khun ch th E.coli v Salmonella bng cc cht c sinh ra trong qu trnh trao i cht ca cc chng LAB.

51

+ Nghim thc 2 : xc nh hot tnh da trn s c ch vi khun ch th E.coli v Salmonella bng cc cht sinh ra trong qu trnh trao i cht ca cc chng LAB nhng c loi b yu t acid lactic bng cch trung ha bng dung dch NaOH 1N. * Chun b : Dch nui cy E.coli v Salmonella ( 21h, 370C) pha long 10-2 tng ng nng t bo 105 t bo/ml. Dch nui cy LAB trong MRS broth k kh 18- 24h, 37oC , mang 1 na i trung ha v pH 6 bng NaOH 1N, thanh trng 80oC trong 10 pht, sau ly tm 4000 vng/ pht trong 15 pht, loi b sinh khi. *Cch thc hin: - ng th nghim : gm 1 ml dch nui cy E.coli hoc Salmonella nng 105 t bo/ml vi 1 ml dch ly tm LAB (trung ha hoc khng trung ha), b sung 8 ml mi trng cao tht-peptone. hiu kh 37oC, 24 gi . - ng i chng : ging ng th nghim, nhng 1 ml dch ly tm LAB c thay bng 1ml MRS broth tit trng. - ng trng : gm 9 ml mi trng cao tht-peptone v 1ml MRS broth tit trng. Tin hnh o c ca tt c cc ng bng my quang ph k bc sng 610nm. * Cng thc tnh ton: T l sng st ca VSV ch th (%) = ODTN/ODC*100 % OD = ODC / ODTN * 100% Nh vy vi %OD cng nh th kh nng khng vi sinh vt ca chng cng cao. Trong : ODC : gi tr OD ca ng i chng ODTN : gi tr OD ca ng th nghim.

52

% vi sinh vt ch th b c ch c tnh nh sau: D% = 100% - %OD , th hin kh nng khng vi sinh vt ca chng , D% cng ln, kh nng khng vi sinh vt cng cao. Ch : kt qu thu c l s liu trung bnh ca 3 ln lp li vi mc ngha thng k 95%. Chng 4: KT QU V BIN LUN Chng 5: KT LUN V KIN NGH

53