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International audience; ContextGut inflammation constitutes a growing health concern in developed countries. It may consist in spontaneous ailments of the gut, involving both the host immune system and microbiota, such as IBD, including ulcerative colitis and Crohn’s disease. It may be caused by a medical treatment, such as mucositis induced by cancer chemotherapy and/or radiotherapy. It coincides with a dysbiosis including a lack of anti-inflammatory bacteria. As an example, propionibacteria are lacking in the microbiota of newborns that develop necrotizing enterocolitis.Methods and resultsWe thus focused on the immunomodulatory properties of GRAS propionibacteria. Selected strains of Propionibacterium freudenreichii induced the regulatory IL-10 cytokine in human immune cells (Foligné et al., 2010, 2013), depending on surface proteins (Le Marechal et al., 2015). Mutation of the slpB gene suppressed this immunomodulatory effect and the resulting slpB mutant induced a rather proinflammatory response (Deutsch et al., 2017). Consumption of wild-type P. freudenreichii protected from colitis induced by both TNBS and by DSS. It alleviated severity of symptoms, modulated local and systemic inflammation, as well as colonic oxidative stress and epithelial cell damages (Plé et al., 2015, 2016; Rabah et al., 2020). Accordingly, consumption of Lactococcus lactis NCDO 2118 harboring pXIES-SEC:slpB and expressing the propionibacterial SlpB reduced severity of colitis, lowered weight loss, disease activity index, shortening of the colon length, and histopathological score, compared with mice treated with L. lactis NCDO 2118 wild-type strain.In the context of mucositis induced by the chemotherapy 5-FU, P. freudenreichii prevented weight loss, reduced inflammation and consequently intestinal damages. It regulated key markers, including Claudin-1 and IL-17a genes, as well as IL-12 and IL-1β cytokines levels (Cordeiro et al., 2018). Mutant strain slpB displayed opposite regulatory effect on cld1 expression and on IL-12 levels, and failed to afford protection towards 5-FU-mucositis (do Carmo et al., 2019).ConclusionThis work emphasizes the importance of SlpB in P. freudenreichii ability to reduce both mucositis and colitis inflammation. It opens perspectives for the development of probiotic products aimed at decreasing side effects of chemotherapy and at helping treatment of colitis, thanks to GRAS bacteria.ReferencesCordeiro, B. F., Oliveira, E. R., Silva, D., H, S., Savassi, B. M., Acurcio, L. B., et al. (2018). Whey Protein Isolate-Supplemented Beverage, Fermented by Lactobacillus casei BL23 and Propionibacterium freudenreichii 138, in the Prevention of Mucositis in Mice. Front. Microbiol. 9. doi:10.3389/fmicb.2018.02035.Deutsch, S.-M., Mariadassou, M., Nicolas, P., Parayre, S., Le Guellec, R., Chuat, V., et al. (2017). Identification of proteins involved in the anti-inflammatory properties of Propionibacterium freudenreichii by means of a multi-strain study. Sci Rep 7, 46409. doi:10.1038/srep46409.do Carmo, F. L. R., Rabah, H., Cordeiro, B. F., da Silva, S. H., Pessoa, R. M., Fernandes, S. O. A., et al. (2019). Probiotic Propionibacterium freudenreichii requires SlpB protein to mitigate mucositis induced by chemotherapy. Oncotarget 10, 7198–7219. doi:10.18632/oncotarget.27319.Foligné, B., Breton, J., Mater, D., and Jan, G. (2013). Tracking the microbiome functionality: focus on Propionibacterium species. Gut 62, 1227–1228.Foligné, B., Deutsch, S. M., Breton, J., Cousin, F. J., Dewulf, J., Samson, M., et al. (2010). Promising immunomodulatory effects of selected strains of dairy propionibacteria as evidenced in vitro and in vivo. Appl.Environ.Microbiol. 76, 8259–8264.Le Marechal, C., Peton, V., Ple, C., Vroland, C., Jardin, J., Briard-Bion, V., et al. (2015). Surface proteins of Propionibacterium freudenreichii are involved in its anti-inflammatory properties. J.Proteomics. 113C, 447–461.Plé, C., Breton, J., Richoux, R., Nurdin, M., Deutsch, S.-M., Falentin, H., et al. (2016). Combining selected immunomodulatory Propionibacterium freudenreichii and Lactobacillus delbrueckii strains: Reverse engineering development of an anti-inflammatory cheese. Mol Nutr Food Res 60, 935–948. doi:10.1002/mnfr.201500580.Plé, C., Richoux, R., Jardin, J., Nurdin, M., Briard-Bion, V., Parayre, S., et al. (2015). Single-strain starter experimental cheese reveals anti-inflammatory effect of Propionibacterium freudenreichii CIRM BIA 129 in TNBS-colitis model. Journal of Functional Foods 18, 575–585. doi:10.1016/j.jff.2015.08.015.Rabah, H., do Carmo, F. L. R., Carvalho, R. D. de O., Cordeiro, B. F., da Silva, S. H., Oliveira, E. R., et al. (2020). Beneficial Propionibacteria within a Probiotic Emmental Cheese: Impact on Dextran Sodium Sulphate-Induced Colitis in Mice. Microorganisms 8, 380. doi:10.3390/microorganisms8030380.- PF induces IL-10 in human PBMCs- Surface layer extraction reduces IL-10 induction- Extracted surface layer induces IL-10- Mutation of the SlpB gene reduces IL-10 induction- PF prevents colitis- PF prevents mucositis- PF slpB mutant does not prevent mucositis- Lactococcus lactis expressing SlpB prevents colitis

International audience; IntroductionGut inflammation constitutes a growing health concern in developed countries. It may consist in spontaneous ailments of the gut, involving both the host immune system and microbiota, such as IBD, including ulcerative colitis and Crohn’s disease. It may be caused by a medical treatment, such as mucositis induced by cancer chemotherapy and/or radiotherapy. It coincides with a dysbiosis including a lack of anti-inflammatory bacteria. As an example, propionibacteria are lacking in the microbiota of newborns that develop necrotizing enterocolitis.Methods Dairy propionibacteria strains were tested in vitro on human PBMCs with respect to their ability to induce immunomodulatory cytokines. Selected strains were then tested in vitro with respect to their ability to protect mice from TNBS-induced colitis, from DSS-induced colitis, and from 5FU-induced mucositis.ResultsWe thus focused on the immunomodulatory properties of GRAS propionibacteria. Selected strains of Propionibacterium freudenreichii induced the regulatory IL-10 cytokine in human immune cells (Foligné et al., 2010, 2013), depending on surface proteins (Le Marechal et al., 2015). Mutation of the slpB gene suppressed this immunomodulatory effect and the resulting slpB mutant induced a rather proinflammatory response (Deutsch et al., 2017). Consumption of wild-type P. freudenreichii protected from colitis induced by both TNBS and by DSS. It alleviated severity of symptoms, modulated local and systemic inflammation, as well as colonic oxidative stress and epithelial cell damages (Plé et al., 2015, 2016; Rabah et al., 2020). Accordingly, consumption of Lactococcus lactis NCDO 2118 harboring pXIES-SEC:slpB and expressing the propionibacterial SlpB reduced severity of colitis, lowered weight loss, disease activity index, shortening of the colon length, and histopathological score, compared with mice treated with L. lactis NCDO 2118 wild-type strain (Belo et al., 2021).In the context of mucositis induced by the chemotherapy 5-FU, P. freudenreichii prevented weight loss, reduced inflammation and consequently intestinal damages. It regulated key markers, including Claudin-1 and IL-17a genes, as well as IL-12 and IL-1β cytokines levels (Cordeiro et al., 2018). Mutant strain slpB displayed opposite regulatory effect on cld1 expression and on IL-12 levels, and failed to afford protection towards 5-FU-mucositis (do Carmo et al., 2019).P. freudenreichii was further shown to produce extracellular vesicles (EVs), which mimic the immunomodulatory features of propionibacteria in vitro by modulating NF-κB transcription factor activity and IL-8 release in cultured human intestinal epithelial cells (Rodovalho et al., 2020). Proteomic analysis revealed presence of SlpB in these EVs.DiscussionThis work emphasizes the importance of SlpB in P. freudenreichii ability to reduce both mucositis and colitis inflammation. It opens perspectives for the development of probiotic products aimed at decreasing side effects of chemotherapy and at helping treatment of colitis, thanks to GRAS bacteria.

International audience; Introduction and Aims. Inflammatory Bowel Diseases (IBD), including Ulcerative Colitis (UC), coincide with alterations in the gut microbiota. Consumption of immunomodulatory strains of probiotic bacteria may induce or prolong remission in UC patients. Fermented foods, including cheeses, constitute major vectors for bacteria consumption. New evidences revealed anti-inflammatory effects of selected strains within the species P.freudenreichii, S.thermophilus and L.delbrueckii. We thus hypothesized that consumption of a functional cheese, fermented by selected strains of these three species, may exert a positive effect on IBD.Methods. Strains of each bacterial species were screened based on the ability to induce regulatory IL-10 in human immune PBMC cells, or to downregulate nuclear factor kappa B (NF-κB) activation in a cultured HT-29 human intestinal epithelial cell line. We then investigated the effect of monostrain experimental cheese. We further investigated the impact two-strains experimental cheese. Finally, we produced in industrial conditions an Emmental cheese using one selected strain of each of the three species, i.e. P. freudenreichii CIRM-BIA 129 in combination with Lactobacillus delbrueckii CNRZ327 and Streptococcus thermophilus LMD-9. Consumption of all cheeses was investigated with respect to prevention of chemically induced colitis in mice.Results. Consumption of the experimental cheeses, or of the industrial Emmental, reduced the severity of subsequent chemically induced colitis, weight loss, disease activity index and histological score, in mice. Furtherrmore, consumption of the Emmental cheese, in a preventive way, reduced small bowel Immunoglobulin A (IgA) secretion, restored occludin gene expression and prevented induction of Tumor Necrosis Factor α (TNFα), Interferon γ (IFNγ) and Interleukin-17 (IL-17).Conclusion. Assembling immunomodulatory strains of both lactic acid and propionic acid starter bacteria leads to an anti-inflammatory Emmental cheese, as revealed in vivo. This opens new perspectives for the development of functional fermented food products for personalised nutrition in the context of IBD.

International audience; Introduction Propionibacterium freudenreichii, is a probiotic bacterium, with an established immunomodulatory role. Selected strains indeed induce the release of regulatory IL-10. Extractable surface proteins of the S-layer type (Slp) are involved in such modulation, but the exact mode of interaction with the host remains unknown. Mucositis is a common side effect of cytotoxic chemotherapy characterized by mucosal injury, inflammation, diarrhea, and weight loss. We hypothesized that immunomodulatory P. freudenreichii may mitigate inflammation and that SlpB may be required for such an effect.Extracellular vesicles (EVs) are nanometric spherical structures involved in intercellular communication, whose production is considered a widespread phenomenon in living organisms. Bacterial EVs are associated with several processes that include survival, competition, pathogenesis, and immunomodulation. We hypothesized that, in addition to surface exposure and secretion of proteins, P. freudenreichii may produce EVs and thus export immunomodulatory proteins to interact with the host.MethodsP. freudenreichii CIRM-BIA 129, previously selected for its ability to induce IL-10 release by PBMCs, was administered orally to mice, prior to colitis induction using TNBS.The slpB gene was inactivated by insertional inactivation. Both parental wild type and mutant strains were administered orally to mice, prior to induction of mucositis using 5-FU. The SlpB protein was purified and its effect on cultured human intestinal epithelial cells investigated with respect to cytokines expression.EVs were purified from cell-free culture supernatants of the probiotic strain P. freudenreichii CIRM-BIA 129, prior to physicochemical and functional characterization.ResultsConsumption of P. freudenreichii CIM-BIA129 strain protected mice against TNBS-induced colitis in mice, alleviating severity of symptoms, modulating local and systemic inflammation, as well as colonic oxidative stress and epithelial cell damages.This strain prevented 5-FU-induced mucositis in mice, regulated key markers, including the expression of Claudin-1 (Cld1) and IL-17a (Il17a), as well as the levels of IL-12 and IL-1β cytokines.The slpB gene was mutated by insertional inactivation and the absence of SlpB protein in the resulting mutant was verified. The mutant strain displayed opposite immunomodulatory effect and failed to mitigate induced mucositis.In HT-29 human intestinal epithelial cells P. freudenreichii reduced expression of IL-8 and TNF-α cytokines in LPS-stimulated cells. P. freudenreichii ΔslpB, lacking the SlpB protein, failed to do so. In the same cells, purified SlpB was shown to induce expression of IL-10 in a dose-dependent manner.Extracellular vesicles (EVs), they were purified from cell-free culture supernatants of P. freudenreichii. They showed typical shapes and sizes of EVs and contained a broad range of proteins, including SlpB. EVs modulated inflammatory responses, IL-8 release and NF-κB activity, in HT-29 human intestinal epithelial cells, as well as the NF-κB pathway.DiscussionThis work emphasizes the importance of SlpB in immunomodulatory P. freudenreichii. It constitutes the first report on identification of P. freudenreichii-derived EVs, alongside their physicochemical, biochemical and functional characterization. It opens perspectives for the development of functional foods and other bioactive products in order to help preventing and/or treating inflammatory diseases, and to decrease side effects of chemotherapy.

The article presents a proteomic data set generated by a comparative analysis of the proteomes of Propionibacterium freudenreichii, comparing the CIRM-BIA 129 and CIRM-BIA 1025 strains. The two strains were cultivated until the beginning of the stationary phase in a chemical defined medium (MMO), and in this medium in the presence of NaCl, with or without glycine betaine. Whole-cell proteins were extracted, trypsinolyzed and analyzed by nano LC-MS/MS, prior to identification and classification by function using the X!Tandem pipeline software and the proteomic data from NCBI.nlm.nigh.gov. Quantification of proteins was then carried out in order to detect change in their expression depending on the culture medium. This article is related to the research article entitled “Benefits and drawbacks of osmotic adjustment in Propionibacterium freudenreichii”. The comparative proteomic analysis of the two strains reveal strain-dependent and medium-dependent stress proteomes in the probiotic P. freudenreichii.

Propionibacterium freudenreichii is a beneficial bacterium consumed in cheeses and in probiotic food supplements. Indeed, selected strains of P. freudenreichii combine both technological and probiotic abilities. Selected strains were shown to adhere to intestinal cells and to modulate the mucosal immune response as well as the proliferation/apoptosis balance. P. freudenreichii are thus used as Swiss-type cheese starters and as probiotics in the industry. For these purposes, P. freudenreichii should be converted into a powdered form to be stabilized, without significant loss of viability. It suffers hyperosmotic constraints in different key steps of its use. This includes dry starter preparation, cheese making and transit through the human digestive tract. Osmotic adaptation, and the corresponding adjustments, are prerequisites to the use of P. freudenreichii. Moreover, during its large-scale industrial production, stabilisation of dried bacteria under a long-lasting live form is a critical point. This process being stressful, pre-treatments should aim at an enhanced tolerance. Culture media constitute a means to adapt bacteria in the aim to increase their survival during technological and digestive processes. In this study, we explored the impact of osmoadaptation in two P. freudenreichii strains, in different conditions, on survival. Method The maximal salt concentrations allowing growth, and inducing osmoadapatation, was selected, using a chemically defined medium. For all experiments, cells were collected at the beginning of the stationary phase. Accumulated osmoprotectants were identified and quantified by C and H NMR. Whole cell proteins were identified and quantified by LC-MS/MS. The different cultures were then submitted to oxidative, heat, acid, bile salts challenges and to freeze-drying. Results During growth, both P. freudenreichii strains accumulated trehalose, glutamate and glycine betaine. Osmoadaptation, strain-dependent, had different effects on multiple stress tolerance, depending on the presence of osmoprotectants. One of the two strains growth was restored by provided glycine betaine (GB). In this strain, salt stress preadaptation enhanced tolerance towards heat, oxidative, acid and freeze-drying challenges in the absence of GB. However, transport and accumulation of provided GB had deleterious effects on stress tolerance, while restoring optimal growth under hyperosmotic constraint. In the other strain, neither salt, nor GB, enhanced stress tolerance, which was constitutively low. Accordingly, whole cell proteomics revealed differences between strains, as well as different adaptive mechanisms triggered by salt in the presence and in the absence of GB. Discussion Osmotic adjustments may thus have positive or deleterious effects on industrial abilities of P. freudenreichii. Tuning of the culture medium composition allows driving bacterial adaptation and leads to better survival during freeze-drying and/or digestion. This, in turn, should lead to high yield and high-quality production of starters and probiotics.

Propionibacterium freudenreichii CIRM-BIA 129 (Pf) is a beneficial bacterium used as a cheese starter. It moreover displays versatile strain-dependent probiotic properties, including the modulation of inflammation. This property results from the production of various metabolites. S-layer surface proteins (Slps), including the major SlpB protein, also play an immunomodulatory role. The "2-in-1" properties, i.e. both fermentative and probiotic properties, make Pf a suitable candidate to develop functional cheeses, in order to prevent inflammatory bowel diseases. The aim of this thesis was to study the impact of the cheese matrix on the immunomodulatory properties of Pf, mediated by its Slps proteins, compared to a culture on milk ultrafiltrate (MUF). In vitro studies suggest that the bacteria from the cheese would have a better ability to tolerate gastric and duodenal stresses, but would have less ability to survive in the colon, compared to bacteria from a MUF culture. In addition, thethe digestive proteolysis of surface proteins improves survival of Pf in the colon. In parallel, the in vitro digestion study showed that proteolysis of surface proteins was only limited by the cheese matrix. This proteolysis leads to the abolition of the anti-inflammatory effects of Slps proteins, which are not de novo expressed in the colonic environment. These results, obtained in vitro, were consistent with the in vivo study, which showed that MUF culture and cheese delivered similar amounts of metabolically active bacteria to the piglets’ colon. This in vivo study showed, however, that t; Propionibacterium freudenreichii CIRM-BIA 129 (Pf) est une bactérie bénéfique utilisée comme levain fromager. Elle présente en outre de nombreuses potentialités probiotiques souche-dépendantes, dont la modulation de l’inflammation. Cette propriété résulte de la production de divers métabolites. Les protéines de surface S-layer (Slps), dont la protéine majoritaire SlpB, y jouent également un rôle immunomodulateur. Les propriétés « 2-en-1 », c’est-à-dire à la fois fermentaires et probiotiques, font de Pf un bon candidat pour développer des fromages fonctionnels, afin de prévenir les maladies inflammatoires intestinales. L’objectif de cette thèse était d’étudier l’impact de la matrice fromagère sur les propriétés immunomodulatrices de Pf, via ses protéines Slps, par comparaison à une culture sur ultrafiltrat de lait (UF). Les études conduites in vitro suggèrent que les bactéries provenant du fromage auraient une meilleure capacité de tolérance aux stress gastriques et duodénaux, mais elleauraient une moindre capacité de survie dans le côlon, par comparaison à des bactéries provenant d’une culture sur UF. De plus, la protéolyse digestive des protéines de surface améliore la survie de Pf dans le côlon. Parallèlement, l’étude de digestion in vitro a montré que la protéolyse des protéines de surface a seulement été réduite par la matrice fromagère. Cette protéolyse conduit à l’abolition des effets anti-inflammatoires des protéines Slps, qui ne sont pas exprimées de novo dans l’environnement colique. Ces résultats obtenus in vitro étaient cohérents avec l’étude in vivo qui a mont

Propionibacterium freudenreichii CIRM-BIA 129 (Pf) is a cheese starter and has several probiotic potentialities, including immunomodulation. This property results from the production of various beneficial metabolites, and from the presence of S-layer surface proteins (Slps). The "2-in-1" property, i.e. both fermentative and probiotic properties, makes Pf a suitable candidate to develop functional cheeses, in order to prevent inflammatory bowel disease. The aim of this thesis was to study the impact of the cheese matrix on the immunomodulatory properties of Pf, mediated by its Slps proteins, compared to a culture on milk ultrafiltrate (MUF). In vitro studies suggest that Pf viability is affected by the delivery vehicle, cheese matrix or MUF during digestion as well as in the colon. We also showed that the cheese matrix limits Slps proteins proteolysis during gastric and duodenal digestion. The in vivo study showed that the two delivery vehicles, cheese and MUF, display different antiinflammatory effects. Finally, an industrial-scale Emmental cheese was manufactured using selected immunomodulatory strains of lactic and propionic acid bacteria. It also highlights the potential of using Emmental probiotic cheese as a lever to prevent inflammatory bowel disease.; Propionibacterium freudenreichii CIRM-BIA 129 (Pf) est un levain fromager et détient plusieurs potentialités probiotiques, dont l’immunomodulation. Cette propriété résulte de la production de divers métabolites bénéfiques et de la présence de protéines de surface S-layer (Slps). Cette propriété « 2-en-1 », c’est-à-dire à la fois fermentaire et probiotique, fait de Pf un bon candidat pour développer des fromages fonctionnels, afin de prévenir les maladies inflammatoires intestinales. L’objectif de cette thèse était d’étudier l’impact de la matrice fromagère sur les propriétés immunomodulatrices de Pf, via ses protéines Slps, par comparaison à une culture sur ultrafiltrat de lait (UF). Les études conduites in vitro suggèrent que la vectorisation par le fromage module la viabilité de Pf lors de la digestion ainsi que dans le côlon. Nous avons montré aussi que la matrice fromagère limite la protéolyse des protéines Slps lors de la digestion gastrique et duodénale. L’étude in vivo a montré que les deux matrices de vectorisation, le fromage et l’UF, présentent des effets anti-inflammatoires différents. Finalement, un emmental a été fabriqué en sélectionnant des souches immunomodulatrices de bactéries lactiques et propioniques. Son ingestion a protégé des souris d’une colite chimiquement induite. Ce travail montre que les propriétés immunomodulatrices de Pf sont matricedépendantes, tout comme elles sont souchedépendantes. Il souligne de plus le potentiel offert par l’utilisation d’un fromage de type emmental probiotique comme un levier pour prévenir les maladies inflammatoires intestinales.

International audience; Propionibacterium freudenreichii is a beneficial bacterium, used as a cheese starter, which presents versatile probiotic properties. These properties are strain-dependent. We hypothesized they may also be delivery vehicle-dependent. In this study, we thus explored in healthy piglets how the cheese matrix affects the immunomodulatory properties of P. freudenreichii. During 2 weeks, three groups of weaned piglets consumed, respectively, P. freudenreichii as a liquid culture (PF-culture), P. freudenreichii under the form of a cheese (PF-cheese), or a control sterile cheese matrix (Cheesematrix). The in vivo metabolic activity of P. freudenreichii was assessed by determining short chain fatty acids (SCFA) concentration and bifidobacteria population in feces. Whatever the delivery vehicle, P. freudenreichii was metabolically active in piglets’ colon and enhanced both bifidobacteria and SCFA in feces. P. freudenreichii consumption decreased the secretion of TNFa and of IL-10 by peripheral blood mononuclear cells (PBMC). It did not alter IL-10, IFNg, IL-17, and TNFa secretion in mesenteric lymph node immune cells (MLNC). PF-cheese enhanced significantly Treg phenotype, while PF-culture decreased significantly Th17 phenotype in PBMC and MLNC. Remarkably, only PF-cheese induced an increase of Th2 phenotype in PBMC and MLNC. Ex vivo stimulation of PBMC and MLNC by Lipopolysaccharides and Concanavalin A emphasized the difference in the immunomodulatory responses between PF-culture and PF-cheese group, as well as between PBMC and MLNC. This study shows the importance to consider the delivery vehicle for probiotic administration. It confirms the anti-inflammatory potential of P. freudenreichii. It opens new perspectives for the use propionibacteria-fermented products as preventive agents for inflammatory bowel diseases and intestinal infectious diseases.

Introduction Propionibacterium freudenreichii (PF) is a Swiss-type cheeses starter and has versatile probiotic properties due to various beneficial metabolites. PF recently emerged as a suitable candidate to develop fermented functional foods. Delivery of viable bacteria to the colon is crucial for in situ beneficial metabolites production. The delivery vehicle was shown to influence P. freudenreichii tolerance towards digestive stresses. It is also suggested to influence PF adaptation to the colon environment. In addition, during digestion, the delivery vehicle may prevent or allow proteolysis of surface proteins. These surface proteins are generally thought to contribute to bacteria tolerance towards environmental stresses. Aim This study aimed at evaluating PF (strain CIRM-BIA 129) adaptation to the caecal environment, according to the delivery vehicle. Besides, we evaluated PF survival in the caecal environment after removal of bacterial surface proteins by enzymatic proteolysis. We address the plausible role of S-layer protein SlpB in PF adaptation and survival. Methods Caecal medium was prepared from caecal contents recovered from piglets. PF pellets were harvested from cultures in Yeast Extract-Lactate (YEL), in Milk ultrafiltrate, or from cheese. Wild-type and SlpB KO mutant PF were further compared with respect to survival in caecal medium. Bacterial pellets were also submitted to surface proteins proteolysis to mimic digestion. Cultures were incubated 50 h at 37°C in anaerobiosis. Survival was monitored by plate counting, optical density and by live/dead fluorescence labeling. Results PF survival decreased in the caecal medium with a similar rate, whatever the growth medium tested. However, optical density was stable and the epifluorecence images did not show dead bacteria. Reduced survival rate was not due to significant cell death.PF seemed rather to enter a viable but nonculturable state in the caecal medium. Surprisingly, proteolytic removal of surface layer proteins enhanced PF culturable state in caecal medium. In addition, the mutation of SlpB did not change PF culturable state but surface proteins proteolysis enhanced also PF KO SlpB s culturable state. Conclusion These results indicate that surface proteins proteolysis during digestion may enhance PF survival and metabolic activity within the caecal medium. A comparative proteomic analysis will address the molecular mechanisms involved in enhanced survival.

Introduction P. freudenreichii is used both as Swiss-type cheese starters and as probiotics. For these purposes, it should be converted into a powdered form to be stabilized, without significant loss of viability. Freeze-drying and spray drying can be implemented in order to obtain dry starters or probiotics. Compared to freeze-drying, spray-drying requires less energy for water removal; however, the use of spray-drying involves exposure to air at high temperatures during the process. It is thus a stressful process, with simultaneous thermal, oxidative and osmotic stresses. To resist to these latter, bacteria should adapt their physiology. Culture media constitute a means to adapt bacteria in the aim to increase their survival during spray drying. Aim In this study, we explored the influence of the growth medium composition on 1) osmoprotectants accumulation and 2) bacteria survival during spray drying. Methods In rich YEL medium, the salt concentration chosen was the highest still allowing growth of P. freudenreichii CIRM BIA 129. For all experiments, cells were collected at the beginning of the stationary phase. Accumulated osmoportectants were identified and quantified by C and H NMR. The different cultures were then submitted to oxidative, heat, and spray drying challenges. Results During growth, P. freudenreichii CIRM-BIA 129 accumulated trehalose, glutamate and glycine betaine. In hyper-osmotic conditions, the accumulation of these osmoprotectants increased.Lactose, which is naturally present in sweet whey, or lactose added to YEL growth medium, triggered acid adaptation, but also increased intracellular trehalose accumulation. The glycine betaine/trehalose ratio accumulated in intracellular content was determined by the culture medium carbon/azote ratio.With an optimized intracellular glycine betaine/trehalose ratio, P. freudenreichii better survived heat and oxidative lethal challenges, as well as spray drying. Conclusion Designing the culture medium composition allows driving bacterial adaptation and leads to better survival after spray drying. This, in turn, should lead to high yield and high-quality production of starters and probiotics.

INTRODUCTION Propionibacterium freudenreichii is an emergent probiotic bacterium, belonging to dairy propionibacteria, within the group of Actinomycetales, and possessing the GRAS (Generally Recognized As Safe, USA, FDA) and QPS (Qualified Presumption of Safety, EFSA, European Union) status. It is a traditional Swiss-type cheeses starter and an emergent probiotic, exerting several beneficial effects, including anti-inflammatory modulation of gut inflammation. This feature relies on several metabolites and on surface proteins including in particular the surface protein SlpB. METHOD In the present study, we assessed first the impact of proteolytic degradation of SlpB on its immunomodulatory effect on colon epithelial HT-29 cells. Intact P. freudenreichii cells, purified surface layer proteins (SLPs), as well as trypsin-proteolyzed SLPs, were used to stimulate HT-29 cells, in the presence or absence of pro-inflamatory LPS. Cytokines were then quantified to evaluate the inflammatory response. We then investigated the potential of cheese matrix as a delivery vehicle of anti-inflammatory P. freudenreichii MAMP to the colon, by comparing dairy liquids matrices to a solid Swiss-type cheese matrix, using two in vitro digestion models. Integrity of SlpB was monitored using western blotting. RESULTS In this study, we firstly investigated the relevance to avoid SlpB digestive proteolysis, by comparing the effect of i) P. freudenreichii CIRM-BIA 129, ii) its native SLPs, or iii) peptides resulting from SLPs stimulations, with respect to modulation of HT-29 cells response to lipopolysaccharide (LPS) challenge. The anti-inflammatory effect exerted by P. freudenreichii CIRM-BIA 129 and by native surface proteins (SLPs) on HT-29 cells was abolished by digestive proteolysis. This result confirmed the importance to protect immunomodulatory surface proteins from digestive proteolysis in order to allow gut immune system modulation. Thus, we examined the effect of dairy matrices on P. freudenreichii viability and on SlpB integrity during digestion. In comparison with liquid matrices, the cheese matrix provided an enhanced tolerance to digestive stresses and protection of SlpB towards proteolysis, during two in vitro digestion models: static and dynamic. CONCLUSION These in vitro results provide new insights into the matrix effect on P. freudenreichii probiotic functionalities. The cheese matrix offers significant protection against the digestion stresses by enhancing survival, and by protecting surface layer proteins from proteolysis. However, in vivo investigations are necessary to study the matrix effect during digestion, to follow SLPs expression and to evaluate the matrix effect on immunomodulation by P. freudenreichii within the gut. Such data accordingly will allow the development of new functional foods for the delivery of P. freudenreichii to the gastrointestinal tract of humans in the aim to help prevention or treatment of life-style related diseases with an inflammatory component.

International audience; Propionibacterium freudenreichii is a well-known ripening starter used in the manufacture of Swiss-type cheeses. It exerts several strain-dependent probiotic features, such as the modulation of apoptosis, of immunity and of microbiota, at the gut level. Promoting fermented dairy products as functional foods is a promising manner to reduce risks of specific life-style related diseases, including colitis. In this aim, the selection of starters should be made on both technological and probiotic criteria. Previously, a single-strain cheese fermented exclusively by P. freudenreichii CIRM-BIA 129 revealed a protective effect against induced colitis in mice. Nonetheless, the effect of delivery matrix on immunomodulatory properties of P. freudenreichii is poorly understood. In addition, data on the effect of fermented cheese on healthy subject or animals are scarce. We thus compared three groups of piglets: 1) fed with a sterilized cheese matrix, 2) fed with a P. freudenreichii CIRM-BIA 129-fermented milk ultrafiltrate and 3) fed with a P. freudenreichii CIRM-BIA 129-single-strain cheese. The immune cells from mesenteric lymph nodes (MLN) were extracted and different analysis were carried out: 1) the basal secretion of cytokines TNFα and IFNγ were assessed by ELISA; 2) the expression of transcription factors Tbet and GATA3, marker of lymphocytes Th1 and Th2 respectively were assessed by RT-PCR, 3); then cells were stimulated ex vivo with several stimuli (LPS and Concanavalin A (ConA) and surface proteins of P. freudenreichii (Slps)), TNFα and IFNγ secretion was measured. The ingestion of P. freudenreichii, whatever the delivery vehicle, doesn’t influence the basal secretion of TNFα and IFNγ. However, the ingestion of P. freudenreichii in the form of cheese enhances Th2 response (tolerance response) by increasing GATA3 expression in MLN cells. In addition, MLN cells from different piglet groups don’t respond similarly to ex vivo stimuli. The ingestion of P. freudenreichii in the form of cheese inhibites the TNFα secretion initiated by LPS simulation and enhanced IFNγ response during co-stimulation LPS and Slps or ConA and Slps. Altogether, in healthy piglets, the cheese matrix modulated the immunomodulatory properties of P. freudenreichii in intestinal immunity which lead to a better reaction to exogenous stimulus.

Some Gram-positive bacteria, including probiotic ones, are covered with an external proteinaceous layer called a surface-layer. Described as a paracrystalline layer and formed by the self-assembly of a surface-layer-protein (Slp), this optional structure is peculiar. The surface layer per se is conserved and encountered in many prokaryotes. However, the sequence of the corresponding Slp protein is highly variable among bacterial species, or even among strains of the same species. Other proteins, including surface layer associated proteins (SLAPs), and other non-covalently surface-bound proteins may also be extracted with this surface structure. They can be involved a various functions. In probiotic Gram-positives, they were shown by different authors and experimental approaches to play a role in key interactions with the host. Depending on the species, and sometime on the strain, they can be involved in stress tolerance, in survival within the host digestive tract, in adhesion to host cells or mucus, or in the modulation of intestinal inflammation. Future trends include the valorization of their properties in the formation of nanoparticles, coating and encapsulation, and in the development of new vaccines.

Propionibacterium freudenreichii is an emergent probiotic, presenting several beneficial effects including anti-inflammatory properties, mediated by surface layer proteins (SLPs) belonged to the S-layer lattice, in particularly the protein SlpB. We hypothesize cheese matrix may be the best delivery vehicle for P. freudenreichii’s anti-inflammatory potential, by enhancing propionibacteria survival to digestives stresses, and by allowing undamaged SLPs to reach the digestive tract. Firstly, we compared the immunomodulatory effects of P. freudenreichii and intact SLPs, to SLPs digested by trypsin, i.e SLPs-peptides on HT29-cells. In contrast to P. freudenreichii and SLPs, SLPs peptides don’t reduce pro-inflammatory cytokines expression during cells co-stimulation with lipopolysaccharide. This result confirmed the importance to protect SLPs from proteolysis to permit them to trigger gut immune system. Secondly, we evaluated propionibacteria resistance and slpB proteolysis during in vitro static digestion, in different delivery vehicles increasingly concentrated in dairy proteins: milk ultrafiltrate, milk and cheese. The experiment clearly showed the protective effect of the Slp proteins by the cheese matrix and better bacterial viability. Then, we carried out in vitro a dynamic digestion using Didgi® system. We selected two matrices only: the milk ultrafiltrate and the cheese. We decided to apply fixed digestion parameters for both matrices: the digestive parameters of cheese. The results show a protective effect of the cheese matrix on the viability of the propionic bacteria and on SLPs against digestive proteolysis. Taken together, those results show that cheese is an adequate delivery vehicle for P. freudenreichii immunomodulatory proteins.

The intestinal microbiota composition has a great impact on physiology and health, since commensal bacteria are crucial to maintain homeostasis and immune regulation of the gut. Consequently, disturbances of this microbiota, a process known as dysbiosis, have severe implications for the host health such as the rise of many gastrointestinal (GI) problems; including inflammatory disorders like the Inflammatory Bowel Diseases (IBD), mucositis, as well as colorectal cancer (CRC). The consumption of probiotics with beneficial effects is a promising tool to help treating such disorders. Indeed, they modulate diverse biological mechanisms involved in GI homeostasis and have been commonly used to reduce such disorders. In this chapter, we present the molecular mechanisms triggered by probiotic bacteria to modulate the gut physiology during gastrointestinal disorder and the importance of the gastrointestinal stresses tolerance as a limiting factors for probiotic application. Moreover, we focus on the emergence of functional probiotic foods, which can act as excellent vehicles, by enhancing stress tolerance and providing a protective matrix towards digestive stresses.

Propionibacterium freudenreichii is a beneficial Actinobacterium, (GRAS & QPS status). It is exploited in the food industry for vitamins production and in Swiss type cheeses manufacturing, such as Emmental (1). Dairy propionibacteria have several probiotic features, including strain-dependent anti-inflammatory properties (Interleukine 10 induction by immune cells) (2). Previous studies showed surface protein (S- layer proteins) are involved in these properties (2), particularly the protein SlpB (Data no published).A monoxenic cheese, fermented by P.freudenreichii only, was developed. Consumption of this cheese reduced severity of chemically induced colitis in mice model (3). This shows the probiotic potential of P.freudenreichii for helping of Inflammatory Bowel Diseases (IBD) treatment. We assume cheese may be the best delivery vehicle of anti-inflammatory potential of P.freudenreichii to digestive tract. Indeed, we presume cheese enhances propionibacteria resistance to stress conditions during digestion. The presence of caseins matrix may exert a protective effect, allowing undamaged bacteria and surface proteins to reach the digestive tract.The experimental strategy chosen was to evaluate propionibacteria resistance and slpB protein proteolysis during in vitro digestion, in different delivery vehicles increasingly concentrated in dairy proteins: culture on milk ultrafiltrate (UF), culture on milk and a monoxenic cheese. P.freudenreichii viability and S-layer proteins susceptibility to proteolysis was followed in the different matrix during in vitro digestion. Two anti-inflammatory strains were used for this experiment: CIRM-BIA 129 and CIRM-BIA 118 (4).This will be correlated with in vivo experiments in the perspective to promote dairy products fermented by propionibacteria as probiotics for target populations.

Propionibacterium freudenreichii is widely used as cheese ripening starter. Its probiotic effects have also been reported at the gut level, including modulation of bifidobacteria, of colon epithelial cells proliferation and apoptosis and of intestinal inflammation. They rely on active metabolism in situ. Growth media and growth conditions determine stress tolerance acquisition in P. freudenreichii, thereby determining its probiotic efficacy in the final products. A simplified process from growth to spray drying of propionibacteria was developed using hyperconcentrated sweet whey as a 2-in-1 medium to both culture and protect P. freudenreichii from spray drying, without harvesting and washing step. The hypertonic stress in hyperconcentrated sweet whey led to overexpression of key stress proteins, accumulation of intracellular storage molecules, compatible solutes, enhanced multistress tolerance acquisition, as well as enhanced survival upon spray-drying. This work opens new perspectives for the sustainable development of new starter and probiotic preparations with enhanced robustness.