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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.
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Betaína/metabolismo , Dessecação , Propionibacterium freudenreichii/fisiologia , Trealose/metabolismo , Adaptação Fisiológica , Carbono/análise , Queijo/microbiologia , Proteção Cruzada , Meios de Cultura/química , Dessecação/métodos , Liofilização , Viabilidade Microbiana , Pressão Osmótica , Probióticos , Propionibacterium freudenreichii/crescimento & desenvolvimento , Propionibacterium freudenreichii/metabolismo , Cloreto de Sódio/análiseRESUMO
UNLABELLED: Propionibacterium freudenreichii is used as a cheese-ripening starter and as a probiotic. Its reported physiological effects at the gut level, including modulation of bifidobacteria, colon epithelial cell proliferation and apoptosis, and intestinal inflammation, rely on active metabolism in situ Survival and activity are thus key factors determining its efficacy, creating stress adaptation and tolerance bottlenecks for probiotic applications. Growth media and growth conditions determine tolerance acquisition. We investigated the possibility of using sweet whey, a dairy by-product, to sustain P. freudenreichii growth. It was used at different concentrations (dry matter) as a culture medium. Using hyperconcentrated sweet whey led to enhanced multistress tolerance acquisition, overexpression of key stress proteins, and accumulation of intracellular storage molecules and compatible solutes, as well as enhanced survival upon spray drying. A simplified process from growth to spray drying of propionibacteria was developed using sweet whey as a 2-in-1 medium to both culture P. freudenreichii and protect it from heat and osmotic injury without harvesting and washing steps. As spray drying is far cheaper and more energy efficient than freeze-drying, this work opens new perspectives for the sustainable development of new starter and probiotic preparations with enhanced robustness. IMPORTANCE: In this study, we demonstrate that sweet whey, a dairy industry by-product, not only allows the growth of probiotic dairy propionibacteria, but also triggers a multitolerance response through osmoadaptation and general stress response. We also show that propionibacteria accumulate compatible solutes under these culture conditions, which might account for the limited loss of viability after spray drying. This work opens new perspectives for more energy-efficient production of dairy starters and probiotics.
Assuntos
Meios de Cultura/metabolismo , Propionibacterium freudenreichii/fisiologia , Soro do Leite/metabolismo , Meios de Cultura/química , Propionibacterium freudenreichii/crescimento & desenvolvimento , Estresse Fisiológico , Soro do Leite/químicaRESUMO
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.
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Extracellular vesicles (EVs) are nanometric spherical structures involved in intercellular communication, whose production is considered to be a widespread phenomenon in living organisms. Bacterial EVs are associated with several processes that include survival, competition, pathogenesis, and immunomodulation. Among probiotic Gram-positive bacteria, some Propionibacterium freudenreichii strains exhibit anti-inflammatory activity, notably via surface proteins such as the surface-layer protein B (SlpB). We have 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. In order to demonstrate their production in this species, EVs were purified from cell-free culture supernatants of the probiotic strain P. freudenreichii CIRM-BIA 129, and their physicochemical characterization, using transmission electron microscopy and nanoparticle tracking analysis (NTA), revealed shapes and sizes typical of EVs. Proteomic characterization showed that EVs contain a broad range of proteins, including immunomodulatory proteins such as SlpB. In silico protein-protein interaction predictions indicated that EV proteins could interact with host proteins, including the immunomodulatory transcription factor NF-κB. This potential interaction has a functional significance because EVs modulate inflammatory responses, as shown by IL-8 release and NF-κB activity, in HT-29 human intestinal epithelial cells. Indeed, EVs displayed an anti-inflammatory effect by modulating the NF-κB pathway; this was dependent on their concentration and on the proinflammatory inducer (LPS-specific). Moreover, while this anti-inflammatory effect partly depended on SlpB, it was not abolished by EV surface proteolysis, suggesting possible intracellular sites of action for EVs. This is the first report on identification of P. freudenreichii-derived EVs, alongside their physicochemical, biochemical and functional characterization. This study has enhanced our understanding of the mechanisms associated with the probiotic activity of P. freudenreichii and identified opportunities to employ bacterial-derived EVs for the development of bioactive products with therapeutic effects.
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BACKGROUNDS 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 in selected strains of Propionibacterium freudenreichii. We thus hypothesized that consumption of a functional cheese, fermented by such a strain, may exert a positive effect on IBD. METHODS: We investigated the impact of cheese fermented by P. freudenreichii on gut inflammation. We developed an experimental single-strain cheese solely fermented by a selected immunomodulatory strain of P. freudenreichii, CIRM-BIA 129. We moreover produced, in industrial conditions, an Emmental cheese using the same strain, in combination with Lactobacillus delbrueckii CNRZ327 and Streptococcus thermophilus LMD-9, as starters. Consumption of both cheeses was investigated with respect to prevention of Dextran Sodium Sulphate (DSS)-induced colitis in mice. RESULTS: Consumption of the single-strain experimental cheese, or of the industrial Emmental, both fermented by P. freudenreichii CIRM-BIA 129, reduced severity of subsequent DSS-induced colitis, weight loss, disease activity index and histological score. Both treatments, 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). CONCLUSIONS: A combination of immunomodulatory strains of starter bacteria can be used to manufacture an anti-inflammatory cheese, as revealed in an animal model of colitis. This opens new perspectives for personalised nutrition in the context of IBD.
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This review deals with beneficial bacteria, with a focus on lactobacilli, propionibacteria, and bifidobacteria. As being recognized as beneficial bacteria, they are consumed as probiotics in various food products. Some may also be used as starters in food fermentation. In either case, these bacteria may be exposed to various environmental stresses during industrial production steps, including drying and storage, and during the digestion process. In accordance with their adaptation to harsh environmental conditions, they possess adaptation mechanisms, which can be induced by pretreatments. Adaptive mechanisms include accumulation of compatible solutes and of energy storage compounds, which can be largely modulated by the culture conditions. They also include the regulation of energy production pathways, as well as the modulation of the cell envelop, i.e., membrane, cell wall, surface layers, and exopolysaccharides. They finally lead to the overexpression of molecular chaperones and of stress-responsive proteases. Triggering these adaptive mechanisms can improve the resistance of beneficial bacteria toward technological and digestive stresses. This opens new perspectives for the improvement of industrial processes efficiency with regard to the survival of beneficial bacteria. However, this bibliographical survey evidenced that adaptive responses are strain-dependent, so that growth and adaptation should be optimized case-by-case.
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Propionibacterium freudenreichii is a beneficial bacterium used as a cheese starter and as a probiotic. Indeed, selected strains of P. freudenreichii combine both technological and health-promoting abilities. Moreover, during large-scale industrial production of dried bacteria and during consumption, P. freudenreichii may undergo different stressful processes. Osmotic adaptation was shown to enhance P. freudenreichii tolerance towards stresses, which are encountered during freeze-drying and during digestion. In this report, we compared the osmoadaptation molecular mechanisms of two P. freudenreichii strains. Both osmotolerance and osmoadaptation were strain-dependent and had different effects on multiple stress tolerance, depending on the presence of osmoprotectants. Availability of glycine betaine (GB) restored the growth of one of the two strains. In this strain, osmotic preadaptation enhanced heat, oxidative and acid stresses tolerance, as well as survival upon freeze-drying. However, addition of GB in the medium 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 that mechanisms triggered by salt in the presence and in the absence of GB are different between strains. Osmotic adjustment may thus have deleterious effects on industrial abilities of P. freudenreichii. BIOLOGICAL SIGNIFICANCE: Propionibacteria are found in various niches including fodder, silage, rumen, milk and cheeses. This means adaptation towards different ecological environments with different physicochemical parameters. Propionibacterium freudenreichii, in particular, is furthermore used both as dairy starter and as probiotic and is thus submitted to high scale industrial production. Production and subsequent stabilization still need optimization. Drying processes like freeze-drying are stressful. Osmotic adjustments may modulated tolerance towards drying. However, they are strain-dependent, medium-dependent and may either reduce or increase stress tolerance. A case-by-case study, for each strain-medium thus seems necessary. In this work, we identify key proteins involved in osmoadaptation and give new insights into adaptation mechanisms in P. freudenreichii. This opens new perspectives for the selections of strains and for the choice of the growth medium composition.
Assuntos
Adaptação Fisiológica , Proteínas de Bactérias/metabolismo , Queijo/microbiologia , Microbiologia de Alimentos , Pressão Osmótica , Propionibacterium freudenreichii/metabolismo , LiofilizaçãoRESUMO
Propionibacterium freudenreichii is a beneficial bacterium, used both as a probiotic and as a cheese starter. Large-scale production of P. freudenreichii is required to meet growing consumers' demand. Production, drying and storage must be optimized, in order to guarantee high P. freudenreichii viability within powders. Compared to freeze-drying, spray drying constitutes the most productive and efficient, yet the most stressful process, imposing severe oxidative and thermal constraints. The aim of our study was to provide the tools in order to optimize the industrial production of dry P. freudenreichii. Bacterial adaptation is a well-known protective mechanism and may be used to improve bacterial tolerance towards technological stresses. However, the choice of bacterial adaptation type must consider industrial constraints. In this study, we combined (i) modulation of the growth medium composition, (ii) heat-adaptation, and (iii) osmoadaptation, in order to increase P. freudenreichii tolerance towards technological stresses, including thermal and oxidative constraints, using an experimental design. We further investigated optimal growth and adaptation conditions, by monitoring intracellular compatible solutes accumulation. Glucose addition, coupled to heat-adaptation, triggered accumulation of trehalose and of glycine betaine, which further provided high tolerance towards spray drying and storage. This work opens new perspectives for high quality and fast production of live propionibacteria at the industrial scale.
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Propionibacterium freudenreichii is a beneficial bacterium with documented effects on the gut microbiota and on inflammation. Its presence within the animal and human intestinal microbiota was correlated with immunomodulatory effects, mediated by both propionibacterial surface components and by secreted metabolites. It is widely implemented, both in the manufacture of fermented dairy products such as Swiss-type cheeses, and in the production of probiotic food complements, under the form of freeze-dried powders. The bottleneck of this drying process consists in the limited survival of bacteria during drying and storage. Protective pre-treatments have been applied to other bacteria and may, in a strain-dependent manner, confer enhanced resistance. However, very little information was yet published on P. freudenreichii adaptation to freeze-drying. In this report, an immunomodulatory strain of this probiotic bacterium was cultured under hyperosmotic constraint in order to trigger osmoadaptation. This adaptation was then combined with acid or thermal pre-treatment. Such combination led to accumulation of key stress proteins, of intracellular compatible solute glycine betaine, to modulation of the propionibacterial membrane composition, and to enhanced survival upon freeze-drying. This work opens new perspectives for efficient production of live and active probiotic propionibacteria.
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Propionibacterium freudenreichii CIRM-BIA 129 (P. freudenreichii wild type, WT) is a probiotic bacterium, which exerts immunomodulatory effects. This strain possesses extractable surface proteins, including SlpB, which are involved in anti-inflammatory effect and in adhesion to epithelial cells. We decided to investigate the impact of slpB gene mutation on immunomodulation in vitro and in vivo. In an in vitro assay, P. freudenreichii WT reduced expression of IL-8 (p<0.0001) and TNF-α (p<0.0001) cytokines in LPS-stimulated HT-29 cells. P. freudenreichii ΔslpB, lacking the SlpB protein, failed to do so. Subsequently, both strains were investigated in vivo in a 5-FU-induced mucositis mice model. Mucositis is a common side effect of cytotoxic chemotherapy with 5-FU, characterized by mucosal injury, inflammation, diarrhea, and weight loss. The WT strain prevented weight loss, reduced inflammation and consequently histopathological scores. Furthermore, it regulated key markers, including Claudin-1 (cld1, p<0.0005) and IL-17a (Il17a, p<0.0001) genes, as well as IL-12 (p<0.0001) and IL-1ß (p<0.0429) cytokines levels. Mutant strain displayed opposite regulatory effect on cld1 expression and on IL-12 levels. This work emphasizes the importance of SlpB in P. freudenreichii ability to reduce mucositis inflammation. It opens perspectives for the development of probiotic products to decrease side effects of chemotherapy using GRAS bacteria with immunomodulatory surface protein properties.
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Propionibacterium freudenreichii is a traditional Swiss-type cheeses starter and constitutes an emergent probiotic, exerting several beneficial effects, including anti-inflammatory modulation of gut inflammation. This feature relies on several metabolites and on surface proteins, with a prominent role of the surface protein SlpB. 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 digestive proteolysis, with respect to modulation of HT-29 cells response to a lipopolysaccharide (LPS) challenge. The anti-inflammatory effect exerted by P. freudenreichii CIRM-BIA 129 and by its 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 towards digestive stresses and protection of SlpB towards proteolysis, during two in vitro digestion models: static and dynamic. Taken together, these results show that cheese is an adequate delivery vehicle for P. freudenreichii immunomodulatory proteins. This opens perspectives for the development of fermented dairy functional foods aimed at target populations at high risk for diet-related diseases with an inflammatory component.
Assuntos
Proteínas de Bactérias/metabolismo , Queijo/microbiologia , Produtos Fermentados do Leite/microbiologia , Digestão , Microbiologia de Alimentos/métodos , Proteínas de Membrana/metabolismo , Probióticos , Propionibacterium freudenreichii/metabolismo , Proteínas de Bactérias/imunologia , Colo/imunologia , Colo/metabolismo , Colo/microbiologia , Células HT29 , Humanos , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Cinética , Proteínas de Membrana/imunologia , Propionibacterium freudenreichii/imunologia , ProteóliseRESUMO
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 (Cheese-matrix). 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 TNFα and of IL-10 by peripheral blood mononuclear cells (PBMC). It did not alter IL-10, IFNγ, IL-17, and TNFα 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.
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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.
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Propionibacterium freudenreichii is a beneficial Gram-positive bacterium, traditionally used as a cheese-ripening starter, and currently considered as an emerging probiotic. As an example, the P. freudenreichii CIRM-BIA 129 strain recently revealed promising immunomodulatory properties. Its consumption accordingly exerts healing effects in different animal models of colitis, suggesting a potent role in the context of inflammatory bowel diseases. This anti-inflammatory effect depends on surface layer proteins (SLPs). SLPs may be involved in key functions in probiotics, such as persistence within the gut, adhesion to host cells and mucus, or immunomodulation. Several SLPs coexist in P. freudenreichii CIRM-BIA 129 and mediate immunomodulation and adhesion. A mutant P. freudenreichii CIRM-BIA 129ΔslpB (CB129ΔslpB) strain was shown to exhibit decreased adhesion to intestinal epithelial cells. In the present study, we thoroughly analyzed the impact of this mutation on cellular properties. Firstly, we investigated alterations of surface properties in CB129ΔslpB. Surface extractable proteins, surface charges (ζ-potential) and surface hydrophobicity were affected by the mutation. Whole-cell proteomics, using high definition mass spectrometry, identified 1,288 quantifiable proteins in the wild-type strain, i.e., 53% of the theoretical proteome predicted according to P. freudenreichii CIRM-BIA 129 genome sequence. In the mutant strain, we detected 1,252 proteins, including 1,227 proteins in common with the wild-type strain. Comparative quantitative analysis revealed 97 proteins with significant differences between wild-type and mutant strains. These proteins are involved in various cellular process like signaling, metabolism, and DNA repair and replication. Finally, in silico analysis predicted that slpB gene is not part of an operon, thus not affecting the downstream genes after gene knockout. This study, in accordance with the various roles attributed in the literature to SLPs, revealed a pleiotropic effect of a single slpB mutation, in the probiotic P. freudenreichii. This suggests that SlpB may be at a central node of cellular processes and confirms that both nature and amount of SLPs, which are highly variable within the P. freudenreichii species, determine the probiotic abilities of strains.
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Dairy propionibacteria are used as cheese ripening starters, as biopreservative and as beneficial additives, in the food industry. The main species, Propionibacterium freudenreichii, is known as GRAS (Generally Recognized As Safe, USA, FDA). In addition to another dairy species, Propionibacterium acidipropionici, they are included in QPS (Qualified Presumption of Safety) list. Additional to their well-known technological application, dairy propionibacteria increasingly attract attention for their promising probiotic properties. The purpose of this review is to summarize the probiotic characteristics of dairy propionibacteria reported by the updated literature. Indeed, they meet the selection criteria for probiotic bacteria, such as the ability to endure digestive stressing conditions and to adhere to intestinal epithelial cells. This is a prerequisite to bacterial persistence within the gut. The reported beneficial effects are ranked according to property's type: microbiota modulation, immunomodulation, and cancer modulation. The proposed molecular mechanisms are discussed. Dairy propionibacteria are described as producers of nutraceuticals and beneficial metabolites that are responsible for their versatile probiotic attributes include short chain fatty acids (SCFAs), conjugated fatty acids, surface proteins, and 1,4-dihydroxy-2-naphtoic acid (DHNA). These metabolites possess beneficial properties and their production depends on the strain and on the growth medium. The choice of the fermented food matrix may thus determine the probiotic properties of the ingested product. This review approaches dairy propionibacteria, with an interest in both technological abilities and probiotic attributes.
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Propionibacterium freudenreichii is a beneficial bacterium traditionally used as a cheese ripening starter and more recently for its probiotic abilities based on the release of beneficial metabolites. In addition to these metabolites (short-chain fatty acids, vitamins, and bifidogenic factor), P. freudenreichii revealed an immunomodulatory effect confirmed in vivo by the ability to protect mice from induced acute colitis. This effect is, however, highly strain-dependent. Local action of metabolites and of immunomodulatory molecules is favored by the ability of probiotics to adhere to the host cells. This property depends on key surface compounds, still poorly characterized in propionibacteria. In the present study, we showed different adhesion rates to cultured human intestinal cells, among strains of P. freudenreichii. The most adhesive one was P. freudenreichii CIRM-BIA 129, which is known to expose surface-layer proteins. We evidenced here the involvement of these proteins in adhesion to cultured human colon cells. We then aimed at deciphering the mechanisms involved in adhesion. Adhesion was inhibited by antibodies raised against SlpB, one of the surface-layer proteins in P. freudenreichii CIRM-BIA 129. Inactivation of the corresponding gene suppressed adhesion, further evidencing the key role of slpB product in cell adhesion. This work confirms the various functions fulfilled by surface-layer proteins, including probiotic/host interactions. It opens new perspectives for the understanding of probiotic determinants in propionibacteria, and for the selection of the most efficient strains within the P. freudenreichii species.
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Dairy propionibacteria are actinomycetales found in various fermented food products. The main species, Propionibacterium freudenreichii, is generally recognized as safe and used both as probiotic and as cheese starter. Its probiotic efficacy tightly depends on its tolerance towards digestive stresses, which can be largely modulated by the ingested delivery vehicle. Indeed, tolerance of this bacterium is enhanced when it is consumed within a fermented dairy product, compared to a dried probiotic preparation. We investigated both stress tolerance and protein neosynthesis upon growth in i) chemically defined or ii) aqueous phase of Emmental cheeses. Although the same final population level was reached in both media, a slower growth and an enhanced survival of CIRM BIA 1 strain of P. freudenreichii subsp. shermanii was observed in Emmental juice, compared to chemically defined medium. This was accompanied by differences in substrates used and products released as well as overexpression of various early stress adaptation proteins in Emmental juice, compared to chemically defined medium, implied in protein folding, in aspartate catabolism, in biosynthesis of valine, leucine and isoleucine, in pyruvate metabolism in citrate cycle, in the propionate metabolism, as well as in oxidoreductases. All these changes led to a higher digestive stress tolerance after growth in Emmental juice. Mechanisms of stress adaptation were induced in this environment, in accordance with enhanced survival. This opens perspectives for the use of hard and semi-hard cheeses as delivery vehicle for probiotics with enhanced efficacy.