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Trng i hc M Tp.

HCM Khoa cng ngh sinh hc Mn: Seminar chuyn ngnh

Tng quan: BO QUN THC PHM BNG PHNG PHP SINH HC: NHNG CHIN LC HA HN TRONG S DNG BACTERIOCIN, BACTERIOPHAGE, ENDOLYSIN.

GVHD: Th.S Nguyn Vn Minh Thc hin: V Ngc Yn Nhi Hunh Th Xun o V Th Thanh Tho Nguyn Th Ngc Trm L Th Ngc Trang Nguyn Khc Vn

S quan tm n cht bo qun sinh hc thc y tm kim nhng hp cht chng khun t nhin t nhiu sinh vt khc nhau. Bacteriocin c bit n rng ri nh l cht bo qun thc phm t nhin, gn y nhng tin b trong nghin cu bacteriocin m ra hng nghin cu mi. Tri li, vic s dng bacteriophage v endolysin ch c xem xt trong 5 nm gn y v cho thy nhng vin cnh ha hn. Bi im bo ny cung cp mt ci nhn tng qut v nhng ng dng trong hin ti, tng lai ca bacteriocin, bacteriophage, endolysin dc theo chui thc phm v nhn mnh nhng ti nghin cu cn ch trng trong tng lai. INTRODUCTION Ng c thc phm l vn nghim trng v gy tn km cho y t cng ng, l mi quan tm trn ton th gii, tr thnh nguyn nhn chnh ca bnh tt. Mc d vi cng ngh k thut hin i, thc tin sn xut tt, qun l cht lng v v sinh v khi nim an ton cng nh nh gi s ri ro v h thng qun l an ton thc phm (HACCP), nhng s lng ca cc ca ng c thc phm vn gia tng trong thp k qua. cc nc lin minh Chu u (EU), nguyn nhn ng c thc phm ph bin nht l do vi khun, c th l Campylobacter, Salmonella, Listeria v virus. Chng nh hng n hn 380,000 ngi dn Chu u mi nm (EFSA, 2009). Ton cu ha th trng thc phm, gii thiu cc thc phm mi l, quy trnh sn xut mi, nhu cu t phi ch bin ngy cng gia tng, thc phm sng v sn phm thc n sn c th i hi mt dy chuyn thc phm di hn, phc tp hn, iu ny lm tng nguy c nhim vi sinh. Theo , cng ngh bo qun thc phm mi l v c b sung tun theo nhng yu cu t farm to fork (t trang tri n sn phm) vn ang c tm kim. Mt trong cc cng ngh bo qun thc phm, c bit ch n s bo qun bng phng php sinh hc nng cao cht lng sn phm v thi hn s dng, gim thiu cc tc ng n gi tr dinh dng v tnh cht cm quan ca cc sn phm d b h hng. Bo qun bng phng php sinh hc hp l khai thc c cc tim nng khng khun ca vi sinh vt t nhin trong thc phm v/hoc cc cht chuyn ha ca chng gip cho vic s dng an ton trong mt thi gian di (nng cao thi gian s dng). Bacteriocin, bacteriophage v nhng enzyme do bacteriophage m ha c xp vo nhm cht bo qun ny. Bi im bo ny s tm tt kin thc c bn v ng dng hin ti ca cc khng sinh t nhin theo dy chuyn thc phm. Da trn state-of-theart, cc xu hng trong tng lai v lnh vc nghin cu cn c quan tm v cp nhiu hn. A. BACTERIOCIN 1. Cu to v phng php hot ng ca Bacteriocin Cu to:

Bacteriocins c bn cht l peptide hay l cc protein c hot tnh khng khun c tng hp t vi khun. Chng c m t u tin Escherichia coli (colicins). Hu ht cc colicins l cc protein tng i ln (ln n 80 kDa), chng c th git cht vi khun lin quan khi kt hp vi mng t bo bn trong hoc cc bo tng mc tiu khc (Cascales v ctv., 2007). Ngy nay, thut ng bacteriocin ch yu c s dng m t cc cation peptide nh, bn nhit c tng hp bi vi khun Gram dng, c th l vi khun lactic (LAB), vi khun ny c s c ch kh rng (ph c ch rng) (Cotter, Hill, & Ross, 2005). T khi sn phm ln men truyn thng s dng vi khun LAB ln men th ngi ta ch trng n cng dng bo qun thc phm bng phng php sinh hc ca bacteriocin do vi khun LAB sinh ra. Bacteriocin l mt nhm khng ng nht v cu trc tin thn, thnh phn v tnh cht ha l. Vic phn loi bao qut bacteriocin vn l mt vn cn bn lun. Mt h thng c xut gn y bi Heng v Tagg (2006) c suy ra t cc h thng phn loi trc s a vo bn m t bn cht ca colicins. Theo , bacteriocin c chia thnh bn lp chnh : Lp 1 hay l Lanbiotic l nhng peptide nh (<5 kDa), bn nhit v tc ng ln cu trc mng. Mt thnh vin ni ting ca nhm ny l nisin (Parada v ctv, 2007). Bao gm post-translationally peptide c th bin i c trng bi cc thioether c bit, vi nn tng l ni phn t vng lanthionine v beta-methyllanthionine (Xie & van der Donk, 2004). o Phn lp Ia Phn lp Ia gm cc peptide dng thun di, linh hot v tch in dng, chng hot ng bng cch hnh thnh cc l trong mng t bo cht ca cc loi vi khun nhm n (Ouwehand v Vesterlund, 2004) o Phn lp Ib Phn lp Ib gm cc peptide c dng hnh cu, cu trc khng linh ng, tch in m hoc khng tch in. Chng th hin hot ng bng cch gy nhiu vi cc phn t enzyme thit yu ca vi khun nhy cm (Parada v ctv, 2007).

Lp II Cn c gi l lp Non-Lanbiotic, bao gm cc bacteriocin c trng lng phn t nh hn 10 kDa, bn nhit v khng cha lanthionine khng bin i v l lp ln nht trong s cc bacteriocins gram dng. Ni chung, chng l cc cation peptide ngn vi im ng in cao. S lin quan c bit cho s bo qun thc phm bng phng php sinh hc l c hot tnh khng Listeria tim nng th hin bi cc bacteriocins c cu trc ging nh pediocin (hng IIa). o Lp IIa Lp IIa l lp ln nht, gm cc peptide hot ng chng Listeria, i din c trng cho nhm ny l pediocin PA-1 v sakacin P. Cc bacteriocin nhm ny ha hn cho nhiu ng dng cng nghip nh vo hot ng khng Listeria mnh ca chng. Thm ch chng cn l cc tc nhn khng Listeria c ch nhiu hn l bacteriocin lp I (nisin) v chng khng c ph c ch rng v v vy, chng khng tiu dit cc ging khi ng (Ouwehand v Vesterlund, 2004). o Lp IIb

Lp IIb hnh thnh bi mt phc hp ca 2 peptide ring bit, nhng peptide ny t hoc khng hot ng. Cc bacteriocin c trng cho nhm ny l lactococcin G, plantaricin EF v plantaricin JK (Parada v ctv, 2007). o Lp IIc Lp IIc l nhng peptide nh, bn nhit, gm nhng bacteriocin khng ng nht nn phng thc hot ng ca chng cng khc nhau. Trong phn lp ny ch tm thy cc bacteriocin nh divergicin A v acidocin B (Parada v ctv, 2007).

Lp III ny bao gm phn ln cc protein khng bn nhit c tim nng khim tn trong bo qun thc phm bng phng php sinh hc. Ngoi tr colicin V v microcin th cc bacteriocin Gram m u nm trong lp ny.

Cui cng, cc peptide dng vng do cc peptide lin kt gia C - v N tn cng tp trung ti lp IV. Chng hn, cc bacteriocin m c tnh da trn cc hot ng phi hp ca hai peptide c lp th c tm thy trong c hai lp I v II. Hu ht bacteriocins LAB c p dng trong bo qun thc phm bng phng php sinh hc thuc lp Ia, II v IV (bng 1).

Cng nh ribosomally tng hp peptide, bacteriocin c m ha bi mt plasmid hoc nhim sc th to cu trc gen m thng c nhm vi gen m ha cho cc protein min dch v vn chuyn. Mt v d c bit l gen ch nh cho vic bin i enzyme v gen iu ha c th cng hin din. Nhng vi khun lactic ny to ra cc protein min dch c hiu t bo v, chng li tc ng khng khun ca bacteriocin do chng tit ra. Phng thc hot ng ca bacteriocin Bacteriocin do vi khun LAB tng hp c nghin cu rng ri, nghin cu tin phong thc hin ch yu vi cc nisin- bacteriocin gram dng c m t u tin. Da trn bn cht cation v tnh k nc, hu ht cc peptide hot ng nh mng t bo thm. S hnh thnh l chn lng khin ton b hoc mt phn proton ng lc b hao mn, cui cng lm cht t bo. S hnh thnh l chn lng bacteriocin dng nh l ch trung gian. Nisin v cc lantibiotic khc s dng vch t bo tin thn ca lipid II nh mt phn t bm (Breukink v ctv., 1999). Bng cch , hai ch hot ng gm s c ch sinh tng hp vch t bo v hnh thnh l chn lng, c kt hp trong mt phn t c hot tnh khng sinh vt (Wiedemann v ctv., 2001). Cch thc ny cng c s dng bi cc lantibiotic v bacteriocin khng to l chn lng nh non- lantibiotic Lcn972 (Martnez v ctv., 2008). Gn y, mt s bacteriocin lp II c nghin cu s dng thnh phn lin kt mng ca h thng mannosephosphotransferase nh l th th c th trong cc t bo mc tiu. (Diep, Skaugen, Salehian, PeterCartwright, & Nes, 2007) Nhiu bacteriocin LAB c hot tnh chng li vic to vi sinh vt v cc vi khun gram dng gy hng trong thc phm, k c cc vi khun khng khng sinh. Vi khun gram m c kh nng khng c nh vai tr bo v ca mng t bo bn ngoi. Tuy nhin, mt s c th hot ng khi kt hp vi tc cht lm bt n mng t bo( nh EDTA). 2. Cc ng dng hin ti ca Bacteriocin trong thc phm Vai tr ca LAB l sinh ra bacteriocin trong thc phm v thc phm ln men truyn thng, l nhn t chnh trong bo qun thc phm bng phng php sinh hc. LAB v bacteriocin thu ht s ch trong mt thi gian di. Ph c ch, dit khun, kh nng tc ng, ph thuc cc iu kin: pH, NaCl, nhit ; nhng n khng hot tnh tc ng ln cc t bo eukaryote nn c th s dng n nh cht bo qun sinh hc. Thi gian u nisin c dng trong 50 giy c ch s pht trin ca Clostridium tyrobutyricum trong phomat, c nhiu bo co v nhiu ng dng ca LAB bacteriocin, hu ht trong ch bin thc phm. Mt s v d v ng dng ca bacteriocin trong sn xut thc phm c tm thy u tin trong lnh vc th y, nng nghip v nui trng thu sn. Nisin v Lacticin 3147 c kt hp cht ch vo trong sn phm sa thng mi chng li bnh vim v. Bacteriocin c xut nh mt s thay th khng sinh v thnh cng trong vic lm gim s ly lan ca cc tc nhn gy bnh trong h tiu ho ca ng vt (Calo-Mata et al., 2008; Diez-Gonzalez, 2007; Line et al.,2008).Trong nui trng thu sn, hu ht cc tc nhn gy bnh l gram m v bacteriocin colicin-like l bin php tt nht phng bnh bng bin php sinh hc. Mt s vi

khun trn cy trng c kh nng tng hp Bacteriocin hn ch s u c ca cc tc nhn gy bnh thc vt (Holtsmark, Eijsink, and Brurberg,2008). ng dng ca bacteriocin l lnh vc ln, nghin cu s c ch cc tc nhn gy bnh v cc vi khun gy h hng thc phm trong sut qu trnh ch bin (Hnh 1). Bacteriocin c kho st trit l Lantibiotic nisin v Lacticin 3147, mt s lp IIa ca bacteriocin pediocinlike. Trong s cc peptide vng, Enterocin AS-48 c chng minh rt hiu qu chng li s gy h hng vi phm vi rng ca cc tc nhn gy bnh trong mt s thc phm bao gm c sa, tht v sn phm rau c. V c bn bacteriocin c ng dng trong 3 dng khc nhau: 1.Trong sn xut ti ch v bo v m nui cy. 2. Nh mt thnh phn (mt ging vi khun c dng ln men). 3.Nh mt cht ph gia trong khu tinh sch hoc bn tinh sch. Trong sn xut in situ, bacteriocin th ph hp v mt cng ngh sn xut v d sinh li nhun. Staphylococcus aureus trong phomat ng t acid v C. tyrobutyricum trong phomat bn cng b c ch bi nisin trong sn xut sa (Rilla, Martinez, Delgado, Rodriguez, 2003; Rilla, Martinez Rodriguez, 2004). C th bo v m nui cy vi khun, n khng gp phn vo tnh cm quan ca thc phm m ch yu c ng dng nng cao cht lng v sinh ca tht cha qua x l v sn phm c (Devlieghere, Vermeiren, and Debevere, 2004). Vic s dng bacteriocin nh mt thnh phn hay cht ph gia i hi phi c chin lc mi sn xut quy m ln ph hp vi chi ph thp. Mt v d, Lacticin 3147 v Enterocin AS-47 c sn xut trong sa gy nh l mt thnh phn sa (Ananou et al., 2008; Morgan, Galvin, Kelly, Ross, and Hill, 1999). Vic s dng whey nh mt c cht l mt s la chn hp dn bi v n cng gp phn ti s dng sn phm ph ca ngnh cng nghip sa. Bn cnh bo qun thc phm, Bacteriocin cn y nhanh s chn phomat bng cch thc y gii phng enzyme trong t bo vo khi phomat v sau s bay hi lm gia tng nng cc hp cht lm phomat chn v tng tnh cm quan (Martinez-Cuesta, Requena and Pelaez, 2006). Bacteriocin to ra cng gi li h vi sinh vt t nhin v m bo cc sn phm ln men ng nht (Ryan, Ross, and Hill, 2001). Thc phm c nh du da trn protein min dich Bacteriocin c kh nng thay th k thut gen chon lc cc khng sinh ca thc phm lin quan n Bacteriocin.

(theo th t t trn xung: bacteriocin, bacteriophage, endolysin) B. BACTERIOPHAGE


1. Th thc khun v chu k khng khun ca chng

Th thc khn hoc thc khun l cc vi sinh vt kh ph bin trn th gii (10 31 ht) v lan truyn rng ri trong cc loi thc phm c ngun gc khc nhau (Brussow & Kutter, 2005). Th thc khun l virus ly nhim c bit v nhn ln trong vi khun. V vy, chng v hi cho con ngi, ng vt v thc vt. Cc thc khun phn thnh 13 h da trn cc hnh dng, kch thc, loi axt nucleic v s hin din/vng mt ca mng hoc lipid trong cu trc. Hu ht chng thuc v trnh t Caudovirales (5360 hoc 5568 bo co n nay) vi mt u l icosahedral (cu trc a din) bc b gene, mt ui v mt DNA si i (hnh 2a). Theo cc tnh nng hnh thi ca ui, chng c phn chia thnh ba h: Myoviridae (ui co c), Siphoviridae (ui di khng co), v Podoviridae (ui rt ngn). Phn cn li ca thc khun l cc khi, cc si, hoc thc khun a kiu hnh vi xon i DNA, DNA xon n, RNA xon i, hoc RNA xon n (Ackermann, 2007).

Ty thuc vo iu kin sng, th thc khun c chia ra thnh thc khun c hi v n ha (hnh 2b). Th thc khun gy c tun theo chu k sinh tan (lytic cycle) theo chng nhn ln trong t bo vi khun gii phng cc thc khun con. Ngc li, cc th thc khun n ha c th i vo chu k tim tan bng cch chn DNA ca n vo nhim sc th ca vi khun (tin thc khun) sao chp nh mt phn b gen ca vt ch cho n khi n c tc ng nhp cc chu k sinh tan. Lu , vi khun lysogenic tr nn min dch chng li s nhim khun ln 2 (bi nhim) vi cng mt th thc khun ging nhau hoc tng t nhau. Chu k sinh tan c chia thnh nhiu giai on (hnh 2b). Trc tin, phage bm vo t bo ch cc im nhn, phn trung gian ca ui lin kt protein (protein ny nhn bit th th c trng ca vi khun). Sau , th thc khun s tim axit nucleic vo t bo ch v n c sao chp nh RNA polymerase ca cc t bo ch. Nh b my di truyn ca t bo ch, b gen ca th thc khun c nhn ln nhiu bn sao v tng hp protein mi sn xut cc protein cu trc nn th thc khun, lp rp vo virion mi, v ly gii protein , cui cng, vi khun ch s b tan. Bc ly gii cui cng l chnh xc, ni c tr cc hot ng khng khun ca th thc khun. Trong thc t, th thc khun c s dng rng ri Lin X c iu tr nhim trng ca con ngi. Kt qu ca h chc chn ch ra rng th thc khun l ph hp vi iu tr lm sng hay d phng bnh truyn nhim gy ra vi khun Gram dng v Gram m (Hanlon, nm 2007; Sulakvelidze & Kutter, 2005) 2. Cc ng dng hin ti ca thc phm da trn vi khun Khi nim v cuc chin chng tc nhn gy bnh trong thc phm bng cch s dng th thc khun ch xut hin gn y, nhng mt vi ng dng dc theo chui thc n c tip cn (bng 2) v mt s cng ty bt u u t vo cng ngh th thc khun (Garc a, Mart nez, Obeso, & Rodr guez, 2008)

Hnh 2:Cu trc ca mt vi khun c ui in hnh (a) v cc bc trong chu k ly gii vi khun v chu k tim tan (b). Cc th thc khun n ha c th thc hin theo cc chu k lysogenic bng cch tch hp vo h gen ca vt ch. Sau khi nhim trng, hoc prophage kch hot.

Th thc khun rt thch hp i) ngn nga hoc gim bt s to dng v bnh trong chn nui ( liu php th thc khun) ii) lm sch xc v cc sn phm th khc, chng hn nh hoa qu ti v rau qu, v kh trng thit b v lin h vi b mt (phage v sinh sinh hc), v iii) tng thi hn s dng, sn xut loi thc phm nh cht bo qun t nhin (phage kim sot sinh hc ) Th thc khun c p dng gim mm bnh chuyn trong chn nui v cng sau khi git m hay vt sa. Mt s nghin cu c thc hin cha tr bnh g vi th khng khun chng li Salmonella (Fiorentin, Vieira, & Barioni, 2005) v Campylobacter (Atterbury v ctv., 2005); cha tr cho ng vt nhai li vi th khng khun nhm mc tiu chng li E. coli gy bnh (Raya v ctv., 2006). Sau khi s dng th thc khun th vi khun gim ng k, c bit l s dng trc khi git m. Th thc khun cng tc ng trn sn phm ct ti (Leverentz v ctv., 2003). Cc th thc khun khc c dng chng li cc bnh thc vt do vi khun, v d nh AgriPhage thng phm (Intralytix) chng li bnh m c chua v tiu. Trong cng mt dng, th khng khun da trn biosanitation c xut

gim s hnh thnh mng sinh hoc (Azeredo & Sutherland, 2008) hoc xa b hoc gim thiu S. aureus mi hoc da trong x l thc phm (Mann, 2008). Bng chng thc nghim cc hot tnh khng khun ca th thc khun trong ch bin thc phm v lu tr vn khan him nhng kt qu ng khch l. Cc c trng chnh ca th thc khun, i khi b gii hn n mt vi chng, t ra mt gnh nng cho vic s dng rng ri th thc khun nh l cht bo qun sinh hc. Vic kt hp th thc khun vo sa nhim Salmonella sn xut ph mai dy gim kh nng sng ca t bo sau khi lu tr (Modi, Hirvi, Hill, & Grifths, 2001). Tng t, S. aureus tng trng trong sa v trong thi gian sn xut curd (sa ng t) b c ch bng th thc khun (Garc a, Madera, Mart nez, & Rodr guez, nm 2007; Garc a, Madera, Mart nez, Rodr guez & Sua rez, 2009) v c ch qu trnh chn, v lu tr ca cc axit coagulated (ng t) v phomt bn cng (cha c cng b kt qu ca mnh). ng dng trn b mt ca phage P100 gy c dit tr hon ton Listeria monocytogenes, ty thuc vo liu lng v cch x l b mt pho mt chn (Carlton, Noordman, Biswas, de Meester, & Loessner, 2005). V d khc ca phage trong bo qun sinh hc l s c ch Enterobacter sakazakii trong hon nguyn sa bt cho tr s sinh (Kim, Klumpp, & Loessner, 2007) v Salmonella typhimurium trn xc xch g mng ca c (frankfurters) (Whichard, Sriranganathan, & Pierson, 2003)). Gn y,hn hp hai th thc khun khng L. monocytogenes, Listex (EBI Food Safety, www.ebifoodsafety.com) v LMP 102 (Intralytics, www.intralytics.com) c Cc Qun l Dc v qun l Thc phm (FDA) ph duyt trn tht. Nm 2007, OmniLytics Inc (www.omnilytics.com) nhn c FDA chp thun cho th thc khun khng-E. coli v khng -Salmonella x l cc ng vt sng trc khi git m. Mt s ng gp ca th thc khun trong an ton thc phm l s dng trong vic pht hin tc nhn gy bnh truyn qua thc phm. Th thc khun t lu c s dng phn loi vi khun v mt s phng php da trn th thc khun c pht trin pht hin cc vi khun trong thc phm (Hagens & Loessner, 2007). Nhng phng php ny v c bn khai thc cc c trng th thc khun.

C. ENDOLYSIN: 1. Cu to v phng php hot ng: Bacteriophage c hai cch gii phng nhng virion mi t t bo vi khun b nhim. bacteriophage dng si y vt cht di truyn ca n vo t bo vi khun m khng lm cht t bo ch (lysogenic cycle). Nhng ngc li, Bacteriophage khng c dng si xm nhp vo t bo tit enzyme ph hy mng (lytic cycle). Nhng on RNA, DNA nh m ha nhng protein cn tr enzyme chu trch nhim sinh tng hp. phn ln DNA phage, endolysin (cng l yu t ph hy t bo c gii hn) c sn xut trong sut giai on truyn t gene thuc pha tr trong chu trnh lytic v chu trch nhim ca enzyme chia ct lp petidoglycan. Endolysin

cng c kh nng lm tan r lp peptidoglycan cu vi khun G(+) khi n tc ng bn ngoi t bo vi khun, bng cch , n tc ng nh tc nhn dit khun. Phn ln Endolysin thiu tn hiu kch thch bi tit v s xm nhp vo lp peptidoglycan ca chng vo t bo th ph thuc vo nhng protein k nc, holins gii hn (term holins), cht cho php phn t endolysin vt qua mng t bo cht v vo c mng t bo. Mt s endolysin khc th c nhng tn hiu peptide c kh nng xm nhp vo t bo ch. Ty thuc vo enzyme c trng, endolysin c chia thnh 5 loi chnh: N-acetylmuramidases (lysozymes) Endo-b-N-acetylglucosaminidases
Lytic transglycosylases, chia i ton b khung ng ca peptidoglycan Endopeptidase, chia i peptide.

N-acetylmuramoyl-L-alanine amidase, ct lin kt amide gia 2 na. ng ch l muramidase v amidase thy phn hu ht cc lin kt bo tn (conserved bond) trong peptidoglycan dng nh ph bin nht. Peptidoglycan b h hi dn n t bo b ph v trong mi trng nhc trng. Vi endolysin cha trnh t C-terminus ging vi vi loi peptide (tch in +) chng khun bng cch ph mng t bo vi khun. Endolysin Gram(+) th hin cu trc phn t gm c t nht l 2 vng chc nng r rng. Vng N-terminal cha hot tnh xc tc, phn ln l hot tnh muralytic nhng vi c hai chc nng cng c miu t. Ti u C-terminus, mt vng ni vch t bo (CBD- cell wall binding domain) a ra vi mc c trng i vi enzyme. Bn ch , CDBs duy tr s kt dnh cht nn ca endolysin mt khi t bo ch b ly gii. Bng cch ny, endolysin khng c gii phng t do vo mi trng. CDBs th khng thng thy t endolysin ca phage Gram(-). Hu ht endolysin th hin ph hot tnh lytic hp thng c s lng loi vi khun ch th ca phase kh hn ch, ngoi tr mt vi ging c bit. Mt ngoi l l yu t tiu hy (lysin) phase cu khun khng nhng tiu gii cu khun rut m cn c Streptococus pyogenes, streptococci nhm B v S. aureus, v th n l lysin c hot tnh rng nht c bit cho n nay. Nhng ng dng trong cng ngh thc phm ca endolysin: Hu ht cc cng trnh chng minh vai tr ca endolysin nh l mt cht dit khun mnh m u tp trung vo phng nga v iu tr mu ng vt nhim khun. Nghin cu trong lnh vc bo qun thc phm vn cn nhng bc khi u. Tuy nhin, s lng endolysin hot ng chng li mm bnh xm nhp thc phm v ng vt c phn lp v m t ngy cng tng v thy c kh nng ng dng trong tng lai. S cp ng gi cho n by gi l cha c s khng endolysin c bo co d n c c cho tip xc vi iu kin mi

trng lp i lp li hay b kch thch t bin. Mc d hi sm hon ton tin chc, nhng khi khng b khng li th (endolysin) thc s l mt li th so vi cc yu t khng khun khc. Mt vi bo co c pht biu gn y v tim nng khng khun ti ch (in situ) ca endolysin trong chui thc phm. nhng bc sn xut u tin, s bo v chng li tc nhn gy bnh trn thc vt Erwinia amylovora c chng minh trong vic chuyn gene T4 lysozyme khoai ty hoc bng ng dng b mt ca endolysin phiEa1h ti t hp trn tri l. Vic chuyn gen biu hin hot tnh endolysin b cng c th hn ch bnh vim v v gim nhim S. aureus trong sa. Nh l mt thc o phng bnh (as a prophylactic measurement), endolysin PlyC bay hi gp phn tiu dit hoc gim Streptococcus equi ca nhiu vt liu khc nhau thm ch n c mt trong cht ty khng ion ha (non ionic detergent), nc cng, hoc vt liu hu c. Tng t, mt endolysin ca t cu khun c th loi b lp mng sinh hc S.aureus. Trong ch bin thc phm, kim sot cc mi nguy sinh hc bng endolysin gn nh l bc c bn trong sn xut. Pioonering tin hnh th nghim vi phage endolysin LysH5 ca t cu. Endolysin tinh khit dit S.aureus trong sa thanh trng, mc d tng s cao hn so vi kt qu in vitro cn thit. LAB ti t hp c th tit ra endolysin Listeria nhng hot tnh trong sa ca chng hoc trong cc thc phm khc th cha c nh gi. Mt vai tr ca endolysin trong bo qun thc phm l nh CBDs (vng ni vch t bo). Nhng vng nhn bit ny thng pht trin nhanh chng, d dng nh danh v pht hin. CBDs ti t hp c c nh trong cc ht Magnetic v c th c thu hi hn 90% ca L.monocytogenes trong mu thc phm.
D. CH CHO TNG LAI:

Mc d vi nhng kin thc v cht khng khun (bacteriocin), th thc khun (bacteriophage) v s quan tm ngy cng tng dnh cho ni tiu t (endolysin), vn cn nhiu iu c bn v vn ng dng lin quan n khai thc y tim lc khng khun ca chng trong an ton thc phm cn tm hiu thm. ( Bng 3 ).

ti nghin cu trn hot cht khng khun, th thc khun v endolysins c hng n trong tng lai. ti Nghin cu c bn C ch khng Bacteriocin Vn c th Phage/endolysin

Truyn ca min dch; khngHot ho thc khun (lysogeny), cho; th quan hot chtc s phn t ca vt ch tnh khng khun (receptor) c hiu v tn s t bin

Cht khng khun K thut protein mi Tnh an ton

Protein khng r phage vi hot tnh khng siu vi /vng dao ng.

nh hng trong phc hpVai tr ca phage trong DNA trao sinh thi (GT, thc phmi v vt ch c lc ; c tnh. ln men) ; c tnh. Bacteriocin/bacteriophage/endolysin nh hng ca khung thc n v ch bin thc phm ( to m hnh ); S dng cng ngh ro chn; Dit mng sinh hc v h thng v sinh sinh hc. S xil, sn xut hu c.

Nghin cu ng dng Ch bin thc phm Lnh vc cha c khai thc

GT, ng d dy - rut.

Nhu cu c bit trong nghin cu c bn c th l tp hp li trong ba lnh vc chnh : i) C ch khng ii) Mi hoc ci tin tnh khng khun iii) Mi quan tm v vn an ton khi s dng cht bo qun sinh hc ny. S khng l mi quan tm chnh trong ngin cu cht bo qun sinh hc mi. Trong iu kin th nghim, s m phng bacteriocin c th thc hin d dng.Tuy nhin, ngi ta cha bit nhiu v s min dch cht khng khun v kh nng chuyn gene. ng ch l, mc d nisin c s dng rng ri nh cht bo qun thc phm bng phng php sinh hc, nhng vn cha xy ra vn khng. Tuy vy, hu qu ca vic thch nghi bacteriocin cn phi c xem xt khi s dng kt hp, hin tng khng cho c th xy ra. S lng ln vt liu cng ngh ( omics ) s lm r cch t bo p li bacteriocin nh th no. Tnh khng cng nh hng n bacteriophage. Tuy nhin, sc khng phage c th gim kh nng sng hoc c lc ca vi khun v vic s dng hn hp thc khun th gim kh nng khng. Hn na, tn s bin i ca phage cao hn ng k so vi vi khun v s chn lc phage mi c th d dng thng c sc khng vi khun. Kh nng to phage phn hu vi khun (lysogeny) cng khin cho vi khun chng li s nhim ln hai, do cc phage chu nhit cn phi ngn nga.

K thut sinh hc phn t v kh nng di truyn a ra hng mi pht trin cht khng khun: bacteriocin, bacteriophage, endolysin. Bacteriocin v endolysin ph hp cho DNA thay i (DNA shuffling) v k thut protein c kh nng to ra bin th rt hiu qu vi ph hot tnh m rng. Th thc khun cng c th bin i gen tho mn yu cu m rng ph k ch. Ngoi ra, thc khun th c m ho bi cc protein hoc peptide khc c ch s sinh trng ca vi khun khi b nhim trng cng nh virion-lin kt enzym thy phn peptidoglycan, mt hp cht chu trch nhim v " s phn gii t bn ngoi " v enzym lin quan n s thoi bin ca polysaccarit b mt. Tt c chng c th l c xem nh khng khun tng lai. Nu xt v vic s dng bacteriocin, bacteriophage, endolysin nh l cht ph gia thc phm, iu quan trng l phi ch n hiu qu hp thu qua ng ming. Th vi ca dng to bacteriocin trong probiotic c vai tr nh sinh thi hc, hot ng phc hp ng d dy rut. Cng c phn t mi nghin cu vi sinh vt ng rut s rt hu ch. Nghin cu chi tit hn v cc cht c i vi t bo v s to min dch cng rt cn thit ( Jasniewski, Cailliez - Grimal, Chevalot, Milliere, & Revol - Junelles, 2009 ). Cho n nay, khng thy s m t nh hng ngc ca th thc khun ng rut-d dy ( Bruttin & Bru ssow, 2005 ). Tri li, khng c d liu c gi tr v endolysin, cho d khng c du hiu ca sc mn cm sau khi vo c th ni chung v mng nhy( Fischetti, 2008 ). Vn an ton khc lin quan n s dng bacteriophage l phi phn tch cn thn trc khi chn cht bo qun sinh hc. iu ny bao gm s hiu bit su v vai tr ca th thc khun gy c v trao i DNA. Th thc khun c th mang gien c hi v thm ch mt s c th thc y chuyn gene.( Novick & Cheng, 2009 ). Cn c theo thc t, s n lc ch yu ci tin hiu qu ca cht bo qun sinh hc trong khung thc n nh: cht c hot tnh khng khun ti ch (in situ). Thnh phn thc n, khi lng vi khun, v cng ngh c trnh by gy tr ngi ln vi hot tnh bacteriocin (reviewed by Ga lvez et al., 2007). N cng p dng cho th thc khun khi b nhim lin quan vi vt ch, gy cn tr trong mi trng bn rn hay rn nh l thc n. Nhiu bin s khc nh l t l s hp th, c ph, thi k tim tng, liu lng thc khun ban u, nng vi trng, v.v cng l c nu ra . Tt c tham s ti hn ny c th l m hnh mu (Cairns, Timms, Jansen, Connerton, &Payne, 2009). Tuy nhin, s hiu bit su sc v ng lc hc ca nhim trng thc khun th trong chui thc n khc nhau vn cn thiu st. Hin nay, ci thin cng ngh bo qun ch yu tp trung vo cng ngh tm ro. Hot cht khng khun bacteriocin c kt hp thnh cng vi cng ngh khc. Chng c kt hp cht ch vo mng bao gi v kt hp vi iu chnh kh(MAP). Bacteriocin cng gip to iu kin t khc nghit hn so vi phng php bo qun truyn thng ( ngha l dng t ho cht cht bo qun hoc x l nhit thp ) gip tit kim nng lng v chi ph. Chng cng h tr cng ngh bo qun mi nh l p sut thy tnh cao v xung in trng cho thy nh hng iu phi ( Ga ' al lvez et., 2007 ). Tri li, s dng bacteriophage v endolysin trong cng ngh tm ro ( hurdle technology) hu nh khng c tm hiu nhng n rt c trin vng. Bacteriophage v endolysin c kt hp thnh cng vi nisin v p sut thy tnh cao

ci tin hot tnh endolysin bng lm peptidoglycan ca Gram m d tip cn (Briers et al., 2008). Bacteriophage v endolysin c ngh nh l thuc kh trng trong mi trng thc phm, bao gm nh sn xut thc n, nhng chin lc phn phi phi c trin khai. Trong chui thc n, c nhiu lnh vc cha c khai thc trong c cht khng khun t nhin. Chng hn nh, ln men xi-l (silage) khi xng s dng bacteriocin. Hn na, hot cht khng khun t nhin ny ph hp vi sn xut thc phm nui trng t nhin, do khuyn khch ngnh cng nghip thc phm bo v mi trng. Hy vng tng lai gn c s phn t ca bacteriocin- bacteriophage- endolysin c hiu bit su v tt hn gip thc n an ton hn. Da trn nn tng tri thc tip cn c, chin lc v cht bo qun sinh hc mi cng nh ng dng cng ngh sinh hc khng khun t nhin c vch ra.

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