Co-culturing Propionibacterium freudenreichii and Bifidobacterium animalis subsp. lactis improves short-chain fatty acids and vitamin B12 contents in soy whey.

The lack of vitamin B12 in unprocessed plant-based foods can lead to health problems in strict vegetarians and vegans. The main aim of this study was to investigate the potential synergy of co-culturing Bifidobacterium animalis subsp. lactis and Propionibacterium freudenreichii in improving production of vitamin B12 and short-chain fatty acids in soy whey. Different strategies including mono-, sequential and simultaneous cultures were adopted. Growth, short-chain fatty acids and vitamin B12 were assessed throughout the fermentation while free amino acids, volatiles, and isoflavones were determined on the final day. P. freudenreichii monoculture grew well in soy whey, whereas B. lactis monoculture entered the death phase by day 4. Principal component analysis demonstrates that metabolic changes in both sequential cultures did not show drastic differences to those of P. freudenreichii monoculture. However, simultaneous culturing significantly improved vitamin B12, acetic acid and propionic acid contents (1.3 times, 5 times, 2.5 times, compared to the next highest treatment [sequential cultures]) in fermented soy whey relative to other culturing modes. Hence, co-culturing of P. freudenreichii and B. lactis would provide an alternative method to improve vitamin B12, acetic acid and propionic acid contents in fermented foods.

Propionibacterium freudenreichii CIRM-BIA 129 mitigates colitis through S layer protein B-dependent epithelial strengthening.

The growing incidence of human diseases involving inflammation and increased gut permeability makes the quest for protective functional foods more crucial than ever. Propionibacterium freudenreichii (P. freudenreichii) is a beneficial bacterium used in the dairy and probiotic industries. Selected strains exert anti-inflammatory effects, and the present work addresses whether the P. freudenreichii CIRM-BIA129, consumed daily in a preventive way, could protect mice from acute colitis induced by dextran sodium sulfate (DSS), and more precisely, whether it could protect from intestinal epithelial breakdown induced by inflammation. P. freudenreichii CIRM-BIA129 mitigated colitis severity and inhibited DSS-induced permeability. It limited crypt length reduction and promoted the expression of zonula occludens-1 (ZO-1), without reducing interleukin-1ß mRNA (il-1ß) expression. In vitro, P. freudenreichii CIRM-BIA129 prevented the disruption of a Caco-2 monolayer induced by proinflammatory cytokines. It increased transepithelial electrical resistance (TEER) and inhibited permeability induced by inflammation, along with an increased ZO-1 expression. Extracellular vesicles (EVs) from P. freudenreichii CIRM-BIA129, carrying the surface layer protein (SlpB), reproduced the protective effect of P. freudenreichii CIRM-BIA129. A mutant strain deleted for slpB (ΔslpB), or EVs from this mutant strain, had lost their protective effects and worsened both DSS-induced colitis and inflammation in vivo. These results shown that P. freudenreichii CIRM-BIA129 daily consumption has the potential to greatly alleviate colitis symptoms and, particularly, to counter intestinal epithelial permeability induced by inflammation by restoring ZO-1 expression through mechanisms involving S-layer protein B. They open new avenues for the use of probiotic dairy propionibacteria and/or postbiotic fractions thereof, in the context of gut permeability.NEW & NOTEWORTHY Propionibacterium freudenreichii reduces dextran sodium sulfate (DSS)-induced intestinal permeability in vivo. P. freudenreichii does not inhibit inflammation but damages linked to inflammation. P. freudenreichii inhibits intestinal epithelial breakdown through S-layer protein B. The protective effects of P. freudenreichii depend on S-layer protein B. Extracellular vesicles from P. freudenreichii CB 129 mimic the protective effect of the probiotic.

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.

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.

Developing a single-stage continuous process strategy for vitamin B12 production with Propionibacterium freudenreichii.

BACKGROUND: Vitamin B12 is a widely used compound in the feed and food, healthcare and medical industries that can only be produced by fermentation because of the complexity of its chemical synthesis. Besides, the use of Generally Recognized as Safe (GRAS) and Qualified Presumption of Safety (QPS) microorganisms, like Propionibacterium freudenreichii, especially non-GMO wild-type producers, are becoming an interesting alternative in markets where many final consumers have high health and ecological awareness. In this study, the production of vitamin B12 using the Propionibacterium freudenreichii NBRC 12391 wild-type strain was characterized and optimized in shake flasks before assessing several scale-up strategies. RESULTS: Initial results established that: (i) agitation during the early stages of the culture had an inhibitory effect on the volumetric production, (ii) 5,6-dimethylbenzimidazole (DMBI) addition was necessary for vitamin B12 production, and (iii) kinetics of vitamin B12 accumulation were dependent on the induction time when DMBI was added. When scaling up in a bioreactor, both batch and fed-batch bioprocesses proved unsuitable for obtaining high volumetric productivities mainly due to carbon source limitation and propionic acid inhibition, respectively. To overcome these drawbacks, an anaerobic single-phase continuous bioprocess strategy was developed. This culture strategy was maintained stable during more than 5 residence times in two independent cultures, resulting in 5.7-fold increase in terms of volumetric productivity compared to other scale-up strategies. CONCLUSION: Overall, compared to previously reported strategies aimed to reduce propionic acid inhibition, a less complex anaerobic single-phase continuous and more scalable bioprocess was achieved.

Methylotrophic bacteria with cobalamin-dependent mutases in primary metabolism as potential strains for vitamin B12 production.

Several bacterial species are known for their ability to synthesize vitamin B12 but biotechnological vitamin B12 production today is restricted to Pseudomonas denitrificans and Propionibacterium freudenreichii. Nevertheless, the rising popularity of veganism leads to a growing demand for vitamin B12 and thereby interest in alternative strains which can be used as efficient vitamin B12 sources. In this work, we demonstrate that methylotrophic microorganisms which utilize the ethylmalonyl-CoA pathway containing B12-dependent enzymes are capable of active vitamin B12 production. Several bacteria with an essential function of the pathway were tested for vitamin B12 synthesis. Among the identified strains, Hyphomicrobium sp. DSM3646 demonstrated the highest vitamin B12 levels reaching up to 17.9 ± 5.05 µg per g dry cell weight. These relatively high vitamin B12 concentrations achieved in simple cultivation experiments were performed in a mineral methanol medium, which makes Hyphomicrobium sp. DSM3646 a new promising cobalamin-producing strain.

Propionibacterium freudenreichii-Assisted Approach Reduces N2O Emission and Improves Denitrification via Promoting Substrate Uptake and Metabolism.

N2O emission is often encountered during biodenitrification. In this paper, a new approach of using microorganisms to promote substrate uptake and metabolism to reduce denitrification intermediate accumulation was reported. With the introduction of Propionibacterium freudenreichii to a biodenitrification system, N2O and nitrite accumulation was, respectively, decreased by 74 and 60% and the denitrification efficiency was increased by 150% at the time of 24 h with P. freudenreichii/groundwater denitrifier of 1/5 (OD600). Propionate, produced by P. freudenreichii, only accelerated nitrate removal and was not the main reason for the decreased intermediate accumulation. The proteomic and enzyme analyses revealed that P. freudenreichii stimulated biofilm formation by upregulating proteins involved in porin forming, putrescine biosynthesis, spermidine/putrescine transport, and quorum sensing and upregulated transport proteins, which facilitated the uptake of the carbon source, nitrate, and Fe and Mo (the required catalytic sites of denitrification enzymes). Further investigation revealed that P. freudenreichii activated the methylmalonyl-CoA pathway in the denitrifier and promoted it to synthesize heme/heme d1, the groups of denitrification enzymes and electron transfer proteins, which upregulated the expression of denitrifying enzyme proteins and enhanced the ratio of NosZ to NorB, resulting in the increase of generation, transfer, and consumption of electrons in biodenitrification. Therefore, a significant reduction in the denitrification intermediate accumulation and an improvement in the denitrification efficiency were observed.

Microaerobic metabolism of lactate and propionate enhances vitamin B12 production in Propionibacterium freudenreichii.

BACKGROUND: Propionibacterium freudenreichii is used in biotechnological applications to produce vitamin B12. Although cultured mainly in anaerobic conditions, microaerobic conditions can greatly enhance biomass formation in P. freudenreichii. Since B12 yields may be coupled to biomass formation, microaerobic conditions show great potential for increasing B12 yields in P. freudenreichii. RESULTS: Here we show biomass formation increases 2.7 times for P. freudenreichii grown in microaerobic conditions on lactate versus anaerobic conditions (1.87 g/L vs 0.70 g/L). Consumption of lactate in microaerobic conditions resulted first in production of pyruvate, propionate and acetate. When lactate was depleted, pyruvate and propionate were oxidised with a concomitant sixfold increase in the B12 titer compared to anaerobic conditions, showing potential for propionate and pyruvate as carbon sources for B12 production. Consequently, a fed-batch reactor with anaerobically precultured lactate-grown cells was fed propionate in microaerobic conditions resulting in biomass increase and production of B12. Vitamin yields increased from 0.3 [Formula: see text] B12 per mmol lactate in anaerobic conditions to 2.4 [Formula: see text] B12 per mmol lactate and 8.4 [Formula: see text] B12 per mmol propionate in microaerobic conditions. Yield per cell dry weight (CDW) increased from 41 [Formula: see text] per g CDW in anaerobic conditions on lactate to 92 [Formula: see text] per g CDW on lactate and 184 [Formula: see text] per g CDW on propionate in microaerobic conditions. CONCLUSIONS: Here we have shown both B12 yield per substrate and per CDW were highest on cells oxidising propionate in microaerobic conditions, showing the potential of propionate for biotechnological production of vitamin B12 by P. freudenreichii.

A Unique Enhancement of Propionibacterium freudenreichii's Ability to Remove Pb(II) from Aqueous Solution by Tween 80 Treatment.

Microbial agents have promise for the bioremediation of Pb(II)-polluted environments and wastewater, the biodecontamination of foods, and the alleviation of toxicity in living organisms. The dairy bacterium Propionibacterium freudenreichii is poorly able to remove Pb(II) from aqueous solution at 25 ppm, ranging from 0 to 10% of initial concentration. Here, we report on an original strong enhancement of this activity (ranging from 75% to 93%, p < 0.01) following the addition of a polysorbate detergent (Tween® 80) during or either shortly after the growth of a P. freudenreichii culture. We evaluated the optimal Tween® 80 concentration for pretreatment conditions, documented the role of other detergents, and explored the possible mechanisms involved. Our results reveal a novel, environmentally friendly, low-cost pretreatment procedure for enhancing the selective removal of lead from water by probiotic-documented bacteria.

Genomic rearrangements in the aspA-dcuA locus of Propionibacterium freudenreichii are associated with aspartase activity.

Propionibacterium freudenreichii is crucial in Swiss-type cheese manufacture. Classic propionic acid fermentation yields the nutty flavor and the typical eyes. Co-metabolism of aspartate pronounces the flavor of the cheese; however, it also increases the size of the eyes, which can induce splitting and reduce the cheese quality. Aspartase (EC 4.3.1.1) catalyzes the deamination of aspartate, yielding fumarate and ammonia. The aspartase activity varies considerably among P. freudenreichii strains. Here, the correlation between aspartase activity and the locus of aspartase-encoding genes (aspA ) and dcuA encoding the C4-dicarboxylate transporter was investigated in 46 strains to facilitate strain selection for cheese culture. Low aspartase activity was correlated with a particular genomic rearrangement: low in vitro aspartase activity always occurred in strains with gene clusters aspA - dcuA where the dcuA was frameshifted, producing a stop codon or was disrupted by an ISL3-like element. The low aspartase activity could be due to the protein sequence of the aspartase or a dysfunctional DcuA. The highest values of aspartase activity were detected in strains with aspA1 - aspA2-dcuA with a DcuA sequence sharing 99.07 - 100% identity with the DcuA sequence of strain DSM 20271 T and an additional C4-dicarboxylate transporter belonging to the DcuAB family.

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.

Metabolic profiling analysis of the vitamin B12 producer Propionibacterium freudenreichii.

Vitamin B12 (VB12 ) is an indispensable cofactor of metabolic enzymes and has been widely used in the food and pharmaceutical industries. In this study, the effects of medium composition on VB12 production by Propionibacterium freudenreichii were evaluated and optimized based on statistical experiments. The results showed that glucose, yeast extract, KH2 PO4 , and glycine have significant effects on VB12 production. The final titer of VB12 reached 8.32 ± 0.02 mg/L, representing a 120% increase over the non-optimized culture medium. We employed a metabolomics approach to analyze the differences of metabolite concentrations in P. freudenreichii cells cultivated in the original medium and optimized fermentation medium. Using multivariate data analysis, we identified a range of correlated metabolites, illustrating how metabolomics can be used to explain VB12 production changes by corresponding differences in the overall cellular metabolism. The concentrations of many metabolic intermediates of glycolysis, the Wood-Werkman cycle, the TCA cycle, and amino acid metabolism were increased, which contributed to the synthesis of propionic acid and VB12 due to an improved supply of energy and precursors.

Propionibacterium freudenreichii thrives in microaerobic conditions by complete oxidation of lactate to CO2.

In this study we show increased biomass formation for four species of food-grade propionic acid bacteria (Acidipropionibacterium acidipropionici, Acidipropionibacterium jensenii, Acidipropionibacterium thoenii and Propionibacterium freudenreichii) when exposed to oxygen, implicating functional respiratory systems. Using an optimal microaerobic condition, P. freudenreichii DSM 20271 consumed lactate to produce propionate and acetate initially. When lactate was depleted propionate was oxidized to acetate. We propose to name the switch from propionate production to consumption in microaerobic conditions the 'propionate switch'. When propionate was depleted the 'acetate switch' occurred, resulting in complete consumption of acetate. Both growth rate on lactate (0.100 versus 0.078 h-1 ) and biomass yield (20.5 versus 8.6 g* mol-1 lactate) increased compared to anaerobic conditions. Proteome analysis revealed that the abundance of proteins involved in the aerobic and anaerobic electron transport chains and major metabolic pathways did not significantly differ between anaerobic and microaerobic conditions. This implicates that P. freudenreichii is prepared for utilizing O2 when it comes available in anaerobic conditions. The ecological niche of propionic acid bacteria can conceivably be extended to environments with oxygen gradients from oxic to anoxic, so-called microoxic environments, as found in the rumen, gut and soils, where they can thrive by utilizing low concentrations of oxygen.

Metabolic Flux Analysis of Simultaneous Production of Vitamin B12 and Propionic Acid in a Coupled Fermentation Process by Propionibacterium freudenreichii.

The metabolic processes involved in simultaneous production of vitamin B12 and propionic acid by Propionibacterium freudenreichii are very complicated. To further investigate the regulatory mechanism of this metabolism, a simplified metabolic network was established. The effects of glucose feeding, propionic acid removal, and 5,6-dimethylbenzimidazole (DMB) addition on the metabolic flux distribution were investigated. The results showed that synthesis of propionic acid can be increased by increasing the metabolic flux through the oxaloacetate and methylmalonyl-CoA branches in the early and middle stages of the coupled fermentation. After DMB addition, the synthesis of vitamin B12 was significantly enhanced via increased metabolic flux through the δ-aminolevulinate branch, which promoted the synthesis of uroporphyrinogen III, a precursor of vitamin B12. Therefore, the analysis of metabolic flux at key nodes can provide theoretical guidance for the optimization of P. freudenreichii fermentation processes. In an experimental coupled fermentation process, the concentrations of vitamin B12 and propionic acid reached 21.6 and 50.12 g/L respectively, increased by 105.71% and 73.91% compared with batch fermentation, which provides a new strategy for industrial production.

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.

Evaluating the effects of Lactobacillus animalis and Propionibacterium freudenreichii on performance and rumen and fecal measures in lactating dairy cows.

Two experiments evaluated the effect of supplementation with a bacterial direct-fed microbial on performance and apparent total-tract nutrient digestion of dairy cows. In experiment 1, 30 multiparous cows (75 ± 32 d in milk) were randomly assigned to 1 of 2 treatments fed for 10 wk. All cows were fed a diet containing 23.8% starch. Treatments were top dressed to rations twice daily and consisted of a combination of Lactobacillus animalis (1 × 109 cfu/d) and Propionibacterium freudenreichii (2 × 109 cfu/d; LAPF) or carrier alone (CON). In experiment 2, 6 ruminally cannulated cows (123 ± 129 d in milk) were randomly assigned to a crossover design with two 6-wk periods. Cows received the same CON or LAPF treatment as in experiment 1. Cows were fed the same 23.8% starch diet as experiment 1 during wk 1 through 5 of each period, and then cows were abruptly switched to a 31.1% starch diet for wk 6. For both experiments, intake and milk yield were measured daily, and milk samples were collected weekly. In experiment 1, fecal grab samples were collected every 6 h on d 7 of experimental wk 1, 2, 4, 6, 8, and 10. Fecal consistency was scored, and fecal starch was measured in daily composite samples. Fecal composites from a subset of 7 cows per treatment were used to measure apparent total-tract nutrient digestion. In experiment 2, rumen pH was continuously recorded during wk 5 and 6. On d 7 of wk 5 (the final day of feeding the 23.8% starch ration), d 1 of wk 6 (the day of diet transition), and d 7 of wk 6 (the final day of feeding the 31.1% starch ration), rumen in situ digestion was determined. Samples of rumen fluid and feces were collected every 6 h on those days for measurement of fecal starch (composited by cow within day), rumen volatile fatty acids, and fecal pH. Rumen and fecal samples were collected at one time point on those days for microbiota assessment. In experiment 1, treatment did not affect intake, milk yield, milk composition, or fecal score. The LAPF treatment decreased fecal starch percentage and tended to increase starch digestion compared with CON, but the differences were very small (0.59 vs. 0.78% and 98.74 vs. 98.46%, respectively). Digestion of other nutrients was unaffected. In experiment 2, LAPF increased rumen pH following the abrupt switch to the high-starch diet, but milk yield was lower for LAPF compared with CON (35.7 vs. 33.2 kg/d). Contrary to the decrease in fecal starch with LAPF observed in experiment 1, fecal starch tended to be increased by LAPF following the abrupt ration change in experiment 2 (2.97 vs. 2.15%). Few effects of treatment on rumen and fecal microbial populations were detectable. Under the conditions used in our experiments, addition of the bacterial direct-fed microbials did not have a marked effect on animal performance, ruminal measures, or total-tract nutrient digestion.

Antiobesity and antidiabetic effects of the dairy bacterium Propionibacterium freudenreichii MJ2 in high-fat diet-induced obese mice by modulating lipid metabolism.

Obesity can cause chronic metabolic disorders such as type 2 diabetes, hyperlipidemia, and nonalcoholic fatty liver diseases. The aim of this study was to investigate the antiobesity and antidiabetic effects of the dairy bacterium P. freudenreichii MJ2 isolated from raw milk using 3T3-L1 cells and high-fat diet (HFD)-induced obese mice. Lipid accumulation and the expression levels of genes related to lipid metabolism, such as preadipocytic gene (Pref-1), adipogenic genes (PPARγ and C/EBPα), and lipogenic genes (FAS, SCD-1, and ACC), significantly decreased in heat-killed P. freudenreichii MJ2 (hkMJ2)-treated adipocytes. Live P. freudenreichii MJ2 (MJ2), hkMJ2, and Lactobacillus plantarum (LP) decreased body weight gain in HFD-induced obese mice compared with the model group. The liver and epididymal white adipose tissue weights in the MJ2-, hkMJ2- and LP-treated groups were significantly lower than those in the model group. The expression levels of genes and proteins related to adipogenesis and lipogenesis significantly decreased and lipolysis (HSL and ATGL) increased in the MJ2-, hkMJ2-, and LP-treated groups. The expression levels of genes related to fatty acid ß-oxidation (CPT-1α and ACOX1) increased in the MJ2-, hkMJ2-, and LP-treated groups. In addition, blood glucose and fasting insulin levels in the MJ2- and hkMJ2-treated groups decreased compared with those in the model group. P. freudenreichii MJ2 ameliorate insulin resistance by obesity. In conclusion, both MJ2 and hkMJ2 alleviate obesity and metabolic syndrome.