Immunomodulation properties of designed fermented milks

L'Unité de Recherche Œnologie (EA4577 de l’Université Bordeaux Segalen et USC1366 INRA, rattachée au département CEPIA - Caractérisation et Elaboration des Produits issus de l'Agriculture), organise la 19ème édition du Club des Bactéries Lactiques.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 probioticproperties but little is still known about their individual and/or synergistic beneficial effects within dairymatrices. In the context of a rising incidence of Inflammatory Bowel Diseases, it has become crucial toevaluate the immunomodulatory potential of bacteria ingested in large numbers via dairy products. Wetherefore selected different strains and combinations of technological LAB and PAB. We determined theirimmunomodulatory potential by IL-10 and IL-12 induction, in human peripheral blood mononuclearcells, on either single or mixed cultures, grown on laboratory medium or directly in milk. Milk wasfermented with selected anti-inflammatory strains of LAB or PAB/LAB mixed cultures and the resultingbacterial fractions were also evaluated for these properties, together with starter viability and optimumtechnological aspects. The most promising fermented milks were evaluated in the context of TNBS- orDSS-induced colitis in mice. The improvement in inflammatory parameters evidenced an alleviation ofcolitis symptoms as a result of fermented milk consumption. This effect was clearly strain-dependent andmodulated by growth within a fermented dairy product. These findings offer new tools and perspectivesfor the development of immunomodulatory fermented dairy products for targeted populations

Immunomodulatory properties of the β-glucan surface polysaccharide of Propionibacterium freudenreichii, a dairy bacterium with probiotic potential

P. freudenreichii (PF) is a Gram + species, with the GRAS (Generally Recognized As Safe) status in the USA and a Qualified Presumption of Safety (QPS) status in Europe. It is widely consumed by humans because it is an essential ripening culture in Swiss type cheeses (Emmental, Leerdammer®…), which contain more than 109 live cells / g of cheese. It is also used as a probiotic in food supplement (Propio-Fidus®). Recently, 30% of the strains of PF were shown shown to produce a (1→3, 1→2)-β-D-glucan surface polysaccharide. A single gene (named gtfF) is responsible for the biosynthesis. Here we investigated the role(s) of this β-glucan

Cold adaptation of the cheese ripening bacterium Protectionniste freudenreichii

Propionibacterium freudenreichii is known to play a key role in the formation of cheese flavor throughout Swiss-type cheese ripening and especially during cold storage of cheese. At every stage of cheese manufacture, P. freudenreichii has to face several stresses-generating conditions and especially a cold-induced stress when Swiss cheeses are transferred from a warm (24°C) ripening room to a cold (4°C) room. The aim of this study was to investigate the adaptation and survival of P. freudenreichii at cold temperature by means of the first global gene expression profile for this species. The temporal transcriptomic response of P. freudenreichii was analyzed during its growth phase at 30°C and then during further incubation at 4°C for 9 days, always preventing any exhaustion of the main carbon source (lactate). As the cells transitioned from the warm to the cold condition, most of the down-expressed genes were involved in cell division, protein turnover, translation, transcription and DNA replication and repair. During incubation at cold temperature, P. freudenreichii adopted multiple strategies for maintaining its viability. It activated a two-component quorum-sensing system (a two-component system sensor kinase and luxR) that can induce population-wide changes in gene expression. It used polyphosphate supplies as energy sources by activating genes coding for Nudix hydrolases and pyrophosphatases. It accumulated carbon supplies by up-regulating genes of lactate, alanine and serine conversion to pyruvate, of aspartate conversion to fumarate, of gluconeogenesis and of glycogen synthesis. Thus, even if its metabolic activity is slowed down at cold temperature, P. freudenreichii remains active, which could explain its ability to continue the production of aroma compounds in cheese during their ripening at low temperature. Understanding the metabolism of P. freudenreichii during cold storage could constitute a real asset for a better optimization of Swiss cheese ripening process thus decreasing the carbon footprint of Swiss-type cheese manufactur

Immunomodulatory properties of the β-glucan surface polysaccharide of Propionibacterium freudenreichii, a dairy bacterium with probiotic potential

P. freudenreichii (PF) is a Gram + species, with the GRAS (Generally Recognized As Safe) status in the USA and a Qualified Presumption of Safety (QPS) status in Europe. It is widely consumed by humans because it is an essential ripening culture in Swiss type cheeses (Emmental, Leerdammer®…), which contain more than 109 live cells / g of cheese. It is also used as a probiotic in food supplement (Propio-Fidus®). Recently, 30% of the strains of PF were shown shown to produce a (1→3, 1→2)-β-D-glucan surface polysaccharide. A single gene (named gtfF) is responsible for the biosynthesis. Here we investigated the role(s) of this β-glucan

Cold adaptation of the cheese ripening bacterium Protectionniste freudenreichii

Propionibacterium freudenreichii is known to play a key role in the formation of cheese flavor throughout Swiss-type cheese ripening and especially during cold storage of cheese. At every stage of cheese manufacture, P. freudenreichii has to face several stresses-generating conditions and especially a cold-induced stress when Swiss cheeses are transferred from a warm (24°C) ripening room to a cold (4°C) room. The aim of this study was to investigate the adaptation and survival of P. freudenreichii at cold temperature by means of the first global gene expression profile for this species. The temporal transcriptomic response of P. freudenreichii was analyzed during its growth phase at 30°C and then during further incubation at 4°C for 9 days, always preventing any exhaustion of the main carbon source (lactate). As the cells transitioned from the warm to the cold condition, most of the down-expressed genes were involved in cell division, protein turnover, translation, transcription and DNA replication and repair. During incubation at cold temperature, P. freudenreichii adopted multiple strategies for maintaining its viability. It activated a two-component quorum-sensing system (a two-component system sensor kinase and luxR) that can induce population-wide changes in gene expression. It used polyphosphate supplies as energy sources by activating genes coding for Nudix hydrolases and pyrophosphatases. It accumulated carbon supplies by up-regulating genes of lactate, alanine and serine conversion to pyruvate, of aspartate conversion to fumarate, of gluconeogenesis and of glycogen synthesis. Thus, even if its metabolic activity is slowed down at cold temperature, P. freudenreichii remains active, which could explain its ability to continue the production of aroma compounds in cheese during their ripening at low temperature. Understanding the metabolism of P. freudenreichii during cold storage could constitute a real asset for a better optimization of Swiss cheese ripening process thus decreasing the carbon footprint of Swiss-type cheese manufactur

The use of targeted mRNA expression as biomarker to track growth and activity of Lactobacillus helveticus in broth and in Swiss cheese

Introduction: Biomarker is defined as a biological characteristic that is objectively measured and evaluated as an indicator of biological processes. Purpose: The aim of this work was to monitor Lactobacillus helveticus mRNA expression levels via biomarkers associated to metabolic activity (tuf gene) and stress response (groL gene) during culture in broth and throughout Swiss cheese manufacture.Methods: Three small scale Swiss cheese were manufactured from microfiltered milk inoculated with Lactobacillus helveticus ITG LH56, Streptococcus thermophilus, Propionibacterium freudenreichii and Lactobacillus paracasei. In parallel, three independent cultures of L. helveticus ITG LH56 were performed in MRS at 37°C. Population was quantified by numeration on MRS agar medium and qPCR. Quantifications of tuf and groL transcripts were expressed as percentage of the total number of targeted transcripts, and considered as indicators of metabolic activity and stress response, respectively. Results: In pure culture, L. helveticus growth occurred with a percentage of mRNA expression levels associated to tuf gene>70%. In contrast, late-exponential and stationary phases were associated to an increasing expression of groL, which reached a maximum of 30% at the beginning of stationary phase. During cheese manufacture, L. helveticus reached maximal population during the acidification step with groL mRNA expression levels higher than 40% yielding a peak at the end of the cold room storage. Entering cold (10 days at 12°C) and warm storage (7 days at 24°C) was associated with tuf expression of 50% even though culturable bacterial counts were decreasing, suggesting that cells were metabolically active and may thus be involved in the formation of aroma compounds. Incidence: This study reports how recording of targeted mRNA expression levels along cheese manufacture and ripening process can give information on a metabolic activity of interest, or more globally, on cell physiological state. The use of biomarkers enables accurate quantifications to better understand and improve online industrial fermentation processe

Identification of the main esterase involved in milk fat hydrolysis in [i]Propionibacterium Freudenreichii[/i]

Free fatty acids (FFA) are important flavor compounds in cheese, where they contribute to pungent,rancid, cheesy, and fruity notes. They mainly result from the lipolytic activity of cheesemicroorganisms. In Swiss cheese, Propionibacterium freudenreichii, a species used as ripeningculture, is the main agent of lipolysis, with 96% of FFA released during ripening resulting from itsactivity. Our aim was to identify the esterase(s) involved in lipolysis by P. freudenreichii. Sincelipolysis in Swiss cheese mainly occurs during P. freudenreichii growth, we hypothesized that it doesnot result from the activity of intracellular enzymes released from lyzed cells, but from surfaceexposedor secreted esterases. Thus, we focused the present study on previously identifiedesterases, one secreted, PF#279, and one putative esterase predicted to be anchored in the plasmamembrane, PF#774. To evaluate the respective role of these two proteins in lipolysis,P. freudenreichii CIRM‐BIA1T was knocked out for the genes encoding these two proteins,separately. Each of these genes was also over‐expressed in the same strain. All genetically modifiedstrains were assessed for their lipolytic activity during their growth in a medium containing milk fatemulsion. In parallel, the lipolytic activity of 22 wild strains was also tested under the sameconditions, and the sequences of these two targeted genes compared among strains. We showedthat mutants over‐expressing either PF#279 or PF#774 released about three times more FFAcompared to the wild‐type strain (3.5 vs 0.9 mg/g fat), demonstrating that both enzymes arelipolytic esterases. However, only the mutant inactivated for PF#279 was affected in its lipolyticactivity. The lipolytic activity of the 22 wild strains tested varied over a large range (FFA netproduction ~ 0.05 to 1.9 mg/g fat). Interestingly, the two non lipolytic strains identified exhibitedthe same single deletion at the beginning of pf279 sequence, whereas pf774 sequence was highlyconserved among strains, confirming the prominent role of PF#279 in lipolysis. Taken together,these results show that PF#279 is the main lipolytic esterase involved in milk fat hydrolysis in P.freudenreichii and is likely a key component in Swiss cheese lipolysis

Identification of the main esterase involved in milk fat hydrolysis in [i]Propionibacterium Freudenreichii[/i]

Free fatty acids (FFA) are important flavor compounds in cheese, where they contribute to pungent,rancid, cheesy, and fruity notes. They mainly result from the lipolytic activity of cheesemicroorganisms. In Swiss cheese, Propionibacterium freudenreichii, a species used as ripeningculture, is the main agent of lipolysis, with 96% of FFA released during ripening resulting from itsactivity. Our aim was to identify the esterase(s) involved in lipolysis by P. freudenreichii. Sincelipolysis in Swiss cheese mainly occurs during P. freudenreichii growth, we hypothesized that it doesnot result from the activity of intracellular enzymes released from lyzed cells, but from surfaceexposedor secreted esterases. Thus, we focused the present study on previously identifiedesterases, one secreted, PF#279, and one putative esterase predicted to be anchored in the plasmamembrane, PF#774. To evaluate the respective role of these two proteins in lipolysis,P. freudenreichii CIRM‐BIA1T was knocked out for the genes encoding these two proteins,separately. Each of these genes was also over‐expressed in the same strain. All genetically modifiedstrains were assessed for their lipolytic activity during their growth in a medium containing milk fatemulsion. In parallel, the lipolytic activity of 22 wild strains was also tested under the sameconditions, and the sequences of these two targeted genes compared among strains. We showedthat mutants over‐expressing either PF#279 or PF#774 released about three times more FFAcompared to the wild‐type strain (3.5 vs 0.9 mg/g fat), demonstrating that both enzymes arelipolytic esterases. However, only the mutant inactivated for PF#279 was affected in its lipolyticactivity. The lipolytic activity of the 22 wild strains tested varied over a large range (FFA netproduction ~ 0.05 to 1.9 mg/g fat). Interestingly, the two non lipolytic strains identified exhibitedthe same single deletion at the beginning of pf279 sequence, whereas pf774 sequence was highlyconserved among strains, confirming the prominent role of PF#279 in lipolysis. Taken together,these results show that PF#279 is the main lipolytic esterase involved in milk fat hydrolysis in P.freudenreichii and is likely a key component in Swiss cheese lipolysis

The long-term survival of [i]Propionibacterium freudenreichii[/i] in a context of nutrient shortage

AimsPropionibacterium freudenreichii is an actinobacterium widely used in dairy industry during the ripening process of Swiss-type cheeses and which presents probiotic properties. PF is reportedly a hardy bacterium, able to survive during the cheese-making process and when subjected to digestive stresses. During this study the long-term survival of P. freudenreichii was investigated for 11 days by means of phenotypic characterization in a culture medium without the addition of any nutrients.Methods and ResultsFor 11 days, in a non-nutrient supplemented culture medium, eight strains were monitored by measuring their optical density, counting colony-forming units (CFU) and using LIVE/DEAD staining and microscopy observation. Under these conditions, all strains displayed high survival rates in the culture medium, their culturability reaching more the 9 log10 CFU/mL after two days. After 11 days, this value ranged from 7.8 to 8.2 log10 CFU/mL depending on the strain, and at least 50% of the P. freudenreichii population displayed an intact envelope. Since lysis of part of a bacterial population may be a microbial strategy to recover nutrients, in CIRM-BIA 138 (the strain with the highest population at day 11), cell lysis was assessed by quantifying intact bacterial cells using qPCR targeting the housekeeping gene tuf. No lysis was observed.ConclusionTaken together, our results suggest that P. freudenreichii strains use a viable but nonculturable (VBNC) state to adapt to the long term survival phase.Significance and Impact of the studyAssessing the viability of P. freudenreichii and understanding their mechanisms for survival should be of great interest regarding their potential probiotic applications