Deciphering the metabolism of Lactobacillus delbrueckii subsp. delbrueckii during soy juice fermentation using phenotypic and transcriptional analysis.

In the context of sustainable diet, the development of soy-based yogurt fermented with lactic acid bacteria is an attractive alternative to dairy yogurts. To decipher the metabolism of Lactobacillus delbrueckii subsp. delbrueckii during soy juice (SJ) fermentation, the whole genome of the strain CIRM-BIA865 (Ld865) was sequenced and annotated. Then Ld865 was used to ferment SJ. Samples were analyzed throughout fermentation for their cell number, carbohydrate, organic acid, free amino acid, and volatile compound contents. Despite acidification, the number of Ld865 cells did not rise, and microscopic observations revealed the elongation of cells from 3.6 µm (inoculation) to 36.9 µm (end of fermentation). This elongation was observed in SJ but not in laboratory-rich medium MRS. Using transcriptomic analysis, we showed that the biosynthesis genes of peptidoglycan and membrane lipids were stably expressed, in line with the cell elongation observed, whereas no genes implicated in cell division were upregulated. Among the main sugars available in SJ (sucrose, raffinose, and stachyose), Ld865 only used sucrose. The transcriptomic analysis showed that Ld865 implemented the two transport systems that it contains to import sucrose: a PTS system and an ABC transporter. To fulfill its nitrogen needs, Ld865 probably first consumed the free amino acids of the SJ and then implemented different oligopeptide transporters and proteolytic/peptidase enzymes. In conclusion, this study showed that Ld865 enables fast acidification of SJ, despite the absence of cell division, leads to a product rich in free amino acids, and also leads to the production of aromatic compounds of interest. IMPORTANCE: To reduce the environmental and health concerns related to food, an alternative diet is recommended, containing 50% of plant-based proteins. Soy juice, which is protein rich, is a relevant alternative to animal milk, for the production of yogurt-like products. However, soy "beany" and "green" off-flavors limit the consumption of such products. The lactic acid bacteria (LAB) used for fermentation can help to improve the organoleptic properties of soy products. But metabolic data concerning LAB adapted to soy juice are lacking. The aim of this study was, thus, to decipher the metabolism of Lactobacillus delbrueckii subsp. delbrueckii during fermentation of a soy juice, based on a multidisciplinary approach. This result will contribute to give tracks for a relevant selection of starter. Indeed, the improvement of the organoleptic properties of these types of products could help to promote plant-based proteins in our diet.

Brine salt concentration reduction and inoculation with autochthonous consortia: Impact on Protected Designation of Origin Nyons black table olive fermentations.

Nyons table olives, named after the French city where they are processed, are naturally fermented black table olives. Their specificity relies on the use of the "Tanche" olive variety harvested at full maturity and their slow spontaneous fermentation in 10% salt brine driven by yeast populations. This study aimed at investigating the benefit of inoculating autochthonous consortia to produce Nyons table olives by fermentation in 10% salt brine and in reduced salt conditions (8%). Two strategies were evaluated: inoculation with a defined autochthonous consortium and inoculation by spent brine backslopping. To define the consortium, yeasts were selected among 48 autochthonous isolates and key features included high halotolerance, low pectinolytic and proteolytic activities, however none had ß-glucosidase activities. The consortium included eight yeast strains with distinct technological properties belonging to five dominant species, i.e. Citeromyces nyonsensis, Pichia membranifaciens, Wickerhamomyces anomalus, Zygotorulaspora mrakii and Candida atlantica. Fermentation trials were conducted over a year and compared by evaluating microbial community shifts (16S and ITS metagenetics) and volatile profiles (GC-MS). Regarding fermentations with the defined consortium, four out of five species implanted in early stages while one, Pichia membranifaciens, persisted and largely dominated by the end of the fermentation. Altogether, inoculation with the defined consortium did not disrupt microbial shifts compared to traditional fermentations although minor differences were observed in volatile profiles. The backslopping method yielded the highest impact on microbial populations and olive volatile profiles, with higher ester abundances at the end of fermentation. Finally, reduced salt in brine gave very promising results as no deleterious effects on microbial communities, volatile dynamics but also safety criteria of the olives were observed compared to traditional fermented olives.

Fine-Tuning of Process Parameters Modulates Specific Metabolic Bacterial Activities and Aroma Compound Production in Semi-Hard Cheese.

The formation of cheese flavor mainly results from the production of volatile compounds by microorganisms. We investigated how fine-tuning cheese-making process parameters changed the cheese volatilome in a semi-hard cheese inoculated with Lactococcus (L.) lactis, Lactiplantibacillus (L.) plantarum, and Propionibacterium (P.) freudenreichii. A standard (Std) cheese was compared with three variants of technological itineraries: a shorter salting time (7 h vs 10 h, Salt7h), a shorter stirring time (15 min vs 30 min, Stir15min), or a higher ripening temperature (16 °C vs 13 °C, Rip16°C). Bacterial counts were similar in the four cheese types, except for a 1.4 log10 reduction of L. lactis counts in Rip16°C cheeses after 7 weeks of ripening. Compared to Std, Stir15min and Rip16°C increased propionibacterial activity, causing higher concentrations of acetic, succinic, and propanoic acids and lower levels of lactic acid. Rip16°C accelerated secondary proteolysis and volatile production. We thus demonstrated that fine-tuning process parameters could modulate the cheese volatilome by influencing specific bacterial metabolisms.

Use of metabarcoding and source tracking to identify desirable or spoilage autochthonous microorganism sources during black olive fermentations.

This study aimed at investigating the influence of the process environment and raw materials as sources of microorganisms during Nyons black table olive fermentations. Fermented olives and/or brine from spoiled fermentation tanks were analyzed and compared to good quality samples from fermentations collected during 3 consecutive harvest years. Fresh olives, salt and different process environment samples were also analyzed. Microbial diversity of all samples was analyzed using 16S and ITS2 amplicon sequencing and SourceTracker tool was used to investigate links between environment, raw materials and fermentation samples. First, comparison of microbial diversity in control and most spoiled fermentations revealed striking differences in bacterial composition with an overall higher diversity in spoiled fermentations especially for lactic acid bacteria with Lentilactobacillus buchneri, Lentilactobacillus parafarraginis dominating in brine and Pediococcus parvulus, Pediococcus ethanolidurans dominating in olive fruits. Fungal communities were similar in composition although higher abundances of Pichia membranifaciens and Penicillium carneum/roqueforti were observed in spoiled samples. Secondly, process environment samples were characterized by high bacterial and fungal diversity, especially compared to fresh olive fruits. Overall, dominant fungal species in control fermentations were also found in most environmental samples revealing a "house mycobiota". SourceTracker analysis further highlighted the contribution of brine and water from the optical sorter as a source of fungi. Most interestingly, spoilage fungi and most bacteria were retrieved in brine and environmental samples while others such as P. ethanolidurans were only found in environmental samples indicating that the studied spoilage originated from a fermentation deviation rather than a punctual contamination. Taken altogether, these results highlighted the positive and negative influence of the process environment and emphasized the relevance of studying it to better understand microbial vectors occurring during food fermentations, especially natural ones.

Linking Pélardon artisanal goat cheese microbial communities to aroma compounds during cheese-making and ripening.

Pélardon is an artisanal French raw goat's milk cheese, produced using natural whey as a backslop. The aim of this study was to identify key microbial players involved in the acidification and aroma production of this Protected Designation of Origin cheese. Microbial diversity of samples, collected from the raw milk to 3-month cheese ripening, was determined by culture-dependent (MALDI-TOF analysis of 2877 isolates) and -independent (ITS2 and 16S metabarcoding) approaches and linked to changes in biochemical profiles (volatile compounds and acids). In parallel, potential dominant autochthonous microorganism reservoirs were also investigated by sampling the cheese-factory environment. Complex and increasing microbial diversity was observed by both approaches during ripening although major discrepancies were observed regarding Lactococcus lactis and Lacticaseibacillus paracasei fate. By correlating microbial shifts to biochemical changes, Lactococcus lactis was identified as the main acidifying bacterium, while L. mesenteroides and Geotrichum candidum were prevalent and associated with amino acids catabolism after the acidification step. The three species were dominant in the whey (backslop). In contrast, L. paracasei, Enterococcus faecalis, Penicillium commune and Scopulariopsis brevicaulis, which dominated during ripening, likely originated from the cheese-making environment. All these four species were positively correlated to major volatile compounds responsible for the goaty and earthy Pélardon cheese aroma. Overall, this work highlighted the power of MALDI-TOF and molecular techniques combined with volatilome analyses to dynamically follow and identify microbial communities during cheese-making and successively identify the key-players involved in aroma production and contributing to the typicity of Pélardon cheese.

Deciphering Microbial Community Dynamics and Biochemical Changes During Nyons Black Olive Natural Fermentations.

French PDO Nyons black table olives are produced according to a traditional slow spontaneous fermentation in brine. The manufacture and unique sensorial properties of these olives thus only rely on the autochthonous complex microbiota. This study aimed at unraveling the microbial communities and dynamics of Nyons olives during a 1.5-year-long spontaneous fermentation to determine the main microbial drivers and link microbial species to key metabolites. Fermentations were monitored at a local producer plant at regular time intervals for two harvests and two olive types (organically and conventionally grown) using culture-dependent and metabarcoding (ITS2 for fungi, V3-V4 region for bacteria) approaches. Olives and brines were also sampled for volatiles, organic acids and phenolic compounds. No major differences in microbiota composition were observed according to olive type or harvest period. Throughout the fermentation, yeasts were clearly the most dominant. ITS2 sequencing data revealed complex fungal diversity dominated by Citeromyces nyonsensis, Wickerhamomyces anomalus, Zygotorulaspora mrakii, Candida boidinii and Pichia membranifaciens species. Bacterial communities were dominated by the Celerinatantimonas genus, while lactic acid bacteria remained scarce. Clear shifts in microbial communities and biochemical profiles were observed during fermentation and, by correlating metabolites and microbiota changes, four different phases were distinguished. During the first 7 days, phase I, a fast decrease of filamentous fungal and bacterial populations was observed. Between days 21 and 120, phase II, W. anomalus and C. nyonsensis for fungi and Celerinatantimonas diazotrophica for bacteria dominated the fermentation and were linked to the pH decrease and citric acid production. Phase III, between 120 and 183 days, was characterized by an increase in acids and esters and correlated to increased abundances of Z. mrakii, P. membranifaciens and C. boidinii. During the last months of fermentation, phase IV, microbial communities were dominated by P. membranifaciens and C. boidinii. Both species were strongly correlated to an increase in fruity esters and alcohol abundances. Overall, this study provides an in-depth understanding about microbial species succession and how the microbiota shapes the final distinct olive characteristics. It also constitutes a first step to identify key drivers of this fermentation.

Surface properties associated with the production of polysaccharides in the food bacteria Propionibacterium freudenreichii.

This study explores the production of polysaccharides (PS) in the strain Pf2289 of the food species Propionibacterium freudenreichii. Pf2289 presents characteristics atypical of the species: a molar-shaped morphotype upon plating, and cells strongly aggregative in liquid medium. When plating Pf2289, another morphotype was observed with a 4% frequency of appearance: round-shaped colonies, typical of the species. A clone was isolated, designated Pf456. No reversibility of Pf456 towards the molar-shaped morphotype was observed. Pf2289 was shown to produce a surface polysaccharide (PS) bound to the cell wall, mainly during the stationary growth phase. Meanwhile, Pf456 had lost the ability to produce the PS. AFM images of Pf2289 showed that entangled filaments spread over the whole surface of the bacteria, whereas Pf456 exhibited a smooth surface. Adhesion force maps, performed with concanavalin-A grafted probes, revealed twice as much adhesion of Pf2289 to concanavalin-A compared to Pf456. Furthermore, the length of PS molecules surrounding Pf2289 measured at least 7 µm, whereas it only reached 1 µm in Pf456. Finally, the presence of PS had a strong impact on adhesion properties: Pf2289 did not adhere to hydrophobic surfaces, whereas Pf456 showed strong adhesion.

Diversity of the metabolic profiles of a broad range of lactic acid bacteria in soy juice fermentation.

This study explores the ability of lactic acid bacteria (LAB) to ferment soy juice. The ability of 276 LAB strains from 25 species to ferment the principal soy carbohydrates, sucrose, raffinose or stachyose was tested in synthetic media and a soy juice. Fermented soy juices (FSJs) were characterized for their odor. Selected FSJs were characterized by targeted metabolomics. All Streptococcus, 83% of Leuconostoc and Lactobacillus and 41% of Lactococcus strains were sucrose-positive, while only 36% of all the LAB strains tested were raffinose-positive and 6% stachyose-positive. Nearly all (97%) the sucrose-positive strains fermented soy juice, indicating that an ability to use sucrose is a good criterion to select strains for soy juice fermentation. Among the most efficient acidifying strains, 46 FSJs had an odor deemed to be acceptable. FSJ composition was dependent on both species and strains: 17/46 strains deglycosylated soy juice isoflavones, the 27 S. thermophilus strains converted a mean 4.4 ± 0.1 g/L of sucrose into 3.0 ± 0.1 g/L of lactic acid versus 5.2 ± 0.1 g/L into 2.2 ± 0.1 g/L for the 18 Lactobacillus and one Lactococcus strains. This study highlights the diversity of the metabolic profiles of LAB strains in soy juice fermentation.

Identification of proteins involved in the anti-inflammatory properties of Propionibacterium freudenreichii by means of a multi-strain study.

Propionibacterium freudenreichii, a dairy starter, can reach a population of almost 109 propionibacteria per gram in Swiss-type cheese at the time of consumption. Also consumed as a probiotic, it displays strain-dependent anti-inflammatory properties mediated by surface proteins that induce IL-10 in leukocytes. We selected 23 strains with varied anti-inflammatory potentials in order to identify the protein(s) involved. After comparative genomic analysis, 12 of these strains were further analysed by surface proteomics, eight of them being further submitted to transcriptomics. The omics data were then correlated to the anti-inflammatory potential evaluated by IL-10 induction. This comparative omics strategy highlighted candidate genes that were further subjected to gene-inactivation validation. This validation confirmed the contribution of surface proteins, including SlpB and SlpE, two proteins with SLH domains known to mediate non-covalent anchorage to the cell-wall. Interestingly, HsdM3, predicted as cytoplasmic and involved in DNA modification, was shown to contribute to anti-inflammatory activity. Finally, we demonstrated that a single protein cannot explain the anti-inflammatory properties of a strain. These properties therefore result from different combinations of surface and cytoplasmic proteins, depending on the strain. Our enhanced understanding of the molecular bases for immunomodulation will enable the relevant screening for bacterial resources with anti-inflammatory properties.

Adaptation of Propionibacterium freudenreichii to long-term survival under gradual nutritional shortage.

BACKGROUND: Propionibacterium freudenreichii is an Actinobacterium widely used in the dairy industry as a ripening culture for Swiss-type cheeses, for vitamin B12 production and some strains display probiotic properties. It is reportedly a hardy bacterium, able to survive the cheese-making process and digestive stresses. RESULTS: During this study, P. freudenreichii CIRM-BIA 138 (alias ITG P9), which has a generation time of five hours in Yeast Extract Lactate medium at 30 °C under microaerophilic conditions, was incubated for 11 days (9 days after entry into stationary phase) in a culture medium, without any adjunct during the incubation. The carbon and free amino acids sources available in the medium, and the organic acids produced by the strain, were monitored throughout growth and survival. Although lactate (the preferred carbon source for P. freudenreichii) was exhausted three days after inoculation, the strain sustained a high population level of 9.3 log10 CFU/mL. Its physiological adaptation was investigated by RNA-seq analysis and revealed a complete disruption of metabolism at the entry into stationary phase as compared to exponential phase. CONCLUSIONS: P. freudenreichii adapts its metabolism during entry into stationary phase by down-regulating oxidative phosphorylation, glycolysis, and the Wood-Werkman cycle by exploiting new nitrogen (glutamate, glycine, alanine) sources, by down-regulating the transcription, translation and secretion of protein. Utilization of polyphosphates was suggested.

Permanent draft genome sequence of the probiotic strain Propionibacterium freudenreichii CIRM-BIA 129 (ITG P20).

Propionibacterium freudenreichii belongs to the class Actinobacteria (Gram positive with a high GC content). This "Generally Recognized As Safe" (GRAS) species is traditionally used as (i) a starter for Swiss-type cheeses where it is responsible for holes and aroma production, (ii) a vitamin B12 and propionic acid producer in white biotechnologies, and (iii) a probiotic for use in humans and animals because of its bifidogenic and anti-inflammatory properties. Until now, only strain CIRM-BIA1T had been sequenced, annotated and become publicly available. Strain CIRM-BIA129 (commercially available as ITG P20) has considerable anti-inflammatory potential. Its gene content was compared to that of CIRM-BIA1 T. This strain contains 2384 genes including 1 ribosomal operon, 45 tRNA and 30 pseudogenes.

Combining selected immunomodulatory Propionibacterium freudenreichii and Lactobacillus delbrueckii strains: Reverse engineering development of an anti-inflammatory cheese.

SCOPE: Inflammatory bowel disease (IBD) constitutes a growing public health concern in western countries. Bacteria with anti-inflammatory properties are lacking in the dysbiosis accompanying IBD. Selected strains of probiotic bacteria with anti-inflammatory properties accordingly alleviate symptoms and enhance treatment of ulcerative colitis in clinical trials. Such properties are also found in selected strains of dairy starters such as Propionibacterium freudenreichii and Lactobacillus delbrueckii (Ld). We thus investigated the possibility to develop a fermented dairy product, combining both starter and probiotic abilities of both lactic acid and propionic acid bacteria, designed to extend remissions in IBD patients. METHODS AND RESULTS: We developed a single-strain Ld-fermented milk and a two-strain P. freudenreichii and Ld-fermented experimental pressed cheese using strains previously selected for their anti-inflammatory properties. Consumption of these experimental fermented dairy products protected mice against trinitrobenzenesulfonic acid induced colitis, alleviating severity of symptoms, modulating local and systemic inflammation, as well as colonic oxidative stress and epithelial cell damages. As a control, the corresponding sterile dairy matrix failed to afford such protection. CONCLUSION: This work reveals the probiotic potential of this bacterial mixture, in the context of fermented dairy products. It opens new perspectives for the reverse engineering development of anti-inflammatory fermented foods designed for target populations with IBD, and has provided evidences leading to an ongoing pilot clinical study in ulcerative colitis patients.

Immunomodulation properties of multi-species fermented milks.

Dairy propionibacteria (PAB) are used as a ripening starter in combination with Lactic acid bacteria (LAB) for dairy products such as Swiss-type cheese. LAB and PAB have also been studied for their probiotic properties but little is still known about their individual and/or synergistic beneficial effects within dairy matrices. In the context of a rising incidence of Inflammatory Bowel Diseases, it has become crucial to evaluate the immunomodulatory potential of bacteria ingested in large numbers via dairy products. We therefore selected different strains and combinations of technological LAB and PAB. We determined their immunomodulatory potential by IL-10 and IL-12 induction, in human peripheral blood mononuclear cells, on either single or mixed cultures, grown on laboratory medium or directly in milk. Milk was fermented with selected anti-inflammatory strains of LAB or PAB/LAB mixed cultures and the resulting bacterial fractions were also evaluated for these properties, together with starter viability and optimum technological aspects. The most promising fermented milks were evaluated in the context of TNBS- or DSS-induced colitis in mice. The improvement in inflammatory parameters evidenced an alleviation of colitis symptoms as a result of fermented milk consumption. This effect was clearly strain-dependent and modulated by growth within a fermented dairy product. These findings offer new tools and perspectives for the development of immunomodulatory fermented dairy products for targeted populations.

Mutations and genomic islands can explain the strain dependency of sugar utilization in 21 strains of Propionibacterium freudenreichii.

BACKGROUND: Propionibacterium freudenreichii (PF) is an actinobacterium used in cheese technology and for its probiotic properties. PF is also extremely adaptable to several ecological niches and can grow on a variety of carbon and nitrogen sources. The aim of this work was to discover the genetic basis for strain-dependent traits related to its ability to use specific carbon sources. High-throughput sequencing technologies were ideal for this purpose as they have the potential to decipher genomic diversity at a moderate cost. RESULTS: 21 strains of PF were sequenced and the genomes were assembled de novo. Scaffolds were ordered by comparison with the complete reference genome CIRM-BIA1, obtained previously using traditional Sanger sequencing. Automatic functional annotation and manual curation were performed. Each gene was attributed to either the core genome or an accessory genome. The ability of the 21 strains to degrade 50 different sugars was evaluated. Thirty-three sugars were degraded by none of the sequenced strains whereas eight sugars were degraded by all of them. The corresponding genes were present in the core genome. Lactose, melibiose and xylitol were only used by some strains. In this case, the presence/absence of genes responsible for carbon uptake and degradation correlated well with the phenotypes, with the exception of xylitol. Furthermore, the simultaneous presence of these genes was in line the metabolic pathways described previously in other species. We also considered the genetic origin (transduction, rearrangement) of the corresponding genomic islands. Ribose and gluconate were degraded to a greater or lesser extent (quantitative phenotype) by some strains. For these sugars, the phenotypes could not be explained by the presence/absence of a gene but correlated with the premature appearance of a stop codon interrupting protein synthesis and preventing the catabolism of corresponding carbon sources. CONCLUSION: These results illustrate (i) the power of correlation studies to discover the genetic basis of binary strain-dependent traits, and (ii) the plasticity of PF chromosomes, probably resulting from horizontal transfers, duplications, transpositions and an accumulation of mutations. Knowledge of the genetic basis of nitrogen and sugar degradation opens up new strategies for the screening of PF strain collections to enable optimum cheese starter, probiotic and white biotechnology applications.

Surface proteins of Propionibacterium freudenreichii are involved in its anti-inflammatory properties.

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.

Recrystallized S-layer protein of a probiotic Propionibacterium: structural and nanomechanical changes upon temperature or pH shifts probed by solid-state NMR and AFM.

Surface protein layers (S layers) are common constituents of the bacterial cell wall and originate from the assembly of strain-dependent surface layer proteins (Slps). These proteins are thought to play important roles in the bacteria's biology and to have very promising technological applications as biomaterials or as part of cell-host cross-talk in probiotic mechanism. The SlpA from Propionibacterium freudenreichii PFCIRM 118 strain was isolated and recrystallized to investigate organization and assembly of the protein using atomic force microscopy and solid-state (1)H and (13)C-nuclear magnetic resonance. SlpA was found to form hexagonal p1 monolayer lattices where the protein exhibited high proportions of disordered regions and of bound water. The lattice structure was maintained, but softened, upon mild heating or acidification, probably in relation with the increasing mobilities of the disordered protein regions. These results gave structural insights on the mobile protein regions exposed by S layer films, upon physiologically relevant changes of their environmental conditions.

Data from an integrative approach decipher the surface proteome of Propionibacterium freudenreichii.

The surface proteins of the probiotic Propionibacterium freudenreichii were inventoried by an integrative approach that combines in silico protein localization prediction, surface protein extraction, shaving and fluorescent CyDye labeling. Proteins that were extracted and/or shaved and/or labeled were identified by nano-LC-MS/MS following trypsinolysis. This method's combination allowed to confirm detection of true surface proteins involved in host/probiotic interactions. The data, supplied in this article, are related to the research article entitled "Surface proteins of P. freudenreichii are involved in its anti-inflammatory properties" (Le Maréchal et al., 2014 [6]).

The first dairy product exclusively fermented by Propionibacterium freudenreichii: a new vector to study probiotic potentialities in vivo.

Dairy propionibacteria display probiotic properties which require high populations of live and metabolically active propionibacteria in the colon. In this context, the probiotic vector determines probiotic efficiency. Fermented dairy products protect propionibacteria against digestive stresses and generally contain a complex mixture of lactic and propionic acid bacteria. This does not allow the identification of dairy propionibacteria specific beneficial effects. The aim of this study was to develop a dairy product exclusively fermented by dairy propionibacteria. As they grow poorly in milk, we determined their nutritional requirements concerning carbon and nitrogen by supplementing milk ultrafiltrate (UF) with different concentrations of lactate and casein hydrolysate. Milk or UF supplemented with 50 mM lactate and 5 g L(-1) casein hydrolysate allowed growth of all dairy propionibacteria studied. In these new fermented dairy products, dairy propionibacteria remained viable and stress-tolerant in vitro during minimum 15 days at 4 °C. The efficiency of milk fermented by the most tolerant Propionibacterium freudenreichii strain was evaluated in piglets. Viability and SCFA content in the colon evidenced survival and metabolic activity of P. freudenreichii. This work results in the design of a new food grade vector, which will allow preclinical and clinical trials.

Assessment of the probiotic potential of a dairy product fermented by Propionibacterium freudenreichii in piglets.

Dairy propionibacteria, including Propionibacterium freudenreichii , display promising probiotic properties, including immunomodulation. These properties are highly strain-dependent and rarely studied in a fermented dairy product. We screened 10 strains, grown in a newly developed fermented milk ultrafiltrate, for immunomodulatory properties in vitro. The most anti-inflammatory strain, P. freudenreichii BIA129, was further tested on piglets. P. freudenreichii -fermented product improved food intake and growth of piglets. Colonic mucosa explants of treated pigs secreted less interleukin 8 (-25%, P < 0.05) and tumor necrosis factor α (-20%, P < 0.05), either in basal conditions or after a lipopolysaccharide challenge. By contrast, the gut structure, barrier function (measured ex vivo in Ussing chambers), microbial diversity (assessed by 16S rRNA pyrosequencing), and colonic short-chain fatty acid content were unchanged, assuming maintenance of normal intestinal physiology. In conclusion, this work confirms in vivo probiotic properties of dairy propionibacteria-fermented products, which are promising for the prevention or healing of inflammatory bowel diseases.

Contribution of surface ß-glucan polysaccharide to physicochemical and immunomodulatory properties of Propionibacterium freudenreichii.

Propionibacterium freudenreichii is a bacterial species found in Swiss-type cheeses and is also considered for its health properties. The main claimed effect is the bifidogenic property. Some strains were shown recently to display other interesting probiotic potentialities such as anti-inflammatory properties. About 30% of strains were shown to produce a surface exopolysaccharide (EPS) composed of (1→3,1→2)-ß-D-glucan due to a single gene named gtfF. We hypothesized that functional properties of P. freudenreichii strains, including their anti-inflammatory properties, could be linked to the presence of ß-glucan. To evaluate this hypothesis, gtfF genes of three ß-glucan-producing strains were disrupted. These knockout (KO) mutants were complemented with a plasmid harboring gtfF (KO-C mutants). The absence of ß-glucan in KO mutants was verified by immunological detection and transmission electron microscopy. We observed by atomic force microscopy that the absence of ß-glucan in the KO mutant dramatically changed the cell's topography. The capacity to adhere to polystyrene surface was increased for the KO mutants compared to wild-type (WT) strains. Anti-inflammatory properties of WT strains and mutants were analyzed by stimulation of human peripheral blood mononuclear cells (PBMCs). A significant increase of the anti-inflammatory interleukin-10 cytokine production by PBMCs was measured in the KO mutants compared to WT strains. For one strain, the role of ß-glucan in mice gut persistence was assessed, and no significant difference was observed between the WT strain and its KO mutant. Thus, ß-glucan appears to partly hide the anti-inflammatory properties of P. freudenreichii; which is an important result for the selection of probiotic strains.