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.

Microbial enrichment of blackcurrant press residue with conjugated linoleic and linolenic acids.

AIMS: The aim of the study was to investigate the isomerization of linoleic (LA) and linolenic acids (LNAs) into their conjugated isomers by Propionibacterium freudenreichii DSM 20270 and utilize this feature for microbial enrichment of blackcurrant press residue (BCPR) with health-beneficial conjugated fatty acids. METHODS AND RESULTS: First, the ability of P. freudenreichii to isomerize 0·4 mg ml-1 of LA and LNA was studied in lactate growth medium. Free LA and α-LNA were efficiently converted into conjugated linoleic (CLA) and α-linolenic acid (α-CLNA), being the predominant isomers c9,t11-CLA and c9,t11,c15-CLNA, respectively. The bioconversion of α-LNA by P. freudenreichii was more efficient in terms of formation rate, yield and isomer-specificity. Thereafter, free LA and LNAs obtained from hydrolysed BCPR neutral lipids, by lipolytically active oat flour, were subjected to microbial isomerization in BCPR slurries. In 10% (w/v) slurries, a simultaneous enrichment in c9,t11-CLA and c9,t11,c15-CLNA of up to 0·51 and 0·29 mg ml-1 was observed from starting levels of 0·96 mg LA ml-1 and 0·37 mg α-LNA ml-1 respectively. CONCLUSIONS: This study shows that growing cultures of P. freudenreichii DSM 20270 are able to simultaneously enrich BCPR with health-beneficial conjugated isomers of LA and α-LNA. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that microbial isomerization technique can be utilized to enrich lipid-containing plant materials with bioactive compounds and thereby enable valorization of low value plant-based side streams from food industry into value-added food ingredients.

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.

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.

A low-power ultrasound attenuation improves the stability of biofilm and hydrophobicity of Propionibacterium freudenreichii subsp. freudenreichii DSM 20271 and Acidipropionibacterium jensenii DSM 20535.

The main topic of this paper was to study the effect of ultrasound-attenuation (US) on the surface properties of propionibacteria (Acidipropionibacterium jensenii DSM 20535 and Propionibacterium freudenreichii DSM 20271). A preliminary screening was done by using different power levels (40 and 60%) and treatment times (4, 6, and 8 min); immediately after sonication, acidification and viable count were tested. The best combinations to avoid post-acidification after 6 h were the following: A. jensenii DSM 20535: power, 40%; time, 8 min; P. freudenreichii subsp. freudenreichii DSM 20271: power, 60%; time, 4 min. Moreover, the effect of US on the growth patterns, surface properties (biofilm formation and hydrophobicity), resistance to some selected antibiotics, and release of intracellular components was evaluated; the experiments were done immediately after the treatment. US-treatment improved the stability of biofilm after 5-7 days, caused an increase of hydrophobicity (from 15 to 27%) immediately after sonication, and determined an increase of cell permeability, as suggested by the release of intracellular components within 24 h and by the increased sensitivity to some antibiotics. This paper is the first report on US-attenuation on propionibacteria and could the background for future researches to modulate the surface properties of these microorganisms.

Growth engineering of Propionibacterium freudenreichii shermanii for organic acids and other value-added products formation.

Propionic acid production from glucose was studied using Propionibacterium freudenreichii shermanii. Conditions were optimized for high yields of propionic acid and total organic acids by sequential optimization of parameters like pH, inoculum age, inoculum volume and substrate concentration. Near-theoretical yield (0.54 ± 0.023 g/g) was achieved for propionic acid with fermentation of 1% glucose using 20% (v/v) of 48 hr old P. shermanii at 30°C, pH maintained at 5.5. Total organic acid yield under these conditions was 0.74 ± 0.06 g/g. The study resulted in achieving 98% and 95% theoretical yields of propionic acid and total organic acids, respectively. Under optimized conditions, along with organic acids, P. shermanii also produced vitamin B12 and trehalose intracellularly, showing its potential to be used as a cell factory.

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.

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.

Hyperconcentrated Sweet Whey, a New Culture Medium That Enhances Propionibacterium freudenreichii Stress Tolerance.

UNLABELLED: Propionibacterium freudenreichii is used as a cheese-ripening starter and as a probiotic. Its reported physiological effects at the gut level, including modulation of bifidobacteria, colon epithelial cell proliferation and apoptosis, and intestinal inflammation, rely on active metabolism in situ Survival and activity are thus key factors determining its efficacy, creating stress adaptation and tolerance bottlenecks for probiotic applications. Growth media and growth conditions determine tolerance acquisition. We investigated the possibility of using sweet whey, a dairy by-product, to sustain P. freudenreichii growth. It was used at different concentrations (dry matter) as a culture medium. Using hyperconcentrated sweet whey led to enhanced multistress tolerance acquisition, overexpression of key stress proteins, and accumulation of intracellular storage molecules and compatible solutes, as well as enhanced survival upon spray drying. A simplified process from growth to spray drying of propionibacteria was developed using sweet whey as a 2-in-1 medium to both culture P. freudenreichii and protect it from heat and osmotic injury without harvesting and washing steps. As spray drying is far cheaper and more energy efficient than freeze-drying, this work opens new perspectives for the sustainable development of new starter and probiotic preparations with enhanced robustness. IMPORTANCE: In this study, we demonstrate that sweet whey, a dairy industry by-product, not only allows the growth of probiotic dairy propionibacteria, but also triggers a multitolerance response through osmoadaptation and general stress response. We also show that propionibacteria accumulate compatible solutes under these culture conditions, which might account for the limited loss of viability after spray drying. This work opens new perspectives for more energy-efficient production of dairy starters and probiotics.

Effect of Direct-Fed Microbial Dosage on the Fecal Concentrations of Enterohemorrhagic Escherichia coli in Feedlot Cattle.

Contamination of beef products by Shiga toxin-producing Escherichia coli is a concern for food safety with a particular subset, the enterohemorrhagic E. coli (EHEC), being the most relevant to human disease. To mitigate food safety risks, preharvest intervention strategies have been implemented with the aim to reduce EHEC in cattle. One class of interventions that has been widely used in feedlots is direct-fed microbials (DFMs), which can contain various dosing rates of probiotic bacteria. Here we compare the use of two different doses of a commercially available DFM on total EHEC load in a commercial feedlot setting. The DFMs used were the standard 10(9) Propionibacterium freudenreichii and 10(6) Lactobacillus acidophilus colony forming units (CFUs)/head/day dose of Bovamine(®) (Nutrition Physiology Company, Guymon, OK) and the higher dose, Bovamine Defend™ (Nutrition Physiology Company), which is dosed at 10(9) P. freudenreichii and 10(9) Lactobacillus acidophilus CFUs/head/day. To analyze the total EHEC fecal concentration, 2200 head of cattle were assigned a DFM feed regimen lasting approximately 5 months. At harvest, 480 head of cattle were sampled using rectoanal mucosal swabs. A quantitative polymerase chain reaction assay targeting ecf1 was used to enumerate the total EHEC fecal concentration for 240 head fed the low-dose DFM and 240 head fed the high-dose DFM. No significant difference (p > 0.05) in the fecal concentration of total EHEC was observed between the two doses. This suggests that using an increased dosage provides no additional reduction in the total EHEC fecal concentration of feedlot cattle compared to the standard dosage.