International Journal of Food Microbiology 140 (2010) 136–145

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International Journal of Food Microbiology

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Probiotic properties of non-conventional lactic acid bacteria: Immunomodulation byOenococcus oeni

Benoît Foligné a,⁎, Joëlle Dewulf a, Jérôme Breton a, Olivier Claisse b, Aline Lonvaud-Funel b, Bruno Pot b

a Institut Pasteur de Lille, Lactic acid Bacteria & Mucosal Immunity, Center for Infection and Immunity of Lille, 1, rue du Pr Calmette, BP 245, F-59019 Lille, France; Univ Lille Nord de France,F-59000 Lille, France; CNRS, UMR 8204, F-59021 Lille, France; Institut National de la Santé et de la Recherche Médicale, U1019, F-59019 Lille, Franceb Université de Bordeaux, UMR 1219 OEnologie, ISVV, .210 Chemin de Leysotte. 33882 Villenave d'Ornon Cedex, France

⁎ Corresponding author. Institut Pasteur de Lille, Lactic acCenter for Infection and Immunity of Lille, 1, rue du Pr CalmeTel.: +33 320871187; fax: +33 320871192.

E-mail address: benoit.foligne@ibl.fr (B. Foligné).

0168-1605/$ – see front matter © 2010 Elsevier B.V. Adoi:10.1016/j.ijfoodmicro.2010.04.007

a b s t r a c t

a r t i c l e i n f o

Article history:Received 5 November 2009Received in revised form 22 March 2010Accepted 2 April 2010

Keywords:OenococcusImmunomodulationSafetyColitisProbiotic

The widely used probiotic bacteria belong to the genera Lactobacillus and Bifidobacterium and have in mostcases been isolated from the human gastrointestinal tract. However, other “less conventional” bacteria, fromallochthonous or extremophilic origin, sharing similar structural or functional features, may also conferspecific health benefits to a host. Firstly, we explored the in vitro immuno-modulatory or immune-stimulatory activities of 25 wine lactic acid bacteria belonging to Oenococcus oeni and Pediococcus parvulus.While cytokines released by peripheral blood mononuclear cells (PBMCs) stimulated by P. parvulus strains,showed little variation, O. oeni strains induced strain-specific cytokine patterns. Some O. oeni strains werethen further analyzed under various conditions for growth, dose and culture medium. In a second phase, weevaluated the oral tolerance and safety of two strains of O. oeni in mice fed a high dose of bacteria for a week.Finally, evidence was gathered on the in vivo anti-inflammatory potential of a selected O. oeni strain using anexperimental 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis mouse model. Although results didnot match the anti-inflammatory levels obtained with certain conventional probiotics, strain IOEB 9115significantly lowered colonic injury and alleviated colitis symptoms. The ‘natural’ tolerance towards acid,ethanol, and phenolic compounds of O. oeni strains combined with a measureable immunomodulatorypotential, suggest a possible use of selected strains isolated from wine as live probiotics.

id Bacteria &Mucosal Immunity,tte, BP 245, F-59019 Lille, France.

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© 2010 Elsevier B.V. All rights reserved.

1. Introduction

The probiotic concept evolved since the end of the 19th centurywhen Döderlein, for the first time suggested that vaginal bacteriaproduced lactic acid that inhibited the growth of pathogens (cited byHolzapfel et al., 2001), Metchnikoff and Tessier's pioneer work(Metchnikoff, 1907) towards the current FAO/WHO definition:“probiotics are live microorganisms which when administered inadequate amounts confer a health benefit on the host” (FAO/WHO,2002). However, the term is often erroneously used to refer to normalcommensal organisms (without substantiation of health effects) orfalsely restricted to bacteria and organisms fromhuman origin. Strainsbelonging to the same species and subspecies can behave differently,providing or not specific health benefit(s) (Sanders, 2008). On theother hand, positive effects on health may also be generated by deadbacteria, metabolites or bacterial derivatives, even though theycannot formally be considered probiotics (Prisciandaro et al., 2009).

The widely used probiotic bacteria are lactobacilli and bifidobacteriamost often isolated from the human gastrointestinal tract. Althoughdata are sometimes scarce, other strains of other lactic acid bacteriasuch as Enterococcus faecalis, Enterococcus faecium, Lactococcus lactis,Streptococcus thermophilus, Leuconostoc mesenteroides and Pediococcusacidilactici were reported to have probiotic potential (Holzapfel et al.,2001, de Vrese and Schrezenmeir, 2008), while non-lactic Gram-positive bacteria such as Propionibacterium freudenreichii or Bacillussubtilis, or Gram-negative strains such as Escherichia coli strain Nissle,and even yeasts like Saccharomyces cerevisiae ssp boulardii could alsobe considered ‘probiotic’ (Lan et al., 2007; Peys et al, 2007; Schultz,2008; Czerucka et al, 2007). Commensal species play a role infunctionality of the intestinal ecosystem and thus exert health-promoting effects, including restoration of dysfunctional immuneresponses. This includes for example bacteria such as Eubacteriumlimosum (Kanauchi et al., 2006), a strain of Faecalibacterium prausnitzii(Sokol et al., 2008), which successfully ameliorated experimentalcolitis, or the human gut symbiont Bacteroides thetaiotamicron (Zoccoet al., 2007).

Importance of the origin of health-promoting strains is actuallypoorly documented. While future developments of functional foodsmay involve a diversification towards other substrates than dairyfoods, the identification and characterization of probiotic strains from

Table 1Bacterial strains and their origin used in the present study.

Strain Identification Origin

BB536 Bifidobacterium longum Morinaga Milk Industry LtdLs33 Lactobacillus salivarius commercial strain/DaniscoNCFM Lactobacillus acidophilus commercial strain/DaniscoMG1363 Lactococcus lactis Cheese starter (Gasson, 1983)TG1 Escherichia coli commensal strain

(Sambrook et al., 1989)Pa-IPL-A12-1 Pediococcus acidilactici IPL collection

(Temmerman et al., 2003)IOEB 8415 Pediococcus parvulus IOEB collectionIOEB 8501 Pediococcus parvulus IOEB collectionIOEB 8508 Pediococcus parvulus IOEB collectionIOEB 8514 Pediococcus parvulus IOEB collectionIOEB 8515 Pediococcus parvulus IOEB collectionIOEB 8608 Pediococcus parvulus IOEB collectionIOEB 9114 Pediococcus parvulus IOEB collectionIOEB 9615 Pediococcus parvulus IOEB collectionIOEB 9646 Pediococcus parvulus IOEB collectionIOEB 8801 Pediococcus parvulus IOEB collectionIOEB 8406 Oenococcus oeni IOEB collectionIOEB 8413 Oenococcus oeni IOEB collectionIOEB 8417 Oenococcus oeni IOEB collectionIOEB 8419 Oenococcus oeni IOEB collectionIOEB 8802 Oenococcus oeni IOEB collectionIOEB 8905 Oenococcus oeni IOEB collectionIOEB 8908 Oenococcus oeni IOEB collectionIOEB 9115 Oenococcus oeni IOEB collectionIOEB 9204 Oenococcus oeni IOEB collectionIOEB 9613 Oenococcus oeni IOEB collectionIOEB 9614 Oenococcus oeni IOEB collectionIOEB 9624 Oenococcus oeni IOEB collectionIOEB 9628 Oenococcus oeni IOEB collectionIOEB 9701 Oenococcus oeni IOEB collectionIOEB 9801 Oenococcus oeni IOEB collection

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diverse habitats may considerably extend the utility of such micro-organisms (de Palencia et al., 2009).

Lactic acid bacteria are also found in other fermented products(cabbage and cereals, pickled vegetables or many Asian food andbeverage types). These strains generally exhibit acid resistance andsurvive very well in the digestive tract, offering sometimes addition-ally immunomodulatory, adherence and/or antimicrobial propertiesthat yeld probiotic opportunities for plant-derived lactic acid bacteria(Kang et al., 2009). A selected Tetragenococcus halophilus strain wasshown to promote in vivo Th1-inducing activity while reducingspecific Th2-immunity in mice (Masuda et al., 2008). Two Pediococcuspentosaceus strains from traditional vegetable pickles had a highcapacity to survive in the gastrointestinal tract, and showed in vitroimmunomodulatory and allergy inhibiting effects (Jonganurakkunet al., 2008) while inhibiting Salmonella invasion in mice (Chiu et al.,2008). In contrast, another P. pentosaceus strain, used as hay preser-vative to prevent farmer's lung pneumonitis, was reported to inducean inflammatory response in mice (Duchaine et al., 1996).

The first study investigating probiotic properties of bacterialstrains from alcoholic fermented beverages focused on a P. parvulusstrain with in vitro evidences of immunomodulatory properties,resistance to gastrointestinal stress and adherence to intestinal cells(de Palencia et al., 2009). However, no attempt has ever been madeto explore such properties for wine-derived micro-organisms invivo, even though the most striking trait of wine lactic acid bacteriais their capacity to cope with a hostile environment (Lonvaud-Funel, 1999; Lonvaud-Funel, 2001; Marcobal et al., 2008; Mills et al.,2005).

Understanding the cytokine patterns elicited by probiotics mayhelp to design probiotics for specific prophylactic or therapeuticpurposes and would enable the development and optimal clinical useof these microbes as health promoting substances (Foligné et al.,2007a,b; Kekkonen et al., 2008). The immunomodulatory effectscould be due to the produced cytokines that further regulate innateand adaptive immune responses and evidences exist for strain-specific ways to direct immune responses to either the Th1 type or theanti-inflammatory side. In vitro models do not take into account thecomplexity of the intestinal barrier and have obvious limitations(Boirivant and Strober, 2007), but offer advantages of high-through-put comparison of intrinsic properties among strains and discriminatehow eukaryotes epithelial or immunocompetent cells may differen-tially sense bacteria. Some immunomodulatory criteria obtained invitro were previously linked with in vivo efficacy (Peran et al., 2005;Foligné et al., 2007a,b; Mileti et al., 2009). Noticeably, the resulting IL-10/IL-12 ratio following cytokine release by stimulated PBMC could berelatively predictive for the strain's performance in the mouse modelof chemically (TNBS) induced colitis (Foligné et al., 2007a,b).

Therefore, we explored the in vitro immuno-modulatory orimmune-stimulatory activities of 25 O. oeni and P. parvulus strainsisolated from wine. Tolerance and some safety aspects linked to theconsumption of high doses of selected O. oeni strains were inves-tigated in mice too. The anti-inflammatory potential of a selectedorally administered O. oeni isolate was evaluated using an in vivoexperimental model of colitis.

2. Materials and methods

2.1. Bacterial strains and growth conditions

Strains usedwere either public strains, commercial isolates, strainsfrom the Institut Pasteur de Lille (IPL) collection or the Collection ofthe UMR 1219 œnologie, (Villenave d'Ornon France), as described inTable 1. All strains, whether O. oeni or P. parvulus were isolated fromred wines, mostly from the wine-producing area of southwest France,during vintages from 1984 to 1998. Strains were typed by theirgenetic profile (genome restriction followed by PFGE). Some bacterial

strains were used as reference strains for immune cell stimulation aspreviously described (Foligné et al., 2007a,b). Lactobacillus strainswere grown under limited aeration at 37°C inMRSmedium (Difco) andBifidobacterium strains were grown anaerobically also at 37°C in MRSsupplemented with 0.05% L-cysteine-hydrochloride (Sigma). L. lactisMG1363 was grown at 30 °C in M17 medium supplemented with 0.5%glucose. E. coli was grown at 37°C with shaking in LB medium (Difco).Pediococcus strains were grown anaerobically at 25°C in MRSsupplemented with 0.05% L-cysteine while for Oenococcus, the latterwas additionally supplemented with 0.6% D-fructose, 0.5% DL-malic acidand 0.1% D-pantothenic acid and adjusted to a final pH of 4.7.Alternatively, a home-made “Leuconosctoc oenos” medium (MLO) wasalso used for the faster propagation and the maintenance of lactic acidbacteria from wine (Zuniga et al., 1993), including the same supple-ments and final pH described above but additionally with 2% steriletomato serum.

Bacterial cells for in vitro immunomodulatory studies weregrown until early stationary phase (unless specified otherwise inthe text) with a minimal absorbance at 600nm (A600) of 1.5, washedtwice in Phosphate Buffered Saline (PBS), resuspended in PBScontaining 20% glycerol using a portable photometer (DensimatBioMerieux) to adjust cell density to McFarland 3 (Araujo et al.,2004). These standardized bacterial preparations, corresponding toapproximately 1×109CFU/mL, were stored at −80°C until furtherused. While one year storage was preliminary shown not to affectboth the microbial cell viability and the magnitude of cytokinerelease (data not provided here), all batches for either the referenceand investigated strains were stored during less than 3 months inthe present study. For in vivo experiments, O. oeni strains weregrown for 4 days in complete MLO, washed twice in sterile PBS (pH7.2) and resuspended at 1.8×1010CFU/mL. Growth curves andcalibration of CFU correspondence were deduced from A600 and

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plating of serial dilutions of the bacterial suspensions on appropriateagar media.

2.2. PBMC isolation and induction of cytokine release

Peripheral bloodmononuclear cells (PBMC) were isolated from bloodof healthy donors as previously described (Foligné et al., 2007a,b). Briefly,after a Ficoll gradient centrifugation (Pharmacia, Uppsala, Sweden),mononuclear cells were collected, washed in RPMI 1640 medium (Livetechnologies, Paisley, Scotland) and adjusted to 2×106cells/mL in RPMI1640 supplemented with gentamicin (150 µg/mL), L-glutamine (2 mM),and 10% foetal calf serum (Gibco-BRL). PBMC (2×106cells/mL) wereseeded in 24-well tissue culture plates (Corning, NY, US). Twentymicroliters of the thawed bacterial suspensions, prepared as describedabove were added. This resulted in a bacteria-to-cell ratio of approxi-mately 10:1. PBS containing 20% glycerol was used as a negative (non-stimulated) control. On the basis of preliminary time-course studies, 24 hstimulation corresponded to the best time point for cytokine responses ofbacteria-stimulated-PBMCs. After 24h stimulation at 37°C in air with 5%CO2, culture supernatants were collected, clarified by centrifugation andstored at−20°Cuntil cytokine analysis. Neithermediumacidification norbacterial proliferation was observed. Cytokines were measured by ELISAusing BD Pharmingen antibody pairs (BD Biosciences, San Jose, Ca, USA)for IL-10, IFN γ and IL-12p70, and R&D systems (Minneapolis, MN, USA)for human TNFα, according to the manufacturer's recommendations andas previously described (Foligné et al., 2007a,b).

2.3. Animals and ethical considerations

BALB/c mice (female, 7 to 8 weeks old) were purchased fromCharles River (St Germain sur l'Arbresle, France). Animals werehoused under temperature-controlled conditions (21 °C) and had freeaccess to water and to a standard mouse chow. The investigation wasconducted in accordance with our institutional guidelines defined bythe European Community guiding principles in the care and use ofanimals (no. 86/609/CEE) and French decree no. 87/848. Experimentswere performed in an accredited establishment (no. A59107; InstitutPasteur de Lille, France) and animal protocols were approved by thelocal ethics committee for animal experiments (CEEA-Nord-Pas deCalais, no. 06-0601).

2.4. Assessment of safety and tolerance to O. oeni strains in orally-fedmice

Bacterial suspensions (200µL), containing 3.5×109CFU of eitherIOEB 8406 and IOEB 9801 in NaHCO3 buffer or vehicle only (NaHCO3

buffer) were daily administered intragastrically to mice (n=10 pergroup) for 7 days. Body weight and general health status of animalswere recorded daily with specific attention for any sign of distress(posture, hair aspect, searching activity, examination of ocularinflammation, fecal character observation and fever which isindirectly assessed by water consumption measurements). Half ofthe mice (n=5) from each group were sacrificed the 7th dayfollowing blood collection by retro-orbital puncture. The abdominalcavity was examined and colon samples were frozen for further assayof Myeloperoxidase (MPO) activity, a neutrophil granule enzymecorrelating inflammatory infiltrates, as reported earlier (Bradley et al.,1982). The rest of mice was maintained for 2 weeks and autopsy (day21) was processed as described above. Mice sera and colon sampleswere stored at −20 °C for subsequent analysis.

Fig. 1. Production of cytokines by peripheral bloodmononuclear cells in response to bacteria.in the supernatants collected from 24h cultures of human PBMCs with reference bacteriaexpressed as mean±SEM (n=4 healthy donors).

2.5. Induction of colitis and inflammation scoring

A standardized murine TNBS colitis model was used in whichmoderate levels of inflammation were induced (Foligné et al., 2006).Briefly, a 50 µL solution of 100 mg/kg TNBS (Sigma) in 50% ethanolwas slowly administered in the colon via a 3.5 F catheter. Bacterialsuspensions (200 µL), containing 3.5×109CFU/mL in NaHCO3 bufferwere administered intragastrically to mice each day, starting 4 daysbefore until the day of TNBS administration while control micereceived the corresponding buffer. Mice were weighed and killed 48hafter TNBS administration. Colons were removed, washed andopened. Inflammation grading was performed blindely by twoindependent observers, using the Wallace scoring method (Wallaceet al., 1989). Results are expressed as % protection, corresponding tothe reduction of the mean macroscopic inflammation score ofbacteria-treated mice (n=10) in comparison to the mean score ofTNBS-treated control mice (NaHCO3 buffer-treated mice, n=10).Histological analysis was performed on May Grünwald–Giemsastained 5µm tissue sections from colon samples fixed in 10% formalinand embedded in paraffin. Tissue lesions were scored according to theAmeho criteria (Ameho et al., 1997). When needed, modification ofthe study design is specified in the text or legend. Additionally, thedegree of polymorphonuclear neutrophil infiltration of the distalcolon was assessed by quantifying myeloperoxidase (MPO), using aslightly modified method (Bradley et al., 1982) as previouslydescribed (Foligné et al, 2007b). A commercial preparation ofprednisone (Cortancyl, Sanofi Aventis, France) was used as positivecontrol of protection and was orally administered for 2 subsequentdays at 10mg/kg starting at the day before TNBS administration.When needed, whole blood was collected by retro-orbital punctureand, after separation the mice sera samples were stored at−20 °C forsubsequent analysis.

2.6. Cytokines and serum amyloid A (SAA) protein assays

Murine IL-6, IL-10 and SAA protein levels were measured by ELISAusing commercial kits from R&D Systems (Minneapolis, MN, USA), BDPharmingen (San Diego, CA, USA) and Biosource International(Camarillo, CA, USA), respectively, with a lower limit of sensitivityof 15pg/mL for IL-6 and 30ng/mL for SAA.

2.7. Data and statistical analysis

All analyses were performed by comparing experimental groups totheir respective controls using the non-parametric one-way analysisof variance Mann–Whitney U-testing, or by Student-t testing whereappropriate. Data are presented as means±SEM. Differences werejudged to be statistically significant when the p value was b0.05. Inaddition, for in vivo experiments, only protection levels exceeding 25%(positive and negative) were considered to be relevant, as previouslydescribed (Foligné et al., 2006). However, P values between 0.05 and0.1 are specified for some experiments to indicate trends.

3. Results

3.1. Immunomodulatory-based screening of wine lactic acid bacteria

The production of cytokines by human PBMCs in response to distinctwine lactic acid bacteria was used as an index of immunomodulatoryactivity. In addition to 5 bacterial reference strains covering the various

IL-10 (A), IL-12 (B), TNFα (C), IFNγ (D) and IL-10/IL-12 ratio (E) were analyzed by ELISA(black bars), Oenococcus (white bars) and Pediococcus (gray bars) species. Data are

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Fig. 2. Growth of O. oeni strains in different media. O. oeni strains IOEB 8406 (blacktriangles), IOEB 9115 (black squares) and IOEB 9801 (empty circles) were either grownin MRS broth (panel A) and in supplemented-MLO broth (panel B). Growth wasrecorded by spectrophotometric absorbance at 600nm (A600) for 8days. Arrowsindicate times when samples were taken for further immunomodulatory studies.

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ranges of cytokines levels (Foligné et al., 2007a,b), fifteen strains ofO. oeni, ten strains of P. parvulus and a commercial isolate of P. acidilacticiwere examined for their ability to induce IL-10, IL-12, INF γ and TNF α(Fig. 1). All these strainswere grownuntil early stationary phase i.e.16 hfor reference strains (the bifidobacterium, L. lactis, E. coli and thelactobacilli), 24 to 30 h for the Pediococcus strainswhileO. oeni required7 days inMRSmedium to reach the stationary phase. The production ofthe 4 cytokineswas significantly induced bymost of the LAB tested after24 h of stimulation, although there were differences between specificstrains for both genera. IL-10 responses differed considerably betweenthe oenococci and pediococci (Fig. 1A). Oenococcal strains IOEB 8406and IOEB 9115 were quite weak inducers of IL-10, similarly as the Lc.lactis reference strain, while strains IOEB 8419 and IOEB 9801 inducedhigh quantities of this cytokine, with values close to the highly anti-inflammatory B. longum reference strain (BB536). The same strain-specificity was also observed for IL-12p70 although differences aresmaller for pediococci. Values of IL-12 were all below those obtainedwith the Lc. lactis, although some responses were still quite elevated, asfor example for strains IOEB 8406 and IOEB 9908, while IL-12 wasundetectable for strains IOEB 9115 and IOEB 9801 (Fig. 1B). Similarresults were obtained for production of IFN γ which usually correlateswell with IL-12 profiles for Grampositive bacteria (Fig. 1C). Consideringreleases of TNF α, usually showing little variation among strains(Foligné et al., 2007a,b), we noted very unusual low levels for theoenococcal strains IOEB 9115 and IOEB 9801 while high values werefound for IOEB 8406 and IOEB 9624 (Fig. 1D).

With the exception of the P. acidilactici isolate, other pediococciexhibited fewer strain-specific differences than the oenococci.Consequently, IL-10/IL-12 ratios are quite similar for all the P. parvulusstrains, with also comparable IFN γ and TNF α for all these strains.

Clear differences between oenococci were obvious, as observedfrom the individual IL-10/IL-12 ratios (Fig. 1E). This anti-inflamma-tory index varied from very low for strain IOEB 8406, to intermediaryfor strain IOEB 9115 and was maximal for strain IOEB 9801.Interestingly, strain IOEB 9115, which showed the same capacity toinduce IL-10 as the “pro-inflammatory” strain IOEB 8406, suggestedthat its higher anti-inflammatory potential is based on a very lowinduction of the pro-inflammatory cytokines IL-12, IFN γ and TNF α.For the next phase, we selected some oenococcal strains based ontheir different in vitro immunogenic response.

3.2. Effects of cultivation period, culture media and cell density oncytokine production

Growth conditions including media composition and cultivationperiods are also well-known to impact on chain length and affect cellwall structure. We thus sought to investigate whether growth phase,culture media or cell density would affect the immunomodulatoryactivity of the 3 O. oeni strains IOEB 8406, IOEB 9115 and IOEB 9801.To this extent we used turbidimetrically standardized samples,obtained at different time intervals, using the ‘slow-growing’ MRSmedium (Fig. 2A) and the ‘fast-growing’ MLO broth (Fig. 2B).

The various set points indicated that both the growth phase andtype of medium have an influence on IL-10 production, with higherquantities induced by cells in the stationary phase, and lower IL-12production by steady state cells (Fig. 3A and C). Depending on thestrain, the medium MLO either abolished the production of IL-10 andIL-12 (strains IOEB 8406 and IOEB 9801), or interestingly, maintainedthe capacity to induce IL-10without changing IL-12 levels (strain IOEB9115) (Fig. 3B and D).

In addition, when samples were taken at the early growth phase,dose–responses also varied with the strain. For IL-10 we observedeither higher or lower levels with an increasing dose of bacteria(Fig. 4A), while TNF α increased in a dose dependent way for allstrains and for both culture media (Fig. 4B). From the 3 Oenococcusstrains tested, strain IOEB 9115 maintained its capacity to induce

relatively high levels of IL-10 in both media MRS and MLO, inducedoverall weak levels of TNF α (Fig. 4B) and yielded extremely lowlevels of IL-12 and IFN γ (data not shown). We therefore selectedthis strain for further in vivo evaluation of its anti-inflammatorypotential.

3.3. Safety and tolerance assessment of O. oeni strains in orally-fed mice

Since safety of LAB strains is a prerequisite for in vivo application(Daniel et al., 2006; Vankerckhoven et al., 2008), we investigated theconsequences of feeding mice for 7 consecutive days with high doses(3.5×109CFU) of the two strains IOEB 8406 and IOEB 9801, grown inMLO broth. The rationale to select these two O. oeni strains used for thesafety assessments inmice is the strainswith themost extreme IL-10/IL-12 ratio (the highest and the lowest), evaluated from the screening ofMRS-cultivated strains (Fig. 1E). Body weight and general health statusof animals were recorded daily for 3 weeks, and no weight loss or signsof distress (posture, hair aspect, searching activity, ocular inflammation,diarrhoea and fever) were observed. On days 7 and 21, the abdominalcavity was examined and showed normal appearance with no sign ofinflammation: color and size of spleen, liver, pancreas and small intes-tinewerenormal, no colon thickening andno spleen overweight). Bloodlevels of IL-6 and SAAwere below the detection level andMPO activitiesin the colonwere similar to the base levels of vehicle-treatedmice (datanot shown).

3.4. Preventive effects of strain O. oeni IOEB 9115 on acute TNBS-colitis

As described above, strain IOEB 9115was further investigated for itsanti-inflammatory capacity in a TNBS-induced experimental colitis inmice. Rectal administration of TNBS causes progressive weight loss innon-treated mice, reaching up to 16% of the initial weight 2 days afterinduction of colitis. The oral 5-days pre-treatment by strain IOEB 9115significantly reduced weight loss to 11.2%, pb0.01 (Fig. 5A). Concom-itantly, colon shortening, which is a strong index of colitis severity, was

Fig. 3. Impact of culture conditions on immunomodulatory responses. The effects of distinct cultivation periods, corresponding to early-, mid-exponential and early stationary phasesrespectively, were evaluated for the 3 O. oeni strains IOEB 8406, IOEB 9115 and IOEB 9801 for IL-10 (left panels, A and B) and IL-12 (right panels, C and D) production on PBMCs(n=3). Oenococciwere either cultivated in MRS (black bars, A and C) or MLO broth (white bars, B and D). IL-10 and IL-12 were analyzed as described in Fig. 1. Data are expressed asmean±SEM (n=3 healthy donors).

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also alleviated in mice fed strain IOEB 9115, showing an intermediarycolon length (9.2±0.28 cm, pb0.05) between TNBS-treated controlmice and non-treated healthy mice (8.5±0.27cm and 10.25±0.11 cmrespectively, pb0.001) (Fig 5B). The prophylactic treatment moreoverled to a 30.4% reduction of the macroscopic inflammation score ascompared to the TNBS-vehicle group (Wallace score 5.1±0.9 versus

3.55±0.69; p=0.12, but not significant, Fig. 5C). In addition to thispositive trend, microscopic assessment also showed lower colonicdamage in the bacteria-treated mice (Figs. 5D and 6C). Histologicallesions were most prominent in the TNBS-control mice, with epithelialerosions and ulcerations of the submucosa and with areas of necrosisextending to the muscular layers (Fig 6B). Additionally, O. oeni

Fig. 4.Dose–response of cytokine production induced byO. oeni strains. The production of IL-10 (A) and TNFα (B)was evaluated for the 3O. oeni strains IOEB 8406, IOEB 9115 and IOEB9801, either cultivated in MRS (black bars) or in MLO broth (white bars) in respect to the reference dose of 10µL of a turbidimetry-standardized suspension, as described in materialand methods. Doses of 5-times less and 5-times more were tested for each growth medium. IL-10 and TNFαwere analyzed as described in Fig. 1. Data are expressed as mean±SEM(n=3 healthy donors).

Fig. 5. Protective effect of 5-days of oral treatment with O. oeni IOEB 9115 on TNBS-colitis. (A): 2-days bodyweight loss (% of initial weight, ±SEM) for healthy untreatedmice (emptycircles), vehicle-TNBS-treated animals (full circles) and O. oeni-fed mice (empty triangles). (B): Changes in colon length (cm), (C): Macroscopic disease scores (Wallace score),(D): Histological damage scores (Ameho score) and (E): Colonic MPO activities, all recorded 48 hours after induction of colitis. Results are expressed as mean±SEM (n=10 per group),*Pb0.05, **Pb0.01, ***Pb0.001 versus No-TNBS healthy control group, #Pb0.05, versus vehicle-TNBS colitis group; (#), 0.05bPb0.1, versus vehicle-TNBS colitis group.

142 B. Foligné et al. / International Journal of Food Microbiology 140 (2010) 136–145

Fig. 6. Representative micrographs of colonic mucosa. Histological sections from anormal (sham) mouse (A) and from mice after induction of TNBS colitis followingtreatment with either vehicle (B) or with O.oeni IOEB 9115 (C); May-Grünwald andGiemsa-stained 5 µm paraffin sections, ×40.

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treatment caused less depletion of goblet cells and smaller loss ofmucosal architecture in the TNBS-control sections as compared tohealthy control mice (Fig 6A). Consistent with this observation, MPOactivity, which follows neutrophil infiltration, was significantly reduced(46%, pb0,05) in the group of mice fed with strain IOEB 9115 (Fig 5E).These results indicate an improvement of inflammation criteria andsupport the prophylactic alleviation of acute colitis by the O. oeni strainIOEB 9115.

4. Discussion

We reported a comparative study where distinct wine LAB were forthe first time considered for their in vitro ability to induce cytokines onhuman PBMCs. Our results demonstrated that certain strains belongingto the species O. oeni

(i) are able to modulate the immune response of immunocompe-tent cells, although attention should be paid to the growthconditions, including the medium composition and thecultivation period of the LAB tested, confirming former datashowing immunostimulatory activities by lactobacilli areaffected by growth phase (Haller et al., 1999, Maassen et al.,2003, Sashihara et al., 2007) and specific medium (Kimoto-Niraet al., 2007, Masuda et al., 2008, Sashihara et al., 2007).

(ii) are well tolerated at high doses in mice, and(iii) exhibit measureable anti-inflammatory activities in vivo by

lowering effects of experimental colitis, suggesting theirpotential as probiotic-like bacteria. So far no relationshipcould be found between these properties and known pheno-typical traits or phylogenetic groups (Bilhere et al., 2009,Renouf et al., 2008). Although immune modulation clearlyplays a role in protection against colitis, the role of theepithelial barrier and possible anti-oxidative mechanismsneeds to be further established.

O. oeni and P. parvulus are part of the indigenous lactic acid bacteria(LAB) flora present on grapes and in wine, responsible for the desirablemalolactic fermentation (MLF) that converts the harsh-tasting malicacid into the much softer lactic acid. The synthesis of variousexopolysaccharides (EPS), glucans, biogenic amines and precursors ofethylcarbamate by O. oeni are considered undesirable in winemaking.However, some of these biochemical or physiological properties as wellas others discussed below, could offer some advantages in other appli-cations and bacteria considered either “beneficial” or “unsuitable” forwinemaking may have health promoting potential:

– Wine LAB are highly resistant to acidic environment, alcoholic andsulfur dioxide stress.

– Some wine LAB can produce EPS (Llauberes et al., 1990; dePalencia et al., 2009; Walling et al., 2005a,b). EPS secretion hasbeen reported to be responsible for the attenuation of colitis byprobiotic strains (Sengül et al., 2006), is involved in resistance tobile salts and low pH (Alp and Aslim, 2009), adhesion to intestinalepithelial cells (Ruas-Madiedo et al., 2006) and can reduce biofilmformation of pathogens (Kim et al., 2009).

– Some wine LAB are able to adhere to epithelial cells (de Palenciaet al., 2009). Although adhesion does not seem to be a prerequisitefor probiotic activity (Blum et al., 1999; Bujalance et al., 2007;Ibnou-Zekri et al., 2003; van der Aa Kühle et al., 2005), and that thein vivo relevance of in vitro adhesion results remains to be illus-trated (Sanders et al., 2008), this characteristic is still believed tohave some relevance in probiotic functionality.

– Some wine LAB are known to secrete bacteriocins (Stiles, 1994;Vescovo et al., 1996), which might have an importance as foodpreservative but could also play a role in human and animal health,with e.g. activities against Listeria monocytogenes (Shin et al., 2008;Trias et al., 2008), or enterococci (Millette et al., 2008).

– Wine LAB can generate biogenic amines (Guerrini et al., 2002;Lonvaud-Funel, 2001).

– Wine LAB, similarly as some other strains of lactobacilli andbifidobacteria (Foligné et al., 2007a,b, He et al., 2002; Helwig et al.,2006; Iwabuchi et al., 2007), do modulate cytokine production bymonocytes and lymphocytes. The intake of these strains in sufficientquantities may divert the immune system into a regulatory ortolerance mode, possibly useful in the prevention or treatment ofatopic disease (Niers et al., 2005) or inflammatory bowel diseases

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(Foligné et al., 2007a,b). The interactions of LAB with regulatory T-cell adds to the knowledge that some strains, releasing larger levelsof pro-inflammatory cytokines such IFNγ and IL-12,maybeuseful totreat an ongoing Th2-skewed immune response such as allergy(Fujiwara et al., 2004;Masuda et al., 2008), while LAB strainswith ananti-inflammatory profile (high IL-10 and IL-10/IL-12 ratio, togetherwith low levels of TNFα) are recommended for lowering andrestoring Th1-skewed immune responses, like observed in IBD(Foligné et al., 2007a,b; Kekkonen et al., 2008; Peran et al., 2005) orirritable bowel syndrome (O'Mahony et al., 2005).

The probiotic status of some LAB strains such L. lactis is still debated,although it has been shown to have immunomodulatory effects(Kimoto-Nira et al., 2007; Mofredj et al., 2007) and to survive thepassage through themouse digestive tract. Other bacteria are generallyaccepted to be useful for health, but may not meet at full the criteriarequested by the current definition of “probiotics”. Clearly the probioticconcept, as defined today, allows for some interpretation and willaccommodate more bacterial strains and preparations than thetraditional text books mention today. Here we have shown, to ourknowledge for the first time, that despite their slow-growing rate,elevated doses ofOenococcusmay exhibit anti-inflammatory potentials.Obviously, the residual bacterial content in a bottle of wine is null or toolow to exert a direct probiotic effect. Consequently, although we couldpropose wine as a (pleasant) vehicle to deliver selected strainspreviously concentrated, with possible synergy with well-known anti-oxidative and anti-inflammatory compounds in wine such Resveratroland other polyphenols (Martin et al, 2006), such use cannot reasonablybe retained. Oenological strains as probiotic may only have a probioticpotential outside their natural habitat and should better exist assupplement rather than in functional food. Thus, it will need to besubstantiated by the necessary clinical tests that validate them as afunctional food additive. In contrast to its relatively slow growth rate,thehigh tolerance toharsh environmental conditions,which is a generaltrait of the O. oeni species, is an argument to promote deeper studies ontheir possible use as probiotics. The optimization of a proper vehicle forO. oeni administration will also need technical development.

In conclusion, wine LAB share structural features with probiotic LAB,offering a wide range of immunomodulation effects. In addition to theirexcellent capacity to resist hostile environments, their specific meta-bolic pathways and their ability to secrete adhesive and antimicrobialcompound, complete their arsenal. With this study, we have tried toillustrate that other than ‘traditional’ probiotic strains can safely beexplored for applications that require specific immune/barrier/meta-bolic functionality and that screening of these collections might yieldstrains with interesting profiles for specific applications envisaged.Preliminary safety tests inmice indicate that higher levels are very welltolerated. Therefore, oenococcimaybe considered for further evaluationin prevention or treatment applied for pathogen-related intestinaldiseases, allergy or inflammation. In the probable perspective thatmultiple mechanisms are involved in the protection against colitis orallergy, the use of selected strains is very likely. Future applications forLAB such as detoxification (Halttunen et al., 2008), anti-oxidativeactivity or sources of beneficial molecules can also be envisaged forthose allochtonous bacteria, applicable for specific probiotic applica-tions in man, animal and aquaculture (Gatesoupe, 2008). History hasshown that microbial biodiversity offers wide perspectives in this senseand, as far as technological and safety challenges can be met, ‘unusual’bacteria, e.g. growing at low temperature-, slow-growing-, extremo-philic or even representatives from thedomain of archeamay qualify forpossible new pharmacological application.

Acknowledgments

This work was supported by funds from Institut Pasteur de Lille,Lille, France.

We thank Thierry Chassat, Globe Ule and Han Vorng for theirassistance in animal handling and human blood sampling. We alsothank the Animal Facility of the “Institut Pasteur de Lille” for logisticassistance.

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