Surface proteins of Propionibacterium freudenreichii MJ2 inhibit RANKL-induced osteoclast differentiation by lipocalin-2 upregulation and lipocalin-2-mediated NFATc1 inhibition.

Osteoclasts degrade bone and osteoclast differentiation has been implicated in bone destruction in rheumatoid arthritis. The dairy bacterium Propionibacterium freudenreichii MJ2 (MJ2) isolated from raw milk inhibits osteoclast differentiation and ameliorates collagen-induced arthritis. This study aimed to investigate the inhibitory effect of the surface proteins of MJ2 on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and explain the underlying mechanism. The murine macrophage cell line RAW 264.7 was used to study the inhibition of osteoclast differentiation. The surface proteins significantly inhibited RANKL-induced osteoclast differentiation in a protein concentration-dependent manner by inhibiting the expression of genes and proteins related to osteoclast differentiation. RNA microarray analysis showed that the surface proteins significantly upregulated lipocalin-2 (lcn2) expression. In addition, they downregulated c-fos and NFATc1 and inhibited the expression of NFATc1-downstream genes Atp6v0d2, Calcr, and Ctsk. siRNA silencing of lcn2 decreased the extent of surface protein inhibition on osteoclast differentiation, suggesting that lcn2 plays an important role in the inhibition of RANKL-induced osteoclast differentiation. In conclusion, surface proteins of MJ2 show inhibitory effects on RANKL-induced osteoclast differentiation by upregulating lcn2 expression, in turn downregulating NFATc1, leading to the inhibition of NFATc1-downstream osteoclastogenesis-related gene expression.

Biochemical, structural, and kinetic characterization of PPi -dependent phosphoenolpyruvate carboxykinase from Propionibacterium freudenreichii.

Phosphoenolpyruvate carboxykinases (PEPCK) are a well-studied family of enzymes responsible for the regulation of TCA cycle flux, where they catalyze the interconversion of oxaloacetic acid (OAA) and phosphoenolpyruvate (PEP) using a phosphoryl donor/acceptor. These enzymes have typically been divided into two nucleotide-dependent classes, those that use ATP and those that use GTP. In the 1960's and early 1970's, a group of papers detailed biochemical properties of an enzyme named phosphoenolpyruvate carboxytransphosphorylase (later identified as a third PEPCK) from Propionibacterium freudenreichii (PPi -PfPEPCK), which instead of using a nucleotide, utilized PPi to catalyze the same interconversion of OAA and PEP. The presented work expands upon the initial biochemical experiments for PPi -PfPEPCK and interprets these data considering both the current understanding of nucleotide-dependent PEPCKs and is supplemented with a new crystal structure of PPi -PfPEPCK in complex with malate at a putative allosteric site. Most interesting, the data are consistent with PPi -PfPEPCK being a Fe2+ activated enzyme in contrast with the Mn2+ activated nucleotide-dependent enzymes which in part results in some unique kinetic properties for the enzyme when compared to the more widely distributed GTP- and ATP-dependent enzymes.

Investigation into the Potential Role of Propionibacterium freudenreichii in Prevention of Colorectal Cancer and Its Effects on the Diversity of Gut Microbiota in Rats.

Colorectal cancer (CRC) accounts for 10% of all cancer diagnoses and cancer-related deaths worldwide. Over the past two decades, several studies have demonstrated the clinical benefits of probiotic supplementation and some studies have shown that certain probiotics can modulate immunity and strengthen gut microbiota diversity. This study aims to assess the impact of the Propionibacterium freudenreichii (PF) probiotic against CRC induced by azoxymethane (AOM), and to investigate its effects on gut microbiota diversity in rats, as well as to evaluate the anti-proliferative activities of PF in HCT116 CRC cells. This experiment was performed using four groups of SD rats: normal control, AOM group, PF group (1 × 109 CFU/mL), and standard drug control (5-fluorouracil, 35 mg/kg). Methylene blue staining of colon tissues showed that the administration of PF significantly reduced the formation of colonic aberrant crypt foci (ACF) compared to the AOM control group. In addition, treated rats had lower levels of malondialdehyde in their colon tissue homogenates, indicating that lipid peroxidation was suppressed by PF supplementation. Furthermore, 16S rRNA gene analysis revealed that probiotic treatment enhanced the diversity of gut microbiota in rats. In vitro study showed that the viability of HCT116 cells was inhibited by the probiotic cell-free supernatant with an IC50 value of 13.3 ± 0.133. In conclusion, these results reveal that consuming PF as probiotic supplements modulates gut microbiota, inhibits the carcinogenic effects of AOM, and exerts anti-proliferative activity against CRC cells. Further studies are required to elucidate the role of PF on the immune response during the development and growth of CRC.

Intracellular osmoprotectant concentrations determine Propionibacterium freudenreichii survival during drying.

Propionibacterium freudenreichii is a beneficial bacterium widely used in food as a probiotic and as a cheese-ripening starter. In these different applications, it is produced, dried, and stored before being used. Both freeze-drying and spray-drying were considered for this purpose. Freeze-drying is a discontinuous process that is energy-consuming but that allows high cell survival. Spray-drying is a continuous process that is more energy-efficient but that can lead to massive bacterial death related to heat, osmotic, and oxidative stresses. We have shown that P. freudenreichii cultivated in hyperconcentrated rich media can be spray-dried with limited bacterial death. However, the general stress tolerance conferred by this hyperosmotic constraint remained a black box. In this study, we modulated P. freudenreichii growth conditions and monitored both osmoprotectant accumulation and stress tolerance acquisition. Changing the ratio between the carbohydrates provided and non-protein nitrogen during growth under osmotic constraint modulated osmoprotectant accumulation. This, in turn, was correlated with P. freudenreichii tolerance towards different stresses, on the one hand, and towards freeze-drying and spray-drying, on the other. Surprisingly, trehalose accumulation correlated with spray-drying survival and glycine betaine accumulation with freeze-drying. This first report showing the ability to modulate the trehalose/GB ratio in osmoprotectants accumulated by a probiotic bacterium opens new perspectives for the optimization of probiotics production.

Cinética de produção de ácidos orgânicos na fermentação de soro lácteo com cultura mista de Propionibacterium freundenreichii subsp ATCC 6207 e Lactobacillus paracasei / Kinetics of production organic acids in the fermentation of milk whey with mixed culture of Propionibacterium freundenreichii subsp ATCC 6207 and Lactobacillus paracasei

Foi estudada a cinética de produção de ácidos orgânicos na fermentação de soro lácteo com cultura mista de Propionibacterium freundenreichii subsp ATCC 6207 e Lactobacillus paracasei. Foram analisados os efeitos das concentrações de células de L. paracasei, de lactose e de CaCO3 sobre a produção de ácidos orgânicos com auxílio de delineamento composto central rotacional (DCCR), totalizando 18 ensaios. Foi verificada existência de uma região ótima usando meio de fermentação contendo concentração acima de 45 g L-1 de lactose e abaixo de 20 g L-1 de CaCO3, com melhor produção dos ácidos orgânicos. A cultura pode eficientemente utilizar soro lácteo para produção de ácidos orgânicos, diminuindo impactos ambientais provocado pelo este subproduto da produção de queijo.

Effect of Propionibacterium freudenreichii on Salmonella multiplication, motility, and association with avian epithelial cells1.

We investigated the effects of a probiotic bacterium, Propionibacterium freudenreichii, on Salmonella multiplication, motility, and association to and invasion of avian epithelial cells in vitro. Two subspecies of P. freudenreichii (P. freudenreichii subsp. freudenreichii and P. freudenreichii subsp. shermanii) were tested against 3 Salmonella serotypes in poultry, namely, S. Enteritidis, S. Typhimurium, and S. Heidelberg, using co-culture-, motility, multiplication, cell association, and invasion assays. Both strains of P. freudenreichii were effective in reducing or inhibiting multiplication of all 3 Salmonella serotypes in co-culture and turkey cecal contents (P ≤ 0.05). P. freudenreichii significantly reduced Salmonella motility (P ≤ 0.05). Cell culture studies revealed that P. freudenreichii associated with the avian epithelial cells effectively and reduced S. Enteritidis, S. Heidelberg, and S. Typhimurium cell association in the range of 1.0 to 1.6 log10 CFU/mL, and invasion in the range of 1.3 to 1.5 log10 CFU/mL (P ≤ 0.05), respectively. Our current in vitro results indicate the potential of P. freudenreichii against Salmonella in poultry. Follow-up in vivo studies are underway to evaluate this possibility.

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.

Possibility of using apple pomaces in the process of propionic-acetic fermentation

Background: In 2014, apple production in EU countries amounted to 11.8 million tonnes. A constant increase in the production of these fruits will lead to the accumulation of thousands of tonnes of apple pomace (production waste). The amount of industrial apples is the highest - their proportiononthe market is estimated at 50-60%, of which over 95% is processed into juice. The proportion of pomace in the traditional pressing method accounts for 20% offruits used. Results: Analysis of the growth dynamics of wild strain Propionibacterium freudenreichii T82 in micro-cultures using different carbon sources showed that the highest bacterial growth occurs in an environment with fructose and the most intense biosynthesis of metabolites was found in medium containing only saccharose. It has been found that P. freudenreichii T82 used apple pomaces as a source of carbon. Propionic acid biosynthesis reached its maximum value in the 120th hour of cultivation (1.771 g/L). At this time, the content of the acetic acid produced reached the level of 7.049 g/L. Conclusions: Utilization of by-products is a significant challenge for manufacturing sites and the natural environment. The solution to this problem may involve the use of pomace as a medium component for microorganism cultivation, which is a source of industrially useful metabolites. This study examined the possibility of using apple pomace as a carbon source in the process of propionic-acetic fermentation via wild strain Propionibacterium freudenreichii T82 bacteria.

The probiotic Propionibacterium freudenreichii as a new adjuvant for TRAIL-based therapy in colorectal cancer.

TNF-Related Apoptosis-Inducing Ligand (TRAIL) is a well-known apoptosis inducer, which activates the extrinsic death pathway. TRAIL is pro-apoptotic on colon cancer cells, while not cytotoxic towards normal healthy cells. However, its clinical use is limited by cell resistance to cell death which occurs in approximately 50% of cancer cells. Short Chain Fatty Acids (SCFA) are also known to specifically induce apoptosis of cancer cells. In accordance, we have shown that food grade dairy propionibacteria induce intrinsic apoptosis of colon cancer cells, via the production and release of SCFA (propionate and acetate) acting on mitochondria. Here, we investigated possible synergistic effect between Propionibacterium freudenreichii and TRAIL. Indeed, we hypothesized that acting on both extrinsic and intrinsic death pathways may exert a synergistic pro-apoptotic effect. Whole transcriptomic analysis demonstrated that propionibacterial supernatant or propionibacterial metabolites (propionate and acetate), in combination with TRAIL, increased pro-apoptotic gene expression (TRAIL-R2/DR5) and decreased anti-apoptotic gene expression (FLIP, XIAP) in HT29 human colon cancer cells. The revealed synergistic pro-apoptotic effect, depending on both death receptors (TRAIL-R1/DR4, TRAIL-R2/DR5) and caspases (caspase-8, -9 and -3) activation, was lethal on cancer cells but not on normal human intestinal epithelial cells (HIEC), and was inhibited by Bcl-2 expression. Finally, milk fermented by P. freudenreichii induced HT29 cells apoptosis and enhanced TRAIL cytotoxic activity, as did P. freudenreichii DMEM culture supernatants or its SCFA metabolites. These results open new perspectives for food grade P. freudenreichii-containing products in order to potentiate TRAIL-based cancer therapy in colorectal cancer.