Dataset Information

Propionibacterium freudenreichii CIRM-BIA 129 Osmoadaptation Coupled to Acid-Adaptation Increases Its Viability During Freeze-Drying.

ABSTRACT: 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.

SUBMITTER: Gaucher F

PROVIDER: S-EPMC6797830 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

<i>Propionibacterium freudenreichii</i> CIRM-BIA 129 Osmoadaptation Coupled to Acid-Adaptation Increases Its Viability During Freeze-Drying.

Gaucher Floriane F Kponouglo Koffigan K Rabah Houem H Bonnassie Sylvie S Ossemond Jordane J Pottier Sandrine S Jardin Julien J Briard-Bion Valérie V Marchand Pierre P Blanc Philippe P Jeantet Romain R Jan Gwénaël G

Frontiers in microbiology 20191009

<i>Propionibacterium freudenreichii</i> 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 o ...[more]

PMID: 31681198

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Project description: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.

Project description: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.

Project description:BACKGROUND: Propionibacterium freudenreichii is essential as a ripening culture in Swiss-type cheeses and is also considered for its probiotic use. This species exhibits slow growth, low nutritional requirements, and hardiness in many habitats. It belongs to the taxonomic group of dairy propionibacteria, in contrast to the cutaneous species P. acnes. The genome of the type strain, P. freudenreichii subsp. shermanii CIRM-BIA1 (CIP 103027(T)), was sequenced with an 11-fold coverage. METHODOLOGY/PRINCIPAL FINDINGS: The circular chromosome of 2.7 Mb of the CIRM-BIA1 strain has a GC-content of 67% and contains 22 different insertion sequences (3.5% of the genome in base pairs). Using a proteomic approach, 490 of the 2439 predicted proteins were confirmed. The annotation revealed the genetic basis for the hardiness of P. freudenreichii, as the bacterium possesses a complete enzymatic arsenal for de novo biosynthesis of aminoacids and vitamins (except panthotenate and biotin) as well as sequences involved in metabolism of various carbon sources, immunity against phages, duplicated chaperone genes and, interestingly, genes involved in the management of polyphosphate, glycogen and trehalose storage. The complete biosynthesis pathway for a bifidogenic compound is described, as well as a high number of surface proteins involved in interactions with the host and present in other probiotic bacteria. By comparative genomics, no pathogenicity factors found in P. acnes or in other pathogenic microbial species were identified in P. freudenreichii, which is consistent with the Generally Recognized As Safe and Qualified Presumption of Safety status of P. freudenreichii. Various pathways for formation of cheese flavor compounds were identified: the Wood-Werkman cycle for propionic acid formation, amino acid degradation pathways resulting in the formation of volatile branched chain fatty acids, and esterases involved in the formation of free fatty acids and esters. CONCLUSIONS/SIGNIFICANCE: With the exception of its ability to degrade lactose, P. freudenreichii seems poorly adapted to dairy niches. This genome annotation opens up new prospects for the understanding of the P. freudenreichii probiotic activity.

Project description: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.

Dataset Information

Propionibacterium freudenreichii CIRM-BIA 129 Osmoadaptation Coupled to Acid-Adaptation Increases Its Viability During Freeze-Drying.

Publications

<i>Propionibacterium freudenreichii</i> CIRM-BIA 129 Osmoadaptation Coupled to Acid-Adaptation Increases Its Viability During Freeze-Drying.

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