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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; 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.

Undissociated lactic acid has been shown to play a major role in complete growth inhibition of Listeria monocytogenes in Gouda cheese. In addition, low water activity conditions may contribute to growth inhibition. In the current study, it was assessed whether the major factors that inhibit growth of L. monocytogenes in Gouda cheese are the factors that determine growth in other types of ready-to-eat cheese as well. Various types of cheeses were selected, some of which had been associated with listeriosis, while others had not. Based on the composition of the different cheese types, the concentrations of undissociated lactic acid were calculated for each type. The ability to support growth of L. monocytogenes was predicted using the Gamma model, based on literature data on total lactic acid content, moisture content, fat content, pH, Aw, and temperature, and optimal growth rates in milk at 30–37 °C. In addition, the actual specific growth rates of L. monocytogenes in the various cheeses were calculated based on available experimental growth data. In 9 out of the 10 RTE cheeses reviewed, the undissociated lactic acid concentrations and aw determined growth/no growth of L. monocytogenes. No growth was correctly predicted for feta, Cheddar and Gouda, and growth was correctly predicted for ricotta, queso fresco, Camembert, high-moisture mozzarella, cottage and blue cheese. Growth of L. monocytogenes was not observed in practice upon inoculation of Emmental, whereas growth in this cheese type was predicted when including the above mentioned factors in the models. Other factors, presumably acetic and propionic acid, are thought to be important to inhibit growth of the pathogen in Emmental. The results from our study show that for cheeses in which lactic acid is a main acid, our model based on undissociated lactic acid, temperature, pH and aw gives a good prediction of potential outgrowth of L. monocytogenes. Implications for L. monocytogenes legislation are discussed per type of RTE cheese reviewed.

La texture des aliments, leur densité contribuent à protéger les bactéries qui viendront enrichir notre flore intestinale. Démonstration avec l’emmental.

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 &alpha, (TNF&alpha, ), Interferon &gamma, (IFN&gamma, ) 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.

Le laboratoire UMR STLO vise à développer une expertise publique sur les composants du lait et de l’œuf et à augmenter la qualité et la sécurité des produits laitiers fermentés, via notamment l’isolement et la caractérisation de souches de bactéries bénéfiques d’intérêt alimentaire. L’une des approches consiste à exploiter les propriétés « 2-en-1 » de certaines souches, c’est-à-dire leurs propriétés techno-fonctionnelles (élaboration de produits laitiers fermentés aux caractéristiques désirées) ainsi que leurs propriétés probiotiques. Certaines bactéries mises en œuvre dans l’élaboration de produits laitiers fermentés peuvent en effet exercer un effet bénéfique sur la santé de l’homme.Les bactéries propioniques laitières, dont P. freudenreichii, sont responsables de l’ouverture et de l’arôme caractéristique de l’Emmental, via la production de CO2 et des acides gras à chaîne courte acétate et propionate. Ces derniers sont à l’origine de l’aptitude de P. freudenreichii à moduler la prolifération et l’apoptose, au niveau de cellules épithéliales digestives. Ainsi, la bactérie, un produit laitier fermenté par cette bactérie, ainsi que ses métabolites, induisent le scénario suivant, au contact de cellules cancéreuses gastriques ou intestinales. Arrêt du cycle cellulaire, dépolarisation des mitochondries, accumulation d’espèces réactives de l’oxygène, déplétion de l’ATP, fragmentation en corps apoptotiques, autant de paramètres qui ont été suivis par cytométrie en flux.Les bactéries propioniques laitières, dont P. freudenreichii, ont une composition de surface (surfaceome) extrêmement variable, d’une souche à l’autre. De même, leur capacité à adhérer aux cellules de l’hôte et leur capacité immunomodulatrice, deux paramètres intimement liés, sont variables. Les protéines de surface impliquées dans ce type d’interactions ont été étudiées par une approche pluridisciplinaire combinat génomique, transcriptomique, protéomique et cytométrie en flux. Ainsi, une protéine de la famille des Surface Layer Proteins (Slp), SlpB, s’est révélée cruciale dans le dialogue entre P. freudenreichii et l’hôte.Ces travaux ouvrent de nouvelles perspectives dans le domaine du développement d’aliments sains, sûrs, hédoniques et fonctionnels (bactéries lactiques et propioniques) issus de la fermentation du lait. Ils peuvent aider, en complément des traitements existants, à prévenir et/ou à soigner des pathologies (cancers, maladies inflammatoires chroniques) dont l’incidence croit dans les pays développés comme le nôtre.

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.

les propionibactéries laitières, des bactéries particulières.. Invitation INRA TOXALIM

Propionibacterium freudenreichii (PF) is a food grade bacterium with “generally recognized as safe” status, most commonly used as a starter for the manufacture of fermented dairy products such as Emmental and Leerdammer cheeses. It is also known for its production of vitamin B12 and propionic acid, and is studied for its remarkable anti-inflammatory properties[1,2]. Exopolysaccharide (EPS) production by food bacteria such as PF[3] is receiving increasing attention for their potential applications in improving both texture and the health properties of foodstuff. In this work, we explored the genetic and phenotypic bases for the surface properties of the EPS-producing PF strain P52. In this purpose, the whole genomes of the EPS-producing PF P52 strain and of its spontaneous mutant, with an altered EPS-phenotype, were analyzed to identify genetic determinants for EPS biosynthesis. Using lectin-grafted AFM probes, single-molecule force spectroscopy was used to stretch EPS polymers over immobilized individual bacteria[4,5] of both strains. Results evidenced that spontaneous mutation could lead to 10-fold shortening of the EPS length. Application of the extended Free Join Chain model indicated up to 30×103 Kuhn segments for the EPS-producing mutant, vs 5×103 for the mutant. Figure 1: Typical deflection images and single-molecule force curves obtained by AFM analysis of the wild-type EPS-producing P. freudenreichii strain (left) and of its spontaneous mutant (right) Combination of genetic data and force spectroscopy observations provided insight on the biosynthesis of EPS with variable lengths, therefore on the control of thickness of the bacterial capsule for specific food applications.

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 favoured by the ability of probiotics to adhere to the host cells. This property depends on key surface compounds, still poorly characterized in propionibacteria. We thus investigated the involvement of a specific propionibacterial surface protein in adhesion of the probiotic Propionibacterium freudenreichii to host cells. We compared several strains of Propionibacterium freudenreichii, with respect to adhesion to cultured human intestinal epithelial cells, using a well-established model. We inactivated genes using a previously developed method, based on the integration of a non-replicative plasmid, harbouring a portion of the target gene. We analysed adhesion of the wild type and the mutant strains using fluorescent-assisted flow cytometry and confocal microscopy. We also sought inhibition of adhesion using antibodies directed against surface layer proteins. 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.

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.

Source : https://www.diplomatie.gouv.fr Brésil - Mai 2018 CBB CAPBIOTEK RENNES; Des scientifiques brésiliens et français travaillent en collaboration pour développer plusieurs projets de recherche autour des bactéries impliquées dans les processus pro et anti-inflammatoires des glandes mammaires et intestinales.Dans le cadre du Laboratoire International Associé (lia) réunissant trois laboratoires de l’UFMG au Brésil (Université Fédérale du Minas Gerais) et deux laboratoires de l’INRA ( UMR STLO de Rennes et Micalis de Jouy-en-Josas - équipe ProbiHote), le projet « Bact-Inflam » a pour objectif de mieux comprendre l’implication des bactéries dans les processus pro ou antiinflammatoires associés aux maladies inflammatoires chroniques de l’intestin (MICI) et aux mammites. Ainsi, plusieurs lignées de bactéries, déjà employées dans la fabrication de produits laitiers, sont utilisées pour faire de l’emmental un aliment probiotique ; le fromage servant de matrice de protection pour ces bactéries. Au total, trois bactéries sont étudiées : Propionibacterium freudenreichii, Streptococcus thermophilus et Lactobacillus delbrueckii. Les résultats mettent en évidence une amélioration de l’inflammation intestinale chez des souris atteintes de colite ulcéreuse après que celles-ci ont ingéré le fromage produit avec ces trois types de bactéries.Source : https://www.diplomatie.gouv.fr Brésil - Mai 2018

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.

Propionibacterium freudenreichii is a beneficial bacterium used as vitamin producer, bio-preservative, cheese ripening starter and probiotic. It adheres to intestinal epithelial cells and mucus and modulates the immune response. Interaction with the host is strain-dependent and relies on key surface proteins, still poorly investigated. We identified surface-exposed proteins in a strain previously selected for its probiotic properties. The role of these proteins in immunomodulatory properties was evidenced. Cheese being a major source of bacteria, we developed an experimental pressed cheese, exclusively fermented by a single strain of P. freudenreichii. Key surface proteins involved in the observed immunomodulatory effects were expressed within the cheese matrix. Consumption of this single-strain cheese protected mice from acute colitis induced by TNBS. P. freudenreichii expressed the key genes of stress tolerance response during growth in the aqueous phase of Emmental cheese, in agreement with enhanced stress tolerance, compared to laboratory culture medium.This work confirms the probiotic potential of P. freudenreichii, it provides a new functional fermented product for preclinical and clinical studies aimed at prevention or treatment of IBD. It constitutes a basis for further studies aimed at the elucidation of mechanisms responsible for its probiotic effects, in a post-genomic context.

Oiling-off is essential for the culinary functional properties of hard- and semi-hard cheeses. However, the parameters implied in the mechanisms leading to oiling-off are not well known. In Emmental cheese, a marked change in the supramolecular organization of fat occurs during the pressing of the warm curd and could be related to the oiling-off of cheese. To check this hypothesis and to determine the relative effects of the temperature and pressing strength, twin-cheeses were submitted to various cooling rates after the curd have been dipped from the vat. These cheeses were either pressed or non-pressed. Using this strategy, we showed that a higher heat load yields to larger inclusions of fat in the cheese matrix, which has been characterised using confocal laser scanning microscopy, and to higher oiling-off. The heat load applied to the curd (integral of the temperature vs. time curve) and oiling-off of cheeses aged of 5 days were linearly related with r2 = 0.89. In contrast, the pressing strength did not influence oiling-off. As a conclusion, the heat load applied during pressing and acidification could be a tool to modulate the oiling-off of Swiss cheese.

La microbiologie du lait et de sa transformation

The production of «Emmental» was unknown in the west of France at the beginning of the 1950s, most of this type of cheese being produced in the eastern part of Central France. Over the following decades, the geography of cheese-production underwent considerable changes. In 2001, 68,8 % of all "Emmental" cheese was produced in western France, while the eastern part of Central France only produced 27,1 %. In 2003, Brittany and the Loire Region increased their production up to 70 % and should soon reach 75 %. At the same time, despite a large decrease in the production of "Emmental , the eastern part of Central France has remained a major, French, cheese-producting area, thanks to the development of other kinds of cheeses, such as "Comté", "Morbier" and "Cancoillote". This article will attempt to throw some light on the vast area of French cheeses on the move., Un début des années 1950, la production d'emmental était inconnue dans l'Ouest de la France alors que l'essentiel de ces fabrications fromagères était concentré dans l'Est central français. Dans les décennies suivantes, cette géographie a connu une mutation considérable ; en 2001, 68,8% de l'emmental produit en France l'est dans l'Ouest alors que l'Est central n'en représente plus que 27, 1 %. En 2003, Bretagne et Pays de la Loire accentuent leur prépondérance (70 %) et devraient rapidement atteindre les 75 %. Dans le même temps, malgré un recul important de la production d'emmental, l'Est central continue à représenter une grande région fromagère nationale grâce au développement d'autres fabrications (comté, morbier, cancoillotte, etc.). Cette étude se propose de mettre en lumière quelques explications de ce vaste «déménagement» du territoire fromager français., Calvez Eugène. Un récent «déménagement» du territoire fromager français: l'exemple de l'emmental. In: L'information géographique, volume 69, n°2, 2005. pp. 184-194.

Mit Hightech ergründen Forscher das Geheimnis des Käses – und machen wieder gut, was die Massenproduktion ihm angetan hatDer Schatz ruht in einer Truhe, hinter schweren Stahltüren. Um dorthin zu gelangen, tippt Anne Thierry an der Kellerwand ihres Instituts einen Sicherheitscode ein und tritt durch eine weiße Doppeltür, die dumpf hinter ihr zufällt. "Hier drinnen ist er", sagt sie und hantiert mit grauen Wollhandschuhen an dem mächtigen Hebel eines Schrankes. Der Schatz ist 15 Zentimeter groß und 800 Gramm schwer – leichter als Gold, aber ähnlich bedeutsam. In Alufolie und eine Plastiktüte gewickelt, lagert hier: Käse. Tiefgefrorener Emmentaler, um genau zu sein.Anne Thierry ist Mikrobiologin. Sie und ihre Kollegen vom Nationalen Institut für Agrarforschung im französischen Rennes ergründen das Innenleben des Käses. Einmal im Jahr lässt Thierry dafür die seltenen Emmentaler-Exemplare in der hauseigenen Käserei unter strengen Vorgaben herstellen. An ihnen untersucht sie, wie sich der Mikrokosmos im Käselaib im Laufe der Reifung verändert und wie bestimmte Bakterien das Aroma prägen. "Das Zusammenspiel der Mikroorganismen ist extrem komplex", sagt Thierry. Sie möchte es sich dennoch zunutze machen. Mithilfe von Mikroben wollen die Käseforscher den Geschmack und die Konsistenz, ja sogar die Form der Löcher steuern. Käse wird zum Hightech-Produkt.Ein großer Holzbottich am Eingang des Institutsgebäudes erinnert daran, wie Käse einst hergestellt wurde. Von Bauern auf der Alm, als liebevoll geformte Einzelstücke. Käse war mal ein urtümliches Naturprodukt und das Ergebnis einer geduldigen Reifung. Heute dagegen stammen die meisten Sorten aus der industriellen Großproduktion, die einförmige Laibe von immer derselben Qualität erschafft, wie aus der Batterie. Auf traditionelle Weise wäre die enorme Käsemenge, die wir jährlich vertilgen, tatsächlich längst nicht mehr zu produzieren. Allein die Deutschen essen 24 Kilogramm Käse pro Kopf im Jahr – noch kräftiger langen in Europa nur die Griechen und die Franzosen zu.Im Laufe der vergangenen Jahrzehnte wurden die Abläufe in der Käseherstellung daher zunehmend standardisiert, die Reinigung der Milch mittels Erhitzung und aufwendiger Filterverfahren wurde optimiert. Vom romantischen Holzbottich fehlt heute jede Spur, dafür tummeln sich keinerlei gesundheitsschädliche Mikroorganismen mehr in der Milch. Die Hygiene ist gut für die Bekömmlichkeit, aber schlecht für den Geschmack. "Die Milch ist heute so sauber, dass daraus kein guter Käse mehr entsteht, wenn wir keine Bakterien von außen hinzufügen", erklärt Thierry. Deshalb braucht es Wissenschaftler wie sie, die dem Käse sein Charisma zurückgeben.Käseproduzenten möchten außerdem neue, originelle Kreationen entwickeln, um die Gaumen ihrer Kunden zu überraschen. Die Hersteller zerbrechen sich die Köpfe, wie sie Prachtexemplare von zarter Textur und nie da gewesenem Aroma erfinden können. Sie setzen dabei auf Bakterien. Anne Thierry widmet sich seit zwei Jahrzehnten vor allem einem Bakterium, das im Emmentaler lebt: Es trägt den schönen Namen "Propionibacterium freudenreichii". Diese Mikroben sind eine Art Tausendsassa: Sie prägen die Bildung des Emmentaler-Aromas, beeinflussen die Reifung und die Lochbildung im Inneren. Thierry kennt das Bakterium gut. Sie weiß, dass es bei kalten und warmen Temperaturen überleben kann, in saurer und salziger Umgebung gedeiht. Und dass schon ein einziges Gramm Emmentaler Milliarden dieser Bakterien beherbergt.Benannt wurden die Mikroben nach dem Berner Bakteriologen Eduard von Freudenreich, der die Käseforschung schon früh prägte. Er las im 19. Jahrhundert die Arbeiten des französischen Mikrobiologen Louis Pasteur, der erkannt hatte, dass die Käsereifung eine Art Gärung darstellt, ähnlich wie bei Bier und Wein. Schon Pasteur wusste, dass Mikroorganismen für diesen Prozess entscheidend sind. Doch welches Bakterium ist maßgeblich? Mehr als 30 Jahre lang stritten Nahrungsmittelforscher darüber, verwarfen eine Theorie über Heubazillen, widerlegten die These, dass sporenbildende Bakterien dafür ausschlaggebend sind. Bis Eduard von Freudenreich den Disput vor rund 100 Jahren beendete. Er identifizierte das Milchsäure-Bakterium als entscheidenden Akteur der ersten Phase in der Käsereifung. Und er entdeckte obendrein noch das Propionibacterium freudenreichii im Emmentaler, als eine Hauptfigur des zweiten Akts.Dabei war Käse schon zu Zeiten Freudenreichs ein uraltes Produkt. Die erste Beschreibung eines Gorgonzola datiert aus dem Jahr 879 nach Christus, die eines Roquefort aus dem Jahr 1070. Und die Historie reicht sogar noch weiter zurück: Schon vor mindestens 7.000 Jahren haben unsere Vorfahren Käse produziert, das berichteten Forscher der University of Bristol kürzlich in der Fachzeitschrift Nature (Salque et. al., 2013). In Polen hatten die Wissenschaftler Scherben und Tongefäße aus jener Zeit gefunden – siebartige Töpfe, zehn Zentimeter im Durchmesser, mit etwa drei mal drei Millimeter großen Löchern. "An 40 Prozent dieser Gefäßscherben fanden wir Fettrückstände", berichtet der Chemiker und Studienautor Richard Evershed. Unsere Vorfahren nutzten die Tontöpfe demnach, um geronnene Milch von der Molke zu trennen und Käse herzustellen.

Ementaler spada u najpoznatije vrste sira u Europi koji se proizvodi već od 13. stoljeća u dolini rijeke Emme u Švicarskoj, po kojoj je i dobio ime. Prepoznatljiv je po svojim šupljinama i slatkastom okusu. Spada u tvrde masne sireve koji zriju najmanje 4 mjeseca, a zbog svoje veličine (100 do 120 kg), mirisa i okusa ponosno nosi titulu „kralj sireva“. Tek od 1953. godine Švicarska ga je uspjela zaštititi oznakom „izvorno švicarsko“ što nije spriječilo da njegova proizvodnja dosegne visoku kvalitetu i u ostalim europskim državama kao što su Njemačka, Francuska, Austrija, Finska… U Švicarskoj se proizvodi još uvijek na autohtoni način od sirovog kravljeg mlijeka koji uključuje dosta truda i ručnog rada što rezultira proizvodom vrhunske kvalitete. Tako proizveden ementaler nosi oznaku AOC te na sebi ima otisnut broj farme na kojoj je proizveden. Kako je potražnja za sirom rasla, nova industrijska postignuća su omogućila olakšanu i bržu proizvodnju ementalera u većim količinama sa zadržanom kvalitetom proizvoda. Tako su u pojedinim fazama ručni rad zamijenili strojevi. Danas se kod obrade gruša umjesto ručnog miješanja koriste različite izvedbe miješalica u kadama za sirenje. Korak naprijed jest i korištenje separatora sirnog zrna od sirutke što je izbacilo iz upotrebe korištenja platna za sir. Kalupljenje je također mehanizirano, dok se za prešanje koriste predpreše i kontinuirane preše koje omogućavaju kontinuiranu proizvodnju bez zastoja. Tradicionalno su se za salamurenje koristili betonski ili drveni bazeni koji su u industriji zamijenjeni praktičnijim sustavima za soljenje koji također omogućavaju kontinuiranost. Rezultat unaprjeđenja proizvodnje jest raširenost i dostupnost ovoga „kralja sireva“ diljem svijeta. Emmental is one of the most famous cheeses in Europe, which is produced from the 13th century in the valley of the Emme River in Switzerland, after which it was named. It is recognizable by its cavities and sweet taste. It belongs to the hard fat cheeses ripened for at least four months, and because of its size (100-120 kg), smell and taste proudly carries the title of "king of cheeses". Only the 1953rd year Switzerland had failed to protect the label "originally Swiss" which did not prevent its production reaches a high quality and in other European countries such as Germany, France, Austria, Finland, etc. In Switzerland, still makes the indigenous way from raw cow's milk, which involves a lot of hard work and manual labor, resulting in a product of superior quality. That produced Emmental AOC labeled and wearing a printed number on the farm where it is produced. As the demand for cheese has grown, new industrial achievements are made possible and facilitated rapid production of Emmental cheese in large quantities while keeping the quality of the product. Thus, in certain phases of handmade replace with machines. Today in curd processing instead of manual mixing using various performance blender in tubs for expansion. A step forward is the use of separator curd from whey which is washed out of the use of using a cheese cloth. Molding is also mechanized, while pressing and continuous use of the pre presses that enable continuous production without downtime. Traditionally used for brining concrete or wooden pools that are in the industry replaced with practical systems for salting which also provide continuity. The result of the production improvement is the spread and availability of this "king of cheeses" around the world.

Propionibacterium freudenreichii is the main Propionibacterium consumed both in traditional cheeses and in innovative probiotic product. A portion of 10 grams of Emmental cheese provides around 10 10 live P. freudenreichii , a major bacterial intake in France and other industrialized countries. Surprisingly, little is known about its effect on human health, compared to lactobacilli or bifidobacteria. However, recent works indicate promising beneficial effects including modulation of intestinal microbiota, metabolic activities and inflammation. In this context, the molecular mechanisms responsible for the immunomodulatory properties of P.freudenreichii , and of fermented dairy product containing it, were investigated. In that aim, an integrative approach was undertaken, in a research program of the French National Agency for Research (ANR). It consisted of the sequencing and annotation of the genomes of 20 strains, followed by comparative genomics, in the design of a pangenomic chip, followed by comparative transcriptomic analysis , in a comparative surface proteomic analysis, and in comparison of the immunomodulatory properties, ex vivo and in vivo , using model cheeses. This investigation reveals, for the first time, the molecular mechanism responsible for modulation of the immune system and of inflammation by P.freudenreichii . These results are in accordance with clinical results obtained by others with the same bacterium

MicrobiologieUn projet de recherche explore le potentiel anti-inflammatoire de certaines bactéries présentes dans l’emmental.Les Français sont de gros mangeurs d’emmental. Ils en consomment environ 20 g par personne et par jour en moyenne. Outre le calcium, nous ingérons grâce à cet emmental des milliards de milliards de bactéries. Depuis 2011, le projet Surfing(1), coordonné par Gwenaël Jan, de l’Inra de Rennes, s’intéresse aux propriétés de certaines d’entre elles : les bactéries propioniques laitières. « Ce sont des levains lactiques. Elles sont bien connues et utilisées par l’industrie agroalimentaire pour leurs aptitudes à apporter un arôme, une texture ou les “yeux” du fromage. Et elles sont ultrarésistantes ! »Une sélection prometteuseCes bactéries peuvent-elles, en plus, avoir des qualités pour notre santé ? « Pour le projet, nous avons choisi Propionibacterium freudenreichii, car nous avions déjà décrypté le génome de vingt souches. » En parallèle, une équipe de l’Inra de Jouy-en-Josas(2) s’est attaquée à la même problématique avec des lactobacilles présents dans le yaourt. « Nous nous sommes focalisés sur la modulation de maladies inflammatoires de l’intestin, qui augmentent en Europe. » Des études précédentes avaient déjà montré que d’autres bactéries alimentaires pouvaient avoir un effet bénéfique dans le traitement de l’une de ces pathologies, la rectocolite hémorragique.« C’est un projet exploratoire, préclinique, précise Gwenaël Jan. Nous avons mené des expérimentations in vitro puis sur des modèles animaux. » Les chercheurs ont d’abord comparé les génomes de vingt souches de bactéries, qui ont ensuite été envoyées à l’Institut Pasteur de Lille, où leur capacité à agir sur la réponse immunitaire, donc sur l’inflammation, ont été testées. Certaines se sont démarquées dès cette étape. « Nous avons confirmé ces différences par d’autres tests in vitro, puis nous avons sélectionné deux souches : la plus et la moins prometteuse, pour les tester sur des souris. » Les résultats ont été à la hauteur des attentes : les bactéries au plus fort potentiel in vitro ont révélé de véritables effets anti-inflammatoires sur les petits rongeurs malades. « Cela ne signifie pas que l’on peut soigner avec ces bactéries, mais qu’elles peuvent avoir un rôle positif dans le traitement. »Du fromage pour les sourisEn parallèle, avec un partenaire industriel(3) et un centre technique(4), l’équipe a fait réaliser des fromages contenant chacun une des deux bactéries testées. « C’était un défi technique car, normalement, il faut plusieurs bactéries différentes pour faire un fromage ! » Les produits obtenus ne ressemblent pas vraiment au beau morceau d’emmental du commerce, mais peu importe. Les chercheurs ont été soulagés de voir que les bactéries n’avaient pas été altérées par cette transformation, par les variations de températures notamment. Quelques échantillons sont partis à l’Institut Pasteur de Lille, pour nourrir les souris malades. « Ces résultats, encourageants, ne sont pas encore publiés », précise le scientifique. Le projet Surfing se termine, mais dès 2015, l’équipe de l’Inra va pouvoir travailler avec des gastroentérologues du CHU de Rennes, dans le cadre d’un projet retenu par le Conseil régional de Bretagne(5), pour tester la souche la plus prometteuse dans une étude clinique.

Propionibacteria derived from dairy products are relevant starter cultures for the production of Swiss and Emmental-type cheeses, and the monitoring of which is mandatory for proper quality control. This study aimed to evaluate an alternative procedure to enumerate propionibacteria, in order to develop a reliable and practical methodology to be employed by dairy industries. 2,3,5-triphenyltetrazolium chloride (TTC) inhibitory activity was tested against five reference strains (CIRM 09, 38, 39, 40 and 116); TTC at 0·0025% (w/v) was not inhibitory, with the exception of one strain (CIRM 116). Subsequently, the four TTC-resistant strains, three commercial starter cultures (PS-1, PB-I, and CHOO) and twelve Emmental-type cheese samples were subjected to propionibacteria enumeration using Lithium Glycerol (LG) agar, and Petrifilm™ Aerobic Count (AC) plates added to LG broth (anaerobic incubation at 30 °C for 7 d). Petrifilm™ AC added to LG broth presented high counts than LG agar (PPPPropionibacteriumspp., despite some limitations observed for specific commercial starter cultures.

Pakistan is amongthe top ten milk producers in the world with 35.5 billion liters annualproduction (FAOSTAT, 2010). It is the 2nd largest buffalo and goatmilk producer in the world with 22.3 and 0.7 billion liters after India andBangladesh, respectively. Buffalo milk’s share is the highest (63%) in thenational milk production with fairly large population of 30.8 million (FAOSTAT,2010). 97% of the total milk production goes into informal sector and only 3%is being processed into UHT, pasteurized and flavoured milk in addition toyoghurt, butter, desi-ghee and cheese. Cheesemanufacturing is very negligible, if some industries are making, they areinterested into pizza cheese or processed one. Even a single ripened cheese isnot being manufactured in such a big milk producing county.The objective of this study was to develop and characterize emmentalcheese with dual purposes; one as pizza cheese and other to create taste forripened cheese in local consumers through their preferred buffalo milk. In thisstudy, emmental of cow milk was used as a reference. To achieve the aim, bothtypes of cheeses were ripened for a period of 70 days and analysed for their physico-chemical,compositional, microbiological, rheological and sensorial characteristics.As function of storage period, pH of buffalo cheese decreased lesssharply than that of cow milk may be due to high buffering capacity whereasacidity of both cheeses was similar. Fat, total protein and non-casein nitrogencontents of buffalo cheese were higher. Non protein nitrogen was almost similarin both cheeses. Ash contents were also higher than that of cow cheese confirmingits more mineral contents as well. Calcium contents were almost twice than thatof cow milk cheese. Magnesium was also higher in cheese from buffalo milk. Totalviable count was lower in cheese from buffalo milk with negative coliforms inboth. Buffalo milk cheese showed more firmness. The judge’s panel gave higheroverall acceptability scores for cheese made from buffalo milk. It might be astart of new era for quality cheese products in the dairy sector of Pakistanand can open new market doors locally as well as internationally on the basisof its uniqueness of buffalo milk.

For Emmental manufacture two kinds of adjunct culture are added: (i) thermophilic lactic acid bacteria (starters) such as Lactobacillus helveticus (LH), and Streptococcus thermophilus (ST) growing the first day of the manufacture and (ii) ripening culture. ST and LH have a key role in curd acidification and proteolysis at the beginning of the manufacture but are considered to be lyzed for a great part of them at the ripening step. The aim of this work was to assess the metabolic activity of these bacteria throughout manufacture and ripening. During Emmental cheesemaking, LH and ST were subjected to i) population quantification by numerations and by quantitative PCR (qPCR) ii) reverse transcription (RT) Temporal Temperature Gel Electrophoresis (TTGE) iii) transcript quantification by RT-qPCR targeting 16S rRNA, tuf and groL mRNAs to evaluate bacterial metabolic activity. During ripening, ST and LH numerations showed a 2.5 log(10) loss of culturability whereas qPCR on pelleted cells revealed only one log(10) of decrease for both of these species. 10(9) ST and 10(8) LH cells/g of cheese still remained. They contained a stable number of 16S transcript and at least 10(6) copies of mRNAs per 10(9) cells until the end of ripening. These results prove the unexpected persistency of thermophilic lactic acid bacteria starters (ST and LH) metabolic activity until the end of ripening and open new perspectives in term of their involvement in the quality of cheeses during ripening.

Cette publication est également parue dans la revue "Journal of Animal Science", Vol.89, E-Suppl.1.; In contrast to other lactic acid bacteria, Lactobacillus helveticus can possess two cell-envelope proteinases (CEPs) called PrtH2 and PrtH (Genay et al 2009, Appl. Env. Microbiol.. 75 (10), 3238-3249; Sadat et al 2011a, in press Int. J. Food Microbiol.). These CEPs exhibit different cleavage specificity on pure s1 -casein (Sadat-Mekmene et al., 2011b, Appl. Env. Microbiol. 77(1) 179-186). The aim of this work was to investigate the proteolytic activity of L. helveticus strains in cheese matrix according to the number of their CEPs and the cheese stretchability. Two strains were selected, ITGLH77 and ITGLH1 which possess one CEP, PrtH2, and two CEPs, PrtH and PrtH2, respectively. The proteolysis was monitored during ripening:i) casein hydrolysis by urea-PAGE; ii) peptide pattern of the cheese aqueous extracts first by SDS-PAGE and second by RP-HPLC. Three chromatographic fractions were collected and peptides identified by RP-HPLC coupled on-line with tandem mass spectrometry. In parallel, the dynamic of stretchability of Emmental cheese was measured in order to determine the contribution of CEPs in its generation, since L. helveticus is known to enhance stretching properties in contrast to other lactobacilli, such as L. delbrueckii subsp. lactis . The microstructure of the strands of Emmental cheeses was observed by confocal laser scanning microscopy. The stretchability of Emmental was significantly higher in cheese manufactured with strain ITGLH77. By using Principal Component Analyses, the stretching properties were found to be correlated with the presence of peculiar peptides derived from primary proteolysis, hydrophobic peptides, containing more than 20 amino acid residues and derived from s1 -casein, which were accumulated in cheese manufactured using strain ITGLH77. The presence of these peptides suggest their less hydrolysis by this strain, in agreement with results observed in vitro (Sadat-Mekmene et al., 2011b), namely different cleavage specificity of CEPs PrtH et PrtH2 on pure s1 -casein.

Mention d'édition : 2ème édition; Propionibacterium ssp.

L’augmentation de l’incidence des maladies inflammatoires chroniques de l’intestin dans les pays industrialisés est un problème de santé émergent. Certaines bactéries ingérées ou commensales peuvent limiter ces maladies par un effets anti-inflammatoire. Les composés de surface de 23 souches de Propionibacterium freudenreichii, une bactérie retrouvée dans l’Emmental qui a démontré des propriétés anti-inflammatoires souche-dépendantes, sont étudiées. Pour ce faire, une extraction des protéines de surface liées de manière non covalente à la paroi bactérienne a été effectuée pour toutes les souches. La séparation de ces extraits indiquait que les protéines de surface extractibles sont variables selon les souches en termes de nature et de quantité. Un marquage se surface in situ à la cyanine 5, suivi d’une extraction des protéines totales a été réalisé. Par la suite, une séparation par 2D-PAGE a permis d’identifier par spectrométrie de masse le protéome de surface et de constater que les protéines marquées présentaient une variabilité souche dépendante. De plus, la présence d’un polysaccharide de surface a été recherchée chez l’ensemble des souches, ce qui a permis de constater qu’environ 35% des souches en possédaient. Enfin, le potentiel zêta, qui caractérise la charge ionique globale en surface, a été mesuré pour chacune des souches. Il semblerait que ce paramètre physico-chimique soit lié à la présence de certaines protéines en surface des bactéries. Ces travaux offrent des perspectives concernant la sélection de protéines candidates pour l’étude des interactions de P. freudenreichii avec son hôte. Des mutants pourront être réalisés afin de vérifier l’impact de ces protéines dans les propriétés physico-chimiques et immunologiques observées.

Stretchability is a techno-functional characteristic of some cheeses such as Emmental, Mozzarella and Cheddar, which gives one of the most desirable cooking properties for consumers. Stretchability would be highly dependent on the proteolytic potential of the bacterial species present in the cheese ecosystem, especially Lactobacillus helveticus (Richoux et al., 2009)1. Proteolytic enzymes include: 1) Cell envelope proteinases located at the bacterial surface which are responsible for the initial degradation of the caseins into peptides and; 2) Intracellular peptidases, released after autolysis of bacteria, which hydrolysed these peptides into smaller ones and amino acids. Aim: The objective was to investigate the dynamic of the stretchability of Swiss-type cheese made with two strains of Lactobacillus helveticus that differed by their ability to release intracellular peptidases in cheese through autolysis.

Lipolysis plays an important role in the formation of cheese flavor. In Emmental cheese, the main part of lipolysis has been associated with the presence of Propionibacterium freudenreichii , a species used as a ripening culture. Our aim was to identify the most probable lipolytic esterase(s) involved in cheese lipolysis by P. freudenreichii . Since cheese lipolysis mainly occurs during P. freudenreichii growth, we hypothesized that P. freudenreichii possesses secreted lipolytic esterase(s). For 12 putative esterase genes previously identified from the genome of P. freudenreichii CIRM1, the level of expression was quantified by real-time reverse transcriptase (RT)-PCR, and the subcellular localization of esterases was predicted in silico . The esterase activity in extracellular and intracellular extracts of P. freudenreichii was characterized by zymography, and the extracellular esterases were identified by mass spectrometry. Finally, the best candidate was overexpressed in the same strain. All of the 12 genes encoding putative esterases were expressed. Esterase PF#279 was predicted to be secreted in the medium, PF#774 to be surface exposed, and the 10 remaining putative esterases to be intracellular. Zymography revealed that esterase activities in culture supernatant differed from the ones detected in intracellular extracts. PF#279 was identified as the sole esterase present in culture supernatant. Transformed P. freudenreichii CIRM1 clones overexpressing PF#279 showed 5 to 8 times more lipolytic activity on milk fat than the wild-type strain. Combining in silico , biochemical, and genetic approaches, we showed that PF#279 is the sole secreted esterase in P. freudenreichii and is active on milk fat. Therefore, it is likely a key component in cheese lipolysis by P. freudenreichii .

Bacterial communities of fermented foods are usually investigated by culture-dependent methods. Real-time quantitative PCR (qPCR) and reverse transcription (RT)-qPCR offer new possibilities to quantify the populations present and their metabolic activity. The aim of this work was to develop qPCR and RT-qPCR methods to assess the metabolic activity and the stress level of the two species used as ripening cultures in Emmental cheese manufacture, Propionibacterium freudenreichii and Lactobacillus paracasei. Three small scale (1/100) microbiologically controlled Emmental cheeses batches were manufactured and inoculated with Lactobacillus helveticus, Streptococcus thermophilus, P. freudenreichii and L. paracasei. At 12 steps of cheese manufacture and ripening, the populations of P. freudenreichii and L. paracasei were quantified by numerations on agar media and by qPCR. 16S, tuf and groL transcript levels were quantified by RT-qPCR. Sampling was carried out in triplicate. qPCR and RT-qPCR assessments were specific, efficient and linear. The quantification limit was 103 copies of cells or cDNA/g of cheese. Cell quantifications obtained by qPCR gave similar results than plate count for P. freudenreichii growth and 0.5 to 1 log lower in the stationary phase. Bacterial counts and qPCR quantifications showed that L. paracasei began to grow during the pressing step while P. freudenreichii began to grow from the beginning of ripening (in the cold room). Tuf cDNA quantification results suggested that metabolic activity of L. paracasei reached a maximum during the first part of the ripening (in cold room) and decreased progressively during ripening (in the warm room). Metabolic activity of P. freudenreichii was maximum at the end of cold ripening room and was stable during the first two weeks in warm room. After lactate exhaustion (after two weeks of warm room), the number of tuf cDNA decreased reflecting reduced metabolic activity. For L. paracasei, groL cDNA were stable during ripening. For P. freudenreichii, groL1 gene was highly-expressed during acidification, while groL2 gene highly expression was only observed at the end of the ripening stage after lactate (carbon substrate of P. freudenreichii) exhaustion. The potential use of 16S and tuf genes for the normalization of cDNA quantification throughout an Emmental cheese manufacture is discussed. For the first time, specific gene expression was performed by RT-qPCR yielding metabolic activity and stress response evaluation for L. paracasei and P. freudenreichii in cheese.

Format du poster : 21 X 29,7; Phosphopetides in hard cooked type cheeses are only partially degraded by peptidases of thermophilic lactic acid bacteria. 4.IDF Dairy Science and Technology Week, 20-24 th April 2009, Rennes, France