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International audience; Propionibacterium freudenreichii is a beneficial bacterium widely used in food as a probiotic and as a cheese-ripening starter. In these different applications, it is produced, dried, and stored before being used. Both freeze-drying and spray-drying were considered for this purpose. Freeze-drying is a discontinuous process that is energy-consuming but that allows high cell survival. Spray-drying is a continuous process that is more energy-efficient but that can lead to massive bacterial death related to heat, osmotic, and oxidative stresses. We have shown that P. freudenreichii cultivated in hyperconcentrated rich media can be spray-dried with limited bacterial death. However, the general stress tolerance conferred by this hyperosmotic constraint remained a black box. In this study, we modulated P. freudenreichii growth conditions and monitored both osmoprotectant accumulation and stress tolerance acquisition. Changing the ratio between the carbohydrates provided and non-protein nitrogen during growth under osmotic constraint modulated osmoprotectant accumulation. This, in turn, was correlated with P. freudenreichii tolerance towards different stresses, on the one hand, and towards freeze-drying and spray-drying, on the other. Surprisingly, trehalose accumulation correlated with spray-drying survival and glycine betaine accumulation with freeze-drying. This first report showing the ability to modulate the trehalose/GB ratio in osmoprotectants accumulated by a probiotic bacterium opens new perspectives for the optimization of probiotics production.

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) 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., 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.

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.

International audience; Introduction Propionibacterium freudenreichii (PF) is a beneficial bacterium, used as Swiss-type cheeses starter. PF exerts several beneficial effects, including anti-inflammatory modulation. This feature relies on several metabolites and on surface proteins. PF emerges as a suitable candidate to develop fermented functional foods. As it is established, the probiotic properties are strain-dependent, however the delivery vehicle effect is barely explored. Aim In this study, our purpose is to explore how the cheese matrix impact its immunomodulatory properties on model of healthy piglets. Methods Three groups of weaned piglets were fed during 2 weeks with PF in form of a culture or of a fermented cheese and compared to a control group fed with sterile cheese. PF’s metabolic activity was assessed by short chain fatty acids dosage and microbiota modulation. Immune cells from small intestinal mesenteric nodes (MLNC) and blood (PBMC) were extracted. The basal cytokines secretion and T lymphocytes phenotypes were evaluated. Then, the immune cells were ex vivo stimulated by pro-inflammatory and anti-inflammatory stimuli and the cytokine response was determined. Results PF was metabolically active in piglets’ colon and enhanced bifidobacteria population and SCFAs production, without significant difference between the two delivery vehicles. PF decreased the secretion of TNFα and IL-10 secretion only by PBMC. In MLNC, PF cheese enhanced Treg and Th2 populations and sustained Th17 population. PF culture enhanced decreased Th17 population in MLNC. In PBMC, PF ingestion enhanced Treg and Th2 populations, without difference between the two delivery vehicles. Ex vivo stimulation of PBMC and MLNC showed a difference in immunomodulation of PF, according the delivery vehicle. Conclusion These results provide new insights into the cheese matrix effect on P. freudenreichii' probiotic functionalities. Such data showed the relevance to take in account the delivery vehicle to develop functionnal foods.

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.

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.