ORIGINAL ARTICLE. P. Ruas-Madiedo 1,2, M. Medrano 2, N. Salazar 1, C.G. de los Reyes-Gavilán 1, P.F. Pérez 2,3 and A.G. Abraham 2,4.

Transcription

1 Journl of Applied Miroiology ISSN ORIGINAL ARTICLE Exopolyshrides produed y Ltoillus nd Bifidoterium strins rogte in vitro the ytotoxi effet of teril toxins on eukryoti ells P. Rus-Mdiedo 1,2, M. Medrno 2, N. Slzr 1, C.G. de los Reyes-Gvilán 1, P.F. Pérez 2,3 nd A.G. Arhm 2,4 1 Deprtment of Miroiology nd Biohemistry of Diry Produts, Instituto de Produtos Láteos de Asturis, Consejo Superior de Investigiones Científis (IPLA-CSIC), Villviios, Asturis, Spin 2 Centro de Investigión y Desrrollo en Criotenologí de Alimentos, CCT L Plt-CONICET, L Plt, Argentin 3 Cátedr de Miroiologí, Fultd de Cienis Exts, L Plt, Argentin 4 Áre Bioquími y Control de Alimentos, Fultd de Cienis Exts, L Plt, Argentin Keywords Billus ereus, Bifidoterium, Co-2, erythroytes, exopolyshrides, Ltoillus, streptolysin-o. Correspondene Ptrii Rus-Mdiedo, Instituto de Produtos Láteos de Asturis, Consejo Superior de Investigiones Científis (IPLA-CSIC), Crreter de Infiesto s n, Villviios, Asturis, Spin. E-mil: rus-mdiedo@ipl.si.es : reeived 4 Mrh 2010, revised 9 July 2010 nd epted 11 August 2010 doi: /j x Astrt Aims: To evlute the pility of the exopolyshrides (EPS) produed y ltoilli nd ifidoteri from humn nd diry origin to ntgonize the ytotoxi effet of teril toxins. Methods nd Results: The ytotoxiity of Billus ereus extrellulr ftors on Co-2 olonoytes in the presene sene of the EPS ws determined y mesuring the integrity of the tissue monolyer nd the dmge to the ell memrne (extrellulr ltte dehydrogense tivity). Additionlly, the protetive effet of EPS ginst the hemolyti tivity of the streptolysin-o ws evluted on rit erythroytes. The EPS produed y Bifidoterium nimlis ssp. ltis nd, Bifidoterium longum nd Ltoillus rhmnosus GG were le to ountert the toxi effet of teril toxins on the eukryoti ells t 1 mg ml )1 EPS onentrtion. The EPS ws the most effetive in ounterting the effet of B. ereus toxins on olonoytes, even t lower doses (0Æ5 mgml )1 ), wheres EPS eliited the highest hemolysis redution on erythroytes. Conlusions: The prodution of EPS y ltoilli nd ifidoteri ould ntgonize the toxiity of teril pthogens, this effet eing EPS nd iologil mrker dependent. Signifine nd Impt of the Study: This work llows gining insight out the mehnisms tht proiotis ould exert to improve the host helth. Introdution Exopolyshrides (EPS) re extrellulr iopolymers tht re produed y mny lti id teri (LAB) nd other teri present in fermented foods nd humn environments, suh s propioniteri nd ifidoteri (Rus-Mdiedo et l. 2009). In ft, the EPS-produing strins re used in the diry industry euse of the ility of their polymers to t s iologil stilizers, visosifiers, emulsifiers nd gelling gents (Arhm et l. 2009; Behre et l. 2009). In ddition, in reent yers, severl helth enefits hve een ttriuted to EPS from LAB (Rus-Mdiedo et l. 2008). The eologil role of EPS for the produing teri is not ompletely understood. It hs een reported tht they ould e involved in ell reognition, dhesion to surfes nd iofilm formtion, whih would enhne the oloniztion of different eosystems (Rus-Mdiedo et l. 2008). In this regrd, it hs een proposed tht EPS produed y intestinl ltoilli nd ifidoteri ould e involved in the dherene to intestinl muus nd lso in the intertion with enteropthogens (Rus-Mdiedo Journl of Applied Miroiology 109, ª 2010 The Soiety for Applied Miroiology 2079

2 Toxin ntgonism of teril exopolyshrides P. Rus-Mdiedo et l. et l. 2006). Also, EPS produed y ifidoteri re le to modulte the intestinl miroiot y ting s fermentle sustrtes (Slzr et l. 2008, 2009). On the other hnd, teril EPS ould ountert the tivity of teriophges (Durlu-Özky et l. 2007) nd of toxi ompounds (Looijesteijn et l. 2001; Kim et l. 2006). The synthesis of EPS in the gut eosystem ould onfer seletive dvntge for the survivl nd oloniztion of this nihe y the produing teri. Indeed, EPS ould improve the survivl throughout the gstrointestinl trt of orlly delivered teri given tht ile is le to indue the synthesis of these polymers in Bifidoterium nimlis (Rus-Mdiedo et l. 2009). In the gut environment, it hs lso een reported tht kefirn, the EPS produed y LAB in the fermented milk kefir, is le to ntgonize the iologil tivity of Billus ereus nd lso their extrellulr ftors on Co-2 olonoytes (Medrno et l. 2008, 2009). However, there is no evidene s to whether EPS produed y ltoilli nd ifidoteri from intestinl origin re le to exert similr protetive effets ginst the tivity of teril toxins. The im of this work ws to evlute the effet of the EPS produed y the strins Ltoillus rhmnosus GG, Bifidoterium longum, Bifidoterium nimlis ssp. ltis nd on the iologil tivity triggered y teril toxins (extrellulr ftors of B. ereus nd streptolysin-o (SLO) from Streptoous pyogenes) on different eukryoti ell models (Co-2 ells nd rit erythroytes). Mterils nd methods Bteril strins nd ulture onditions The teril strins used in this study re shown in Tle 1. Stok ultures of B. ereus B10502 were stored t )80 C in BHI (Brin Hert Infusion; Biokr Dignostis, Beuvis, Frne) using glyerol 1% (v v) s ryo-protetive gent. The strin ws re-tivted in BHI supplemented with gluose 0Æ1% (w v) for 16 h t 32 C inn oritl shker, nd these ultures were used to inoulte (4% v v) 5 ml of fresh BHI-gluose tht ws inuted under the sme onditions. Cell-free superntnts were otined fter entrifugtion (900 g, 10 min) nd filtrtion (0Æ45 lm). Seril dilutions of superntnts were mde in phosphte-uffered sline (PBS: KH 2 PO 4 0Æ144 g l )1,N 2 HPO 4 0Æ795 g l )1 nd NCl 9 g l )1 ). The four EPS-produing ltoilli nd ifidoteri strins (Tle 1) were mintined t )80 C in MRSC roth [MRS (Biokr) + 0Æ25% (w v) l-ysteine (Sigm Chemil Co., St. Louis, MO, USA)] with 20% glyerol. For the isoltion of EPS, frozen stoks were grown overnight t 37 C under neroi tmosphere (10% H 2, 10% CO 2, nd 80% N 2 ) in M500 hmer (Down Whitley Sientifi, West Yorkshire, UK). Cultures (200 ll) were used to inoulte gr-mrsc pltes using sterile glss eds. Pltes were inuted for 5 dys t 37 C in neroi onditions. Isoltion nd hrteriztion of the EPS For the isoltion of the EPS, ell iomss ws olleted from gr-mrsc pltes using 2 ml ultrpure wter per plte nd the resulting volume ws mixed with one volume of 2 mol l )1 NOH. The suspension ws gently stirred overnight t room temperture to promote polymer relese, nd fterwrds ells were removed y entrifugtion (8400 g, 30 min). The EPS frtion ws preipitted using two volumes of old-solute ethnol (4 C, 48 h), followed y entrifugtion, resuspension in ultrpure wter nd dilysis for 3 dys t 4 C with dily hnge of wter using dilysis tues (Sigm) of kd moleulr mss ut off. Finlly, the dilysed EPS were freeze-dried (Rus-Mdiedo et l. 2006). The protein ontent of the EPS frtions ws determined y the BCA protein ssy kit (Piere, Rokford, IL, USA). The molr mss (MM) nd rdius of gyrtion (Rg) of the EPS were mesured y size-exlusion hromtogrphy nd multi-ngle lser light sttering detetion (SEC-MALLS, Slzr et l. 2009). The monoshride omposition ws determined y GC MS (Slzr et l. 2009). The phosphte ontent of eh EPS ws diretly mesured in n tomi emission spetrosopy Perkin Elmer 5500 indutively oupled plsm (Perkin Elmer, Norwlk, CT, USA) t the Centro de Cienis Mediomientles (CCM-CSIC, Mdrid). Speies Strins Origin* Tle 1 Miro-orgnism used in this study Billus ereus B10502 Food poisoning outrek (Minnrd et l. 2004) Ltoillus rhmnosus GG (LMG18243) LMG Culture olletion (humn fees) Bifidoterium nimlis ssp. ltis Isolted from ommeril diry produt Bile-dpted strin from (IPLA olletion) Bifidoterium longum NIZO Culture olletion (infnt fees) *LMG: Belgin Co-ordinted Colletions of Miro-orgnisms (Gent, Belgium) nd NIZO Food Reserh Colletion (Ede, the Netherlnds) Journl of Applied Miroiology 109, ª 2010 The Soiety for Applied Miroiology

3 P. Rus-Mdiedo et l. Toxin ntgonism of teril exopolyshrides Eh EPS ws resuspended in PBS solution t 20 mg ml )1, nd severl dilutions rnging from 0Æ05 to 1mgml )1 were used for experiments. Culture of humn Co-2 ell line Co-2 ells were routinely grown in Duleo s modified Egle s medium (DMEM) ontining 25 mmol l )1 gluose (GIBCO BRL Life Tehnologies, Rokville, MD USA), supplemented with 15% (v v) het-intivted (30 min t 56 C) foetl lf serum (PAA Lortories GmH, Pshing, Austri), 1% (v v) nonessentil mino ids (PAA Lortories GmH), peniillin (12 UI ml )1 ), streptomyin (12 lg ml )1 ), gentmiin (50 lg ml )1 ) nd fungizone (1Æ25 lg ml )1 ). For mintenne purposes, the ell line ws pssed weekly, using 0Æ02% (w v) trypsin (Sigm) in PBS ontining 3 mmol l )1 EDTA. Cell suspensions ( ells per ml) were seeded in 6- or 24-well pltes (Greiner Bio One, Frikenhusen, Germny) nd inuted to form onfluentdifferentited monolyers for 14 ± 1 dys t 37 C in 5% CO 2 tmosphere. The ulture medium ws hnged every 2 dys, nd the experiments were performed with ells mong pssges 56 nd 60. Dethment of Co-2 ells The integrity of the Co-2 monolyers ws determined y mesuring the ell dethment s previously reported y Minnrd et l. (2001). Briefly, differentited ells grown in 24-well pltes were o-inuted with severl dilutions (from 1 2to1 64) of B. ereus B10502 superntnts t 37 C in5%co 2 tmosphere for 150 min in presene of different EPS onentrtions (0, 0Æ05, 0Æ5 nd 1mgml )1 ). After inution, Co-2 ells were wshed with PBS, fixed for 1 min with 2% (w v) formldehyde in PBS nd wshed gin. Stining ws performed with 500 ll of rystl violet solution [0Æ13% rystl violet, 5% ethnol nd 2% formldehyde (w v v) in PBS] y inuting for 20 min. Afterwrds, stin exess ws wshed with PBS, nd smples were treted for 1 h with 50% (v v) ethnol. Asorne of stined ells ws mesured t 590 nm in Metrol 330 spetrophotometer (Metrol, Buenos Aires, Argentin). The perentge of tthed ells ws lulted s the sorne of treted ells with respet to the sorne of untreted-ontrol ells (suspended in PBS nd inuted in the sene of B. ereus superntnt nd EPS). Ltte dehydrogense tivity in Co-2 ells Co-2 ell memrne integrity ws determined y mesuring the extrellulr ltte dehydrogense (LDH) tivity using the LDH-P unitest kit (Weiner L, Rosrio, Argentin), following the mnufturer s speifitions. Differentited Co-2 ells grown in six-well pltes were o-inuted with seril dilutions (1 16 to 1 32) of B. ereus B10502 ell-free superntnts in the presene (1 mg ml )1 ) nd sene of teril EPS t 37 C in 5% CO 2 tmosphere for 2 h. The extrellulr LDH tivity ws determined y mixing 50 ll of Co-2 superntnts with the enzyme sustrte nd inuting t 30 C. Asorne t 340 nm ws mesured ontinuously during 120 s in spetrophotometer Bekmn DU650 (Bekmn, Bre, CA, USA), nd LDH tivity ws lulted s the derese in sorne for 60 s. Hemolysis of erythroytes The puttive protetive effet of EPS ginst the toxi tivity of SLO from Strep. pyogenes upon erythroytes ws ssessed ording to Promdonkoy nd Ellr (2003). Commeril SLO (Sigm) ws diluted in PBS ontining 20 mmol l )1 l-ysteine, djusted to ph 7Æ4, filtered nd mixed with (2% v v) rit erythroytes suspended in PBS. Toxin units used in the experiments were round 300 UI enzyme per ml [one unit is defined s the mount of enzyme le to use 50% lysis of 50 ll of 2% (v v) red lood suspension in PBS, ph 7Æ4 t37 C in 30 min]. Erythroyte suspensions were o-inuted with SLO in the presene (1 mg ml )1 ) nd sene of the EPS t 37 C for 60 min. Two ontrol smples were used: 0% lysis (erythroytes in sene of SLO toxin) nd 100% lysis [erythroytes treted with 0Æ2% Triton X-100 (Sigm) in PBS]. Asorne derese in erythroyte suspensions ws mesured t 600 nm in Metrol 330 spetrophotometer t different smpling points for 60 min. The perentge of remining (not lysed) erythroytes in the smples ws lulted s follows: 100 (sorne of smple ) sorne of 100% lysis ontrol) (sorne of 0% lysis ontrol ) sorne of 100% lysis ontrol). Sttistil nlysis All experiments hve een rried out t lest in triplite. Sttistil nlysis ws performed using the spss-p 11.0 softwre (SPSS In., Chigo, IL, USA). Dt were sujeted to n one-wy nov, nd the lest signifint differene test ws used for the susequent omprison of mens. For the hemolysis dt nd within eh type of EPS smple, pired smples t-test ws performed to ompre the perentge of remining erythroytes fter 60 min of inution with respet to the initil vlues. Journl of Applied Miroiology 109, ª 2010 The Soiety for Applied Miroiology 2081

4 Toxin ntgonism of teril exopolyshrides P. Rus-Mdiedo et l. Results Physio-hemil hrteristis of the teril EPS Tle 2 shows the protein ontent, MM distriution nd hemil omposition of the EPS nlysed. The protein ontent of the EPS ws in generl lower thn 3%, inditing high-rohydrte ontent nd thus good degree of purity of the polymer. Ltoillus rhmnosus GG synthesized unique pek of 2Æ D. However, the EPS synthesized y Bifidoterium strins (, nd ) presented more thn one pek of different MM. The EPS produed y Bif. longum hd two polymer frtions, one of low (4Æ D) MM nd nother of high (4Æ D) MM. Bif. nimlis ssp. ltis nd re losely relted strins. A ileresistnt derivtive strin (dox) ws otined from the prentl y grdul dpttion to ile slts. This iledpted strin onverted spontneously to stle ropy phenotype y suessive suultivtion, resulting in the strin. The ropy phenotype of strin is sent in the prentl, nd these two strins produed polymers differing in MM nd pek distriution (Tle 2). Both strins shre two EPS frtions of low MM (round nd D), ut is le to dditionlly produe, in high mounts (56%), n EPS of high (3Æ D) MM, proly eing responsile for the ropy hrter. The EPS produed y these losely relted strins lso differed in their monoshride rtio omposition. The EPS frtion showed higher gluose nd gltose, nd lower rhmnose, rtio thn. Although EPS nd GG lso hve the sme monomers in their omposition, the rtio mong the four polymers ws different. It is worth noting tht EPS nd lso hve phosphte in their omposition, whih ws not deteted in nd GG. Finlly, vritions in the Rg of the highest MM pek of eh EPS were lso deteted with vlues of 13, 17, 47 nd 67 nm for EPS GG,, nd, respetively. These results show high vriility in the physio-hemil hrteristis of the teril EPS used in this study, whih ould ount for different iologil effets. Effet of teril EPS ginst Billus ereus ytotoxiity on Co-2 ells The protetive effet of the teril EPS ginst the toxi effet of B. ereus extrellulr ftors on Co-2 ells ws determined y testing the integrity of Co-2 ell monolyer. Figure 1 shows the Co-2 ells tht remined dhered to the surfe of the well pltes fter o-inution with seril dilutions (1 16 to 1 64) of B. ereus superntnts in the presene (1 mg ml )1 ) nd sene of EPS. Under these experimentl onditions, ll EPS were le to signifintly (P < 0Æ05) ntgonize the ell dethment triggered y B. ereus B10502 superntnts t the highest toxin onentrtion (dilution 1 16), the EPS eing le to disply the highest protetion. At B. ereus superntnt 1 32 nd 1 64 dilutions, the lower toxin onentrtion did not indue the dethment of the Co-2 monolyers in the ontrol (sene of EPS) smples, nd no differenes with respet to the presene of the EPS were deteted. Higher toxin onentrtions (dilutions 1 2 to 1 8, dt not shown) used the totl dethment of the ells, nd no protetion ws oserved with ny of the EPS ssyed. Tking into ount the previous results, the EPS with the highest (EPS ) nd one of the lowest () ntgonism pility ginst B. ereus superntnt toxiity on Co-2 ells were hosen to study puttive dose-response effet of these EPS (Fig. 2). For EPS, signifint (P <0Æ05) protetion effet ws deteted t onentrtions higher thn 0Æ05 mg ml )1, wheres for EPS ws deteted only t 1 mg ml )1. In ddition, t the highest EPS onentrtion tested (1 mg ml )1 ), the perentge of ells tht remined dhered ws signifintly higher (P <0Æ001) for EPS thn for EPS. Thus, the differentil EPS hrteristis my hve ount for this vriility in the protetive pility etween oth EPS. Tle 2 Physio-hemil hrteristis of the exopolyshrides (EPS) used in this study EPS Protein (%) Molr mss (D) distriution (%)* Monoshride rtio Pek 1 Pek 2 Pek 3 Gl Gl Rhm PO 4 3Æ1 ) 2Æ (44%) 5Æ (56%) 2Æ Æ6 3Æ (56%) 3Æ (21%) 4Æ (23%) 1 1 1Æ5 + 2Æ2 4Æ (47%) ) 4Æ (53%) ) GG 2Æ9 ) 2Æ (100%) ) ) *Perentge lulted with respet to the totl mount (lg) of peks mesured y size-exlusion hromtogrphy (SEC-MALLS). Glu, gluose; Gl, gltose; Rhm, rhmnose Journl of Applied Miroiology 109, ª 2010 The Soiety for Applied Miroiology

5 P. Rus-Mdiedo et l. Toxin ntgonism of teril exopolyshrides Toxin onentrtion Superntnt dilution D1/64 D1/32 D1/16 % ells dhered EPS GG GG GG Figure 1 Perentge of Co-2 ells dhered to the well plte fter tretment with severl dilutions (1 16, 1 32 nd 1 64) of Billus ereus B10502 superntnts in the sene (white rs) nd presene (1 mg ml )1 ) of the exopolyshrides isolted from the strins Bifidoterium nimlis ssp. ltis nd, Bifidoterium longum nd Ltoillus rhmnosus GG (grey rs). The perentge of ells tht remined tthed ws lulted s follows: 100 [590- nm sorne of treted smples sorne of ontrol ells (inuted in sene of B. ereus superntnt)]. Within eh superntnt dilution, the olumns with different letters re sttistilly different (P < 0Æ05) ording to the lest signifint differene men omprison test. % ells dhered *** The four EPS tested showed signifint differenes (P <0Æ05) with respet to the ontrol (sene EPS) smple t oth toxin onentrtions tested. The effet ws lso signifint (P <0Æ001) t low-toxin onentrtion (dilution 1 32). As similrly ourred when ell dethment ws nlysed, the EPS frtion showed the lowest protetive effet of the four EPS nlysed. Thus, this result suggests tht teril EPS exerted protetive effet on Co-2 ells y voiding the loss of memrne integrity nd or the relese of ytoplsmti ontent EPS onentrtion (mg ml 1 ) Figure 2 Perentge of Co-2 ells tht remined dhered to the well plte fter the tretment with the dilution 1 16 of Billus ereus B10502 superntnt in the presene of severl onentrtions of the exopolyshrides (EPS) isolted from the strins Bifidoterium nimlis ssp. ltis nd. Conentrtions: 0 (h), 0Æ05 ( ), 0Æ5 ( ) nd 1 ( )mgml )1. Within eh EPS, the olumns tht do not hve ommon supersript re sttistilly different (P < 0Æ05) ording to the lest signifint differene men omprison test. At the highest onentrtion (1 mg ml )1 ), n one-wy nov test ws performed to nlyse differenes etween oth polymers (***P < 0Æ001). To evlute Co-2 ell memrne integrity, the extrellulr relese of LDH tivity ( ytoplsmti enzyme) ws mesured in the milieu of Co-2 o-ultivted with B. ereus superntnt dilutions 1 16 nd 1 32 in the presene (1 mg ml )1 ) nd sene of teril EPS (Fig. 3). Effet of EPS ginst SLO toxin on erythroyte ells The results presented erlier prompted us to study the effet of these EPS on the iologil tivity of lrge-pore forming toxins. Rit erythroytes were employed to evlute the puttive protetion of teril EPS ginst the hemolyti tivity of the ommeril teril hemolysin SLO. Kinetis of hemolysis performed in the presene (1 mg ml )1 ) nd sene of the EPS under study re depited in Fig. 4. The remining erythroytes of ll smples signifintly diminished with respet to the initil vlues fter 60 min of erythroyte o-inution with the SLO toxin (pired t-test: P < 0Æ001). However, when the EPS were dded, the rte of hemolysis deresed nd signifint differenes were found t the end of the inution period with respet to the smple in sene of EPS (P <0Æ05). It is worth noting tht in the se of hemolysis, the EPS displyed the lowest protetion pility of the four EPS tested, wheres the EPS showed the highest. This represents n opposite tendeny Journl of Applied Miroiology 109, ª 2010 The Soiety for Applied Miroiology 2083

6 Toxin ntgonism of teril exopolyshrides P. Rus-Mdiedo et l. Toxin onentrtion Superntnt dilution D1/32 D1/16 LDH tivity 0 0 EPS GG GG d Figure 3 Extrellulr ltte dehydrogense (LDH) tivity of Co-2 ells fter o-ultivtion with Billus ereus B10502 superntnts (dilutions 1 16 nd 1 32) in the sene (white rs) nd presene (grey rs) of the exopolyshrides (1 mg ml )1 ) isolted from the strins Bifidoterium nimlis ssp. ltis nd, Bifidoterium longum nd Ltoillus rhmnosus GG. The LDH tivity ws lulted s the derese in sorne t 340 nm fter 60 s of retion. Within eh B. ereus superntnt dilution, the olumns tht hve not ommon supersript re sttistilly different (P <0Æ05) ording to the lest signifint differene men omprison test. % remining erythroytes with respet to tht oserved for the model Co-2 nd extrellulr toxins of B. ereus. Disussion Time (min) d d Figure 4 Lysis kinetis of rit erythroytes o-inuted with streptolysin-o toxin (300 UI) without exopolyshrides (EPS) ( ) or with 1mgml )1 of EPS (h), EPS (4), EPS GG (d) nd EPS (s). The perentge of remining erythroytes ws lulted s follows: 100 (600- nm sorne of smple ) sorne of 100% lysis ontrol) (sorne of 0% lysis ontrol ) sorne of 100% lysis ontrol). At the end of the retion period (60 min), the symols tht do not shre ommon letter re sttistilly different (P < 0Æ05) ording to the lest signifint differene men omprison test. The pility of teril EPS to ntgonize the memrne eukryoti ell dmge triggered y B. ereus extrellulr ftors nd y SLO from Strep. pyogenes, ws evluted in two omplementry ell models: olonoytelike ells (Co-2 ellulr line) nd rit erythroytes. These teril toxins produe different effets upon eukryoti ells. B. ereus n trigger in humns diverse symptoms relted to their dhesion to the intestinl epithelium nd invsion of enteroytes (Minnrd et l. 2004) nd or euse of the prodution of wide rnge of extrellulr ftors suh s ytolysins (ereolysin-o), phospholipses nd hemolysins, mong others (Alouf 2000; Minnrd et l. 2007; Stenfors-Arnesen et l. 2008). The SLO elongs to the lrge-pore forming fmily of toxins (Alouf 2000; Thelestm 2000), in whih is inluded the ereolysin-o from B. ereus. In this study, we hve shown tht the EPS produed y strins of ltoilli nd ifidoteri re le to ountert the toxi effet of teril toxins. However, the effet ws different depending on the EPS onsidered nd lso on the iologil (eukryoti ell) mrker studied. In the se of the B. ereus superntnts, the EPS IPLA- R1 nd showed the lowest protetion pility y preventing the Co-2 ell de-tthment nd or the LDH relese. A ommon feture of these two polymers is the prodution of high-mm EPS frtion (>10 6 D). The high-mm frtion is not present in the EPS synthesized y strin (prentl strin of ), whih exerted the highest protetion, suggesting tht the high- MM frtion is not neessry to exert the protetive effet ginst B. ereus toxins. Additionlly, the protetive effet of EPS ws deteted t lower doses (0Æ5 mgml )1 ) thn EPS, nd t the onentrtion of 1mgml )1, the EPS eliited signifintly higher ntgoni effet thn. The medium MM (2Æ D) EPS produed y GG strins hd iologil effet loser to EPS. However, the kefirn isolted from the kefir grins, whih is rnhed gluogltn with n MM higher thn 10 7 D (Piermrí et l. 2008), ws le to signifintly rogte the ytotoxi effet of 2084 Journl of Applied Miroiology 109, ª 2010 The Soiety for Applied Miroiology

7 P. Rus-Mdiedo et l. Toxin ntgonism of teril exopolyshrides B. ereus toxins t similr onentrtions (from 0Æ3 to 1mgml )1, Medrno et l. 2008) thn our EPS. Thus, the MM of polymer is not the only determining ftor involved in its protetive pility. Also the hemil omposition, type of union nd the sptil struture of the EPS ould e involved in the different iologil effet. With respet to the puttive mehnism ginst B. ereus toxins, the teril EPS ould t y loking the toxin reeptors in the eukryoti ell or y pturing the toxin, thus voiding its intertion with the reeptor. In oth ses, the EPS would void the initition of n intrellulr sde response tht ends with the deth, y nerosis or poptosis, of the eukryoti ell (Henderson et l. 1999). Regrding the ntgonism of teril EPS ginst the hemolyti tivity of SLO on erythroytes, our EPS were le to diminish the rte of hemolysis. However, in this se, the EPS showed the lower perentge of protetion nd the EPS the highest; this is just the opposite ehviour of tht deteted for the Co- 2 B. ereus toxins system. This finding suggested tht the mehnism involved in hemolysis protetion might e different to tht involved in ell dethment. One of the min mehnisms leding to hemolysis y hemolysins, suh s SLO (nd lso the ereolysin-o from B. ereus), is the oligomeriztion nd ssemly of toxin monomers into the ytoplsmi memrne of eukryoti ells, thus forming lrge pores (Plmer et l. 1998; Henderson et l. 1999). The EPS produed y our ifidoteri nd ltoilli might disply their ntgonism y hindering the ontt of the toxin with the erythroyte memrne; this will void the toxin-suunits oligomeriztion nd pore formtion, thus EPS ould exert osmoti protetion. In this wy, MM nd Rg re importnt struturl hrteristi of EPS to e onsidered. We oserved tht the lowest protetion on hemolysis ws otined with EPS with low MM nd Rg, this is the EPS. Thus, it seems tht polymers hving smll Rg nd low MM might e le to etter rogte the ytotoxi effet of B. ereus toxins, ut re less effetive for lrge-pore forming toxin suh s SLO. The physil prmeters nd hemil omposition of EPS re relted to the struture of the repeting units tht uild the polymers. In our se, with the exeption of EPS GG (Lndersjö et l. 2002), the repeting units of EPS, nd re still unknown. Thus, we do not hve enough dt to onlude tht the MM nd Rg re the unique prmeters diretly relted to the nti-toxin effet of our EPS nd to extend this onlusion to other teril EPS. However, it is well known tht the physio-hemil hrteristis of teril EPS re diretly orrelted with their tehnologil nd iologil funtionlity (Ruijssenrs et l. 2000; Vningelgem et l. 2004; Mozzi et l. 2009; Slzr et l. 2009). Therefore, it seems tht the physio-hemil properties of the EPS would lso determine the speifi intertions with eukryoti ell reeptors nd or with toxins. Conlusions The EPS synthesized y the humn nd diry origin strins Lt. rhmnosus GG, Bif. longum, Bif. nimlis ssp. ltis, nd its ile-dpted derivtive, re le to exert n in vitro ntgoni effet ginst the ytophti effet on Co-2 ells triggered y B. ereus extrellulr ftors nd ginst the hemolyti tivity of lrge-pore forming toxin, suh s SLO, on rit erythroytes. Differenes mong EPS depended on the iologil mrker studied nd ould e ttriuted to the vriility of the physio-hemil hrteristis of these iopolymers. Although the protetive mehnisms re still not fully understood, it is tempting to hypothesize tht the teril EPS ould t y loking reeptors on the eukryoti ell surfe or y ting s toxin-svenger gents. Nevertheless, the pility to rogte the tivity of teril toxins y EPS produed y ltoilli nd ifidoteri is nother mehnism involved in the ntgonism ginst virulene ftors of teril pthogens exerted y proioti strins. Aknowledgements This work ws finned y joint ollortion projet CSIC (Spin) CONICET (Argentin) of referene 2005AR0047 nd y FEDER funds (Europen Union) nd the Spnish Pln Nionl de I+D+I through the projets AGL nd AGL M. Medrno knowledges her fellowship to CONICET nd N. Slzr ws funded y the FPI fellowship from the Spnish Ministry of Siene nd Innovtion (MICINN). Isel Cuest (IPLA-CSIC) nd Alii Prieto (CIB-CSIC) re knowledged for their exellent tehnil ssistne in the HPLC nd GC MS nlysis. Referenes Arhm, A.G., Medrno, M., Piermri, J.A. nd Mozzi, F. (2010) Novel pplitions of polyshrides from lti id teri: fous on kefirn. In Food Hydroolloids: Chrteristis, Properties nd Strutures ed. Hollingworth, C.S. pp New York: Nov Pulishers. Alouf, J.E. (2000) Bteril protein toxins. In Bteril Toxins. Methods nd Protools. Methods in Moleulr Biology ed. Holst, O. pp New Jersey: Humn Press. Journl of Applied Miroiology 109, ª 2010 The Soiety for Applied Miroiology 2085

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