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Bacterial extracellular vesicles (EVs) are natural lipidic nanoparticles implicated in intercellular communication. Although EV research focused mainly on pathogens, the interest in probiotic-derived EVs is now rising. One example is Propionibacterium freudenreichii , which produces EVs with anti-inflammatory effects on human epithelial cells. Our previous study with P. freudenreichii showed that EVs purified by size exclusion chromatography (SEC) displayed variations in protein content according to bacterial growth conditions. Considering these content variations, we hypothesized that a comparative proteomic analysis of EVs recovered in different conditions would elucidate whether a representative vesicular proteome existed, possibly providing a robust proteome dataset for further analysis. Therefore, P. freudenreichii was grown in two culture media, and EVs were purified by sucrose density gradient ultracentrifugation (UC). Microscopic and size characterization confirmed EV purification, while shotgun proteomics unveiled that they carried a diverse set of proteins. A comparative analysis of the protein content of UC- and SEC-derived EVs, isolated from cultures either in UF (cow milk ultrafiltrate medium) or YEL (laboratory yeast extract lactate medium), showed that EVs from all these conditions shared 308 proteins. This EV core proteome was notably enriched in proteins related to immunomodulation. Moreover, it showed distinctive features, including highly interacting proteins, compositional biases for some specific amino acids, and other biochemical parameters. Overall, this work broadens the toolset for the purification of P. freudenreichii -derived EVs, identifies a representative vesicular proteome, and enumerates conserved features in vesicular proteins. These results hold the potential for providing candidate biomarkers of purification quality, and insights into the mechanisms of EV biogenesis and cargo sorting. [ABSTRACT FROM AUTHOR]

Propionibacterium freudenreichii is a beneficial bacterium that modulates the gut microbiota, motility and inflammation. It is traditionally consumed within various fermented dairy products. Changes to consumer habits in the context of food transition are, however, driving the demand for non-dairy fermented foods, resulting in a considerable development of plant-based fermented products that require greater scientific knowledge. Fermented soymilks, in particular, offer an alternative source of live probiotics. While the adaptation of lactic acid bacteria (LAB) to such vegetable substrates is well documented, little is known about that of propionibacteria. We therefore investigated the adaptation of Propionibacterium freudenreichii to soymilk by comparison to cow's milk. P. freudenreichii grew in cow's milk but not in soymilk, but it did grow in soymilk when co-cultured with the lactic acid bacterium Lactobacillus plantarum. When grown in soymilk ultrafiltrate (SUF, the aqueous phase of soymilk), P. freudenreichii cells appeared thinner and rectangular-shaped, while they were thicker and more rounded in cow's milk utltrafiltrate (MUF, the aqueous phase of cow milk). The amount of extractable surface proteins (SlpA, SlpB, SlpD, SlpE) was furthermore reduced in SUF, when compared to MUF. This included the SlpB protein, previously shown to modulate adhesion and immunomodulation in P. freudenreichii. Tolerance toward an acid and toward a bile salts challenge were enhanced in SUF. By contrast, tolerance toward an oxidative and a thermal challenge were enhanced in MUF. A whole-cell proteomic approach further identified differential expression of 35 proteins involved in amino acid transport and metabolism (including amino acid dehydrogenase, amino acid transporter), 32 proteins involved in carbohydrate transport and metabolism (including glycosyltransferase, PTS), indicating metabolic adaptation to the substrate. The culture medium also modulated the amount of stress proteins involved in stress remediation: GroEL, OpuCA, CysK, DnaJ, GrpE, in line with the modulation of stress tolerance. Changing the fermented substrate may thus significantly affect the fermentative and probiotic properties of dairy propionibacteria. This needs to be considered when developing new fermented functional foods. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Lactobacillus delbrueckii subsp. bulgaricus is a beneficial lactic acid bacterium and constitutes one of the most used, and thus consumed, dairy starters, worldwide. This homofermentative bacterium was the first lactobacillus described and is involved in the fermentation of yogurt and of diverse other fermented products, including cheeses. It has a long history of safe use, as well as documented probiotic lato sensu effects, including alleviation of lactose intolerance. Plant-based fermented products presently experience a considerable development, as a result of evolution of consumers' habits, in a general context of food transition. This requires research and development, and thus scientific knowledge, to allow such transition, including the development of fermented soy milks. These last indeed offer an alternative source of live and active bacteria. The yogurt starters L. delbrueckii subsp. bulgaricus , together with Streptococcus thermophilus , have been implemented to generate yogurt-type fermented soy milks worldwide. While the adaptation of these starters to the dairy environment has been extensively studied, little is known about L. delbrueckii adaptation to the soy environment. We therefore investigated its adaptation to soy milk and compared it to cow's milk. Surprisingly, it did not grow in soy milk, neither alone, nor in co-culture with S. thermophilus. Acidification of soy milk was however faster in the presence of both species. In order to deepen such adaptation, we then compared L. delbrueckii growth and survival in soy milk ultrafiltrate (SUF, the aqueous phase of soy milk) and compared it to cow's milk ultrafiltrate (MUF, the aqueous phase of cow milk). This comparison revealed major differences in terms of cell morphology and proteome composition. Lactobacilli appeared deformed and segmented in soy. Major differences in both the surface and the cellular proteome indicated upregulation of stress proteins, yet downregulation of cell cycle and division machinery. Altogether, these results suggest that soy milk may be a stressing environment for the yogurt starter L. delbrueckii subsp. bulgaricus. • Low populations of lactobacilli are found in commercial fermented soy milks. • Lactobacillus delbrueckii subsp. bulgaricus grows poorly in soy milk. • Soy milk induces major morphological changes in Lactobacillus delbrueckii subsp. bulgaricus. • Lactobacillus delbrueckii subsp. bulgaricus accumulates a series of stress proteins in soy environment. [ABSTRACT FROM AUTHOR]

Propionibacterium freudenreichii is a beneficial bacterium used in the food industry as a vitamin producer, as a bio-preservative, as a cheese ripening starter and as a probiotic. It is known to adhere to intestinal epithelial cells and mucus and to modulate important functions of the gut mucosa, including cell proliferation and immune response. Adhesion of probiotics and cross-talk with the host rely on the presence of key surface proteins, still poorly identified. Identification of the determinants of adhesion and of immunomodulation by P. freudenreichii remains a bottleneck in the elucidation of its probiotic properties. In this report, three complementary proteomic methods are used to identify surface-exposed proteins in a strain, previously selected for its probiotic properties. The role of these proteins in the reported immunomodulatory properties of P. freudenreichii is evidenced. This work constitutes a basis for further studies aimed at the elucidation of mechanisms responsible for its probiotic effects, in a post-genomic context. Biological significance Dairy propionibacteria, mainly the species Propionibacterium freudenreichii , are consumed in high amounts within Swiss type cheeses. These peculiar bacteria are considered both as dairy starters and as probiotics. Their consumption modulates the gut microbiota, which makes them both probiotic and prebiotic. Promising immunomodulatory properties have been identified in these bacteria, in vitro , in animals and in humans. However, the mechanisms responsible for such anti-inflammatory properties are still unknown. In this work, we identify surface proteins involved in adhesion and immunostimulation by P. freudenreichii . This opens new perspectives for its utilization in new functional fermented food products, in clinical trials, and in understanding modulation of gut inflammation by products containing propionibacteria. [ABSTRACT FROM AUTHOR]

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. 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. Unlabelled Image • P. freudenreichii crossprotection by osmoadaptation is strain-dependent. • Glycine betaine is not necessarily beneficial for P. freudenreichii osmoadaptation. • Osmoadaptation increases P. freudenreichii CIRM-BIA 129 viability during freeze-drying. • P. freudenreichii CIRM-BIA 129 freeze-drying tolerance is higher following osmoadaptation in the absence of GB. [ABSTRACT FROM AUTHOR]