Surface-exposed chaperonin 60 derived from Propionibacterium freudenreichii MJ2 inhibits adipogenesis by decreasing the expression of C/EBPα/PPARγ.

Recent studies have shown that the health benefits of probiotics are not limited to those offered by living bacteria. It was reported that both live and killed cells of Propionibacterium freudenreichii MJ2 (MJ2) isolated from raw milk showed antiobesity activity in 3T3-L1 cells and high-fat diet-induced obese mice. This study was aimed at identifying the active component(s) responsible for the antiadipogenic activity of MJ2. Cell wall, surface protein, and cytoplasmic fractions of MJ2 were investigated for their inhibitory effects on adipogenesis in 3T3-L1 cells. Adipocytes treated with the surface protein fraction showed significantly lower lipid accumulation. Using the MASCOT algorithm following LC-MS/MS analysis, 131 surface proteins were identified and they were principally classified into three categories (network clusters related to ribosomes, carbon metabolism, and chaperones). Among them, chaperonin 60 (Cpn60) was selected as a potential candidate protein. Cpn60 inhibited lipid accumulation and adipogenesis during the early period of differentiation (days 0-2) and decreased expression of genes related to adipogenesis (Pparg and Cebpa) and lipogenesis (Fas and Scd1). The expression of Gata2/3, which suppresses adipogenesis, significantly increased in Cpn60-treated cells. Moreover, the nuclear translocation of C/EBPß was inhibited by Cpn60 treatment. In conclusion, Cpn60, a surface protein in MJ2, shows antiadipogenic activity by reducing the expression of C/EBPß through the upregulation of Gata2/3 expression followed by downregulation of Pparg and Cebpa 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.

Co-cultures of Propionibacterium freudenreichii and Bacillus amyloliquefaciens cooperatively upgrade sunflower seed milk to high levels of vitamin B12 and multiple co-benefits.

BACKGROUND: Sunflower seeds (Helianthus annuus) display an attractive source for the rapidly increasing market of plant-based human nutrition. Of particular interest are press cakes of the seeds, cheap residuals from sunflower oil manufacturing that offer attractive sustainability and economic benefits. Admittedly, sunflower seed milk, derived therefrom, suffers from limited nutritional value, undesired flavor, and the presence of indigestible sugars. Of specific relevance is the absence of vitamin B12. This vitamin is required for development and function of the central nervous system, healthy red blood cell formation, and DNA synthesis, and displays the most important micronutrient for vegans to be aware of. Here we evaluated the power of microbes to enrich sunflower seed milk nutritionally as well as in flavor. RESULTS: Propionibacterium freudenreichii NCC 1177 showed highest vitamin B12 production in sunflower seed milk out of a range of food-grade propionibacteria. Its growth and B12 production capacity, however, were limited by a lack of accessible carbon sources and stimulants of B12 biosynthesis in the plant milk. This was overcome by co-cultivation with Bacillus amyloliquefaciens NCC 156, which supplied lactate, amino acids, and vitamin B7 for growth of NCC 1177 plus vitamins B2 and B3, potentially supporting vitamin B12 production by the Propionibacterium. After several rounds of optimization, co-fermentation of ultra-high-temperature pre-treated sunflower seed milk by the two microbes, enabled the production of 17 µg (100 g)-1 vitamin B12 within four days without any further supplementation. The fermented milk further revealed significantly enriched levels of L-lysine, the most limiting essential amino acid, vitamin B3, vitamin B6, improved protein quality and flavor, and largely eliminated indigestible sugars. CONCLUSION: The fermented sunflower seed milk, obtained by using two food-grade microbes without further supplementation, displays an attractive, clean-label product with a high level of vitamin B12 and multiple co-benefits. The secret of the successfully upgraded plant milk lies in the multifunctional cooperation of the two microbes, which were combined, based on their genetic potential and metabolic signatures found in mono-culture fermentations. This design by knowledge approach appears valuable for future development of plant-based milk products.

Use of Propionibacterium freudenreichii T82 Strain for Effective Biosynthesis of Propionic Acid and Trehalose in a Medium with Apple Pomace Extract and Potato Wastewater.

Propionic acid bacteria are the source of many metabolites, e.g., propionic acid and trehalose. Compared to microbiological synthesis, the production of these metabolites by petrochemical means or enzymatic conversion is more profitable. The components of microbiological media account for a large part of the costs associated with propionic fermentation, due to the high nutritional requirements of Propionibacterium. This problem can be overcome by formulating a medium based on the by-products of technological processes, which can act as nutritional sources and at the same time replace expensive laboratory preparations (e.g., peptone and yeast extract). The metabolic activity of P. freudenreichii was investigated in two different breeding environments: in a medium containing peptone, yeast extract, and biotin, and in a waste-based medium consisting of only apple pomace and potato wastewater. The highest production of propionic acid amounting to 14.54 g/L was obtained in the medium containing apple pomace and pure laboratory supplements with a yield of 0.44 g/g. Importantly, the acid production parameters in the waste medium reached almost the same level (12.71 g/L, 0.42 g/g) as the medium containing pure supplements. Acetic acid synthesis was more efficient in the waste medium; it was also characterized by a higher level of accumulated trehalose (59.8 mg/g d.s.). Thus, the obtained results show that P. freudenreichii bacteria exhibited relatively high metabolic activity in an environment with apple pomace used as a carbon source and potato wastewater used as a nitrogen source. This method of propioniate production could be cheaper and more sustainable than the chemical manner.

Improving the drying of Propionibacterium freudenreichii starter cultures.

Propionibacterium freudenreichii is a beneficial food-grade actinobacterium, widely implemented, and thus consumed, in various food products. As the main application, P. freudenreichii is used as a cheese-ripening starter, mostly in hard type cheeses. Indeed, during manufacture of "Swiss-type" cheeses (or opened-body cheeses), the technological process favors propionibacteria growth, as well as the corresponding propionic fermentation. This leads to the characteristic flavor of these cheeses, through the release of short chain fatty acids and through lipolysis, as well as to their specific texture. To fulfil this ripening, massive amounts of propionibacteria are industrially produced, dried and stored, prior to cheese making. Furthermore, P. freudenreichii is commercialized in various probiotic food supplements aiming at preserving intestinal health and comfort, in line with its ability to produce beneficial metabolites (short chain fatty acids, vitamins), as well as immunomodulatory compounds. Other industrial applications of P. freudenreichii include the production of food-grade vitamins of the B group, of trehalose, of conjugated linoleic acid, and of biopreservatives. For these different applications, maintaining survival and activity of propionibacteria during production, drying, storage and finally implementation, is crucial. More widely, maintaining live and active probiotic bacteria represents a challenge as the market for probiotic products increases. Probiotic bacteria are, for a bulk majority, freeze-dried, but spray drying is also more and more considered. Indeed, this process is both continuous and more cost-efficient, as it utilizes less energy compared to freeze-drying; on the other hand, it exposes bacteria to higher heat and oxidative stresses. Apart from process optimization and strain selection, it is possible to enhance the resistance of bacteria by taking advantage of their adaptation capacity. Indeed, P. freudenreichii stress tolerance can be boosted by different pretreatments applied before the drying step, thus considerably increasing its final survival. In particular, adaptation to hyperosmotic conditions improves stress tolerance, while the presence of osmoprotectants may mitigate this improvement. Thermal adaptation also modulates tolerance towards these technological challenges. The composition of the growth medium, including the ratio between the carbohydrates provided and the non-protein nitrogen, plays a key role in driving the accumulation of osmoprotectants. This, in turn, determines P. freudenreichii tolerance towards different stresses, and overall towards both freeze-drying and spray-drying. As an example, the accumulation of trehalose enhances its spray-drying survival, while the accumulation of glycine betaine enhances its freeze-drying survival. Growth of propionibacteria in hyperconcentrated whey was used to trigger multiple stress tolerance acquisition, underpinned by overexpression of key stress protein, accumulation of cytoplasmic storage compounds, and leading to enhanced spray-drying survival. A simplified process, from cultivation to atomization, was developed by using whey as a 2-in-1 medium in which propionibacteria were grown, protected and dried with minimal cell death. This innovative process was then subjected to scaling up at the industrial level. In this aim, a gentle multi-stage drying process offering mild drying conditions by coupling spray drying with belt drying, led to final probiotic survival close to 100% when stress tolerance acquisition was previously implemented. Such innovation opens new avenues for the efficient, cost-effective and sustainable development of new probiotic production technologies, as well as probiotic application in the context of food and feed. KEY POINTS: • Propionibacteria acquire multi-stress tolerance when grown in hyper-concentrated whey. • Spray drying of osmo-adapted probiotic bacteria is possible with limited cell death. • A two-in-one drying method is developed to grow and dry probiotic bacteria in the same matrix.

Optimization of propionic acid production in apple pomace extract with Propionibacterium freudenreichii.

Sequential optimization of propionate production using apple pomace was studied. All experiments were performed in a static flask in anaerobic conditions. Effect of apple pomace as nitrogen source against conventional N sources (yeast extract, peptone) was studied. The double increase was observed in propionic acid production while using yeast extract and peptone (0.29 ± 0.01 g/g), as against the use of only apple pomace extract (APE) (0.14 ± 0.01 g/g). Intensification of propionic acid fermentation was also achieved by increasing the pH control frequency of the culture medium from 24-(0.29 ± 0.01 g/g) to 12-hour intervals (30 °C) (0.30 ± 0.02 g/g) and by increasing the temperature of the culture from 30 to 37 °C (12-hour intervals of pH control) (0.32 ± 0.01 g/g). An important factor in improving the parameters of fermentation was the addition of biotin to the medium. The 0.2 mg/L dose of biotin allowed to attain 7.66 g/L propionate with a yield of 0.38 ± 0.03 g/g (12-hour intervals of pH control, 37 °C).

Effect of crude and pure glycerol on biomass production and trehalose accumulation by Propionibacterium freudenreichii ssp. shermanii 1.

The dairy propionibacteria, which are traditionally used for the production of Swiss cheeses, are able to synthesize valuable biomolecules, e.g. B group vitamins, propionic acid, and trehalose with unique chemical and physical properties. Both, dairy propionibacteria cells and trehalose, have found many applications as attractive and effective components in food, beauty and health care products. This study confirmed the ability of several strains from the Propionibacterium genus to create trehalose from glycerol. The research aimed to investigate the effect of crude and pure glycerol on biomass production and on trehalose accumulation by Propionibacterium freudenreichii ssp. shermanii 1. The results indicated that the capacity for trehalose accumulation by Propionibacterium spp. was strain dependent. Propionibacterium freudenreichii ssp. shermanii 1 was able to grow on crude glycerol. For both, pure and crude glycerol, the highest amount of dry biomass leveled off at about 4 g/L. While the use of crude glycerol had no effect on the final concentration of biomass, it reduced the accumulation of trehalose in the cells. An increase in the concentration of carbon source (2-8%) resulted in more than a 5-fold rise in trehalose production. The highest trehalose concentration of 195.04 mg/L was obtained with cultures of the said strain supplemented to 8% with pure glycerol.