Hélène Falentin

Recent advances in quantitative PCR (qPCR) applications in food microbiology

Molecular methods are being increasingly applied to detect, quantify and study microbial populations in food or during food processes. Among these methods, PCR-based techniques have been the subject of considerable focus and ISO guidelines have been established for the detection of food-borne pathogens. More particularly, real-time quantitative PCR (qPCR) is considered as a method of choice for the detection and quantification of microorganisms. One of its major advantages is to be faster than conventional culture-based methods. It is also highly sensitive, specific and enables simultaneous detection of different microorganisms. Application of reverse-transcription-qPCR (RT-qPCR) to study population dynamics and activities through quantification of gene expression in food, by contrast with the use of qPCR, is just beginning. Provided that appropriate controls are included in the analyses, qPCR and RT-qPCR appear to be highly accurate and reliable for quantification of genes and gene expression. This review addresses some important technical aspects to be considered when using these techniques. Recent applications of qPCR and RT-qPCR in food microbiology are given. Some interesting applications such as risk analysis or studying the influence of industrial processes on gene expression and microbial activity are reported.

The Complete Genome of Propionibacterium freudenreichii CIRM-BIA1T, a Hardy Actinobacterium with Food and Probiotic Applications

Background Propionibacterium freudenreichii is essential as a ripening culture in Swiss-type cheeses and is also considered for its probiotic use [1]. This species exhibits slow growth, low nutritional requirements, and hardiness in many habitats. It belongs to the taxonomic group of dairy propionibacteria, in contrast to the cutaneous species P. acnes. The genome of the type strain, P. freudenreichii subsp. shermanii CIRM-BIA1 (CIP 103027T), was sequenced with an 11-fold coverage. Methodology/Principal Findings The circular chromosome of 2.7 Mb of the CIRM-BIA1 strain has a GC-content of 67% and contains 22 different insertion sequences (3.5% of the genome in base pairs). Using a proteomic approach, 490 of the 2439 predicted proteins were confirmed. The annotation revealed the genetic basis for the hardiness of P. freudenreichii, as the bacterium possesses a complete enzymatic arsenal for de novo biosynthesis of aminoacids and vitamins (except panthotenate and biotin) as well as sequences involved in metabolism of various carbon sources, immunity against phages, duplicated chaperone genes and, interestingly, genes involved in the management of polyphosphate, glycogen and trehalose storage. The complete biosynthesis pathway for a bifidogenic compound is described, as well as a high number of surface proteins involved in interactions with the host and present in other probiotic bacteria. By comparative genomics, no pathogenicity factors found in P. acnes or in other pathogenic microbial species were identified in P. freudenreichii, which is consistent with the Generally Recognized As Safe and Qualified Presumption of Safety status of P. freudenreichii. Various pathways for formation of cheese flavor compounds were identified: the Wood-Werkman cycle for propionic acid formation, amino acid degradation pathways resulting in the formation of volatile branched chain fatty acids, and esterases involved in the formation of free fatty acids and esters. Conclusions/Significance With the exception of its ability to degrade lactose, P. freudenreichii seems poorly adapted to dairy niches. This genome annotation opens up new prospects for the understanding of the P. freudenreichii probiotic activity.

New insights into physiology and metabolism of Propionibacterium freudenreichii

Dairy propionibacteria are Actinobacteria, mainly isolated from dairy environments. Propionibacterium freudenreichii has been used for a long time as a ripening culture in Swiss-type cheese manufacture, and is more and more considered for its potent probiotic effects. This review summarises the knowledge on the main P. freudenreichii pathways and the main features explaining its hardiness, and focuses on recent advances concerning its applications as a cheese ripening agent and as a probiotic for human health. Propionibacteria have a peculiar metabolism, characterised by the formation of propionic acid as main fermentation end-product. They have few nutritional requirements and are able to use a variety of carbon substrates. From the sequence of P. freudenreichii CIRM-BIA1(T) genome, many pathways were reconstituted, including the Wood-Werkman cycle, enzymes of the respiratory chain, synthesis pathways for all amino acids and many vitamins including vitamin B(12). P. freudenreichii displays features allowing its long-term survival. It accumulates inorganic polyphosphate (polyP) as energy reserve, carbon storage compounds (glycogen), and compatible solutes such as trehalose. In cheese, P. freudenreichii plays an essential role in the production of a variety of flavour compounds, including not only propionic acid, but also free fatty acids released via lipolysis of milk glycerides and methyl-butanoic acids resulting from amino acid degradation. P. freudenreichii can exert health-promoting activities, such as a bifidogenic effect in the human gut and promising immunomodulatory effects. Many P. freudenreichii properties involved in adaptation, cheese ripening, bio-preservation and probiotic effects are highly strain-dependent. The elucidation of the molecular mechanisms involved is now facilitated by the availability of genome sequence and molecular tools. It will help in the selection of the most appropriate strain for each application.

Specific metabolic activity of ripening bacteria quantified by real-time reverse transcription PCR throughout Emmental cheese manufacture

Bacterial communities of fermented foods are usually investigated by culture-dependent methods. Real-time quantitative PCR (qPCR) and reverse transcription (RT)-qPCR offer new possibilities to quantify the populations present and their metabolic activity. The aim of this work was to develop qPCR and RT-qPCR methods to assess the metabolic activity and the stress level of the two species used as ripening cultures in Emmental cheese manufacture, Propionibacterium freudenreichii and Lactobacillus paracasei. Three small scale (1/100) microbiologically controlled Emmental cheeses batches were manufactured and inoculated with Lactobacillus helveticus, Streptococcus thermophilus, P. freudenreichii and L. paracasei. At 12 steps of cheese manufacture and ripening, the populations of P. freudenreichii and L. paracasei were quantified by numerations on agar media and by qPCR. 16S, tuf and groL transcript levels were quantified by RT-qPCR. Sampling was carried out in triplicate. qPCR and RT-qPCR assessments were specific, efficient and linear. The quantification limit was 10(3) copies of cells or cDNA/g of cheese. Cell quantifications obtained by qPCR gave similar results than plate count for P. freudenreichii growth and 0.5 to 1 log lower in the stationary phase. Bacterial counts and qPCR quantifications showed that L. paracasei began to grow during the pressing step while P. freudenreichii began to grow from the beginning of ripening (in the cold room). Tuf cDNA quantification results suggested that metabolic activity of L. paracasei reached a maximum during the first part of the ripening (in cold room) and decreased progressively during ripening (in the warm room). Metabolic activity of P. freudenreichii was maximum at the end of cold ripening room and was stable during the first two weeks in warm room. After lactate exhaustion (after two weeks of warm room), the number of tuf cDNA decreased reflecting reduced metabolic activity. For L. paracasei, groL cDNA were stable during ripening. For P. freudenreichii, groL1 gene was highly-expressed during acidification, while groL2 gene highly expression was only observed at the end of the ripening stage after lactate (carbon substrate of P. freudenreichii) exhaustion. The potential use of 16S and tuf genes for the normalization of cDNA quantification throughout an Emmental cheese manufacture is discussed. For the first time, specific gene expression was performed by RT-qPCR yielding metabolic activity and stress response evaluation for L. paracasei and P. freudenreichii in cheese.

Surface proteins of Propionibacterium freudenreichii are involved in its anti-inflammatory properties

UNLABELLED Propionibacterium freudenreichii is a beneficial bacterium used in the food industry as a vitamin producer, as a bio-preservative, as a cheese ripening starter and as a probiotic. It is known to adhere to intestinal epithelial cells and mucus and to modulate important functions of the gut mucosa, including cell proliferation and immune response. Adhesion of probiotics and cross-talk with the host rely on the presence of key surface proteins, still poorly identified. Identification of the determinants of adhesion and of immunomodulation by P. freudenreichii remains a bottleneck in the elucidation of its probiotic properties. In this report, three complementary proteomic methods are used to identify surface-exposed proteins in a strain, previously selected for its probiotic properties. The role of these proteins in the reported immunomodulatory properties of P. freudenreichii is evidenced. This work constitutes a basis for further studies aimed at the elucidation of mechanisms responsible for its probiotic effects, in a post-genomic context. BIOLOGICAL SIGNIFICANCE Dairy propionibacteria, mainly the species Propionibacterium freudenreichii, are consumed in high amounts within Swiss type cheeses. These peculiar bacteria are considered both as dairy starters and as probiotics. Their consumption modulates the gut microbiota, which makes them both probiotic and prebiotic. Promising immunomodulatory properties have been identified in these bacteria, in vitro, in animals and in humans. However, the mechanisms responsible for such anti-inflammatory properties are still unknown. In this work, we identify surface proteins involved in adhesion and immunostimulation by P. freudenreichii. This opens new perspectives for its utilization in new functional fermented food products, in clinical trials, and in understanding modulation of gut inflammation by products containing propionibacteria.

Reverse transcription quantitative PCR revealed persistency of thermophilic lactic acid bacteria metabolic activity until the end of the ripening of Emmental cheese

For Emmental manufacture two kinds of adjunct culture are added: (i) thermophilic lactic acid bacteria (starters) such as Lactobacillus helveticus (LH), and Streptococcus thermophilus (ST) growing the first day of the manufacture and (ii) ripening culture. ST and LH have a key role in curd acidification and proteolysis at the beginning of the manufacture but are considered to be lyzed for a great part of them at the ripening step. The aim of this work was to assess the metabolic activity of these bacteria throughout manufacture and ripening. During Emmental cheesemaking, LH and ST were subjected to i) population quantification by numerations and by quantitative PCR (qPCR) ii) reverse transcription (RT) Temporal Temperature Gel Electrophoresis (TTGE) iii) transcript quantification by RT-qPCR targeting 16S rRNA, tuf and groL mRNAs to evaluate bacterial metabolic activity. During ripening, ST and LH numerations showed a 2.5 log(10) loss of culturability whereas qPCR on pelleted cells revealed only one log(10) of decrease for both of these species. 10(9) ST and 10(8) LH cells/g of cheese still remained. They contained a stable number of 16S transcript and at least 10(6) copies of mRNAs per 10(9) cells until the end of ripening. These results prove the unexpected persistency of thermophilic lactic acid bacteria starters (ST and LH) metabolic activity until the end of ripening and open new perspectives in term of their involvement in the quality of cheeses during ripening.

Bovine Teat Microbiome Analysis Revealed Reduced Alpha Diversity and Significant Changes in Taxonomic Profiles in Quarters with a History of Mastitis

Mastitis is a mammary gland inflammatory disease often due to bacterial infections. Like many other infections, it used to be considered as a host-pathogen interaction driven by host and bacterial determinants. Until now, the involvement of the bovine mammary gland microbiota in the host-pathogen interaction has been poorly investigated, and mainly during the infectious episode. In this study, the bovine teat microbiome was investigated in 31 quarters corresponding to 27 animals, which were all free of inflammation at sampling time but which had different histories regarding mastitis: from no episode of mastitis on all the previous lactations (Healthy quarter, Hq) to one or several clinical mastitis events (Mastitic quarter, Mq). Several quarters whose status was unclear (possible history of subclinical mastitis) were classified as NDq. Total bacterial DNA was extracted from foremilk samples and swab samples of the teat canal. Taxonomic profiles were determined by pyrosequencing on 16s amplicons of the V3-4 region. Hq quarters showed a higher diversity compared to Mq ones (Shannon index: ~8 and 6, respectively). Clustering of the quarters based on their bacterial composition made it possible to separate Mq and Hq quarters into two separate clusters (C1 and C2, respectively). Discriminant analysis of taxonomic profiles between these clusters revealed several differences and allowed the identification of taxonomic markers in relation to mastitis history. C2 quarters were associated with a higher proportion of the Clostridia class (including genera such as Ruminococcus, Oscillospira, Roseburia, Dorea, etc.), the Bacteroidetes phylum (Prevotella, Bacteroides, Paludibacter, etc.), and the Bifidobacteriales order (Bifidobacterium), whereas C1 quarters showed a higher proportion of the Bacilli class (Staphylococcus) and Chlamydiia class. These results indicate that microbiota is altered in udders which have already developed mastitis, even far from the infectious episode. Microbiome alteration may have resulted from the infection itself and or the associated antibiotic treatment. Alternatively, differences in microbiome composition in udders with a history of mastitis may have occurred prior to the infection and even contributed to infection development. Further investigations on the dynamics of mammary gland microbiota will help to elucidate the contribution of this endogenous microbiota to the mammary gland health.

The first dairy product exclusively fermented by Propionibacterium freudenreichii: a new vector to study probiotic potentialities in vivo.

Dairy propionibacteria display probiotic properties which require high populations of live and metabolically active propionibacteria in the colon. In this context, the probiotic vector determines probiotic efficiency. Fermented dairy products protect propionibacteria against digestive stresses and generally contain a complex mixture of lactic and propionic acid bacteria. This does not allow the identification of dairy propionibacteria specific beneficial effects. The aim of this study was to develop a dairy product exclusively fermented by dairy propionibacteria. As they grow poorly in milk, we determined their nutritional requirements concerning carbon and nitrogen by supplementing milk ultrafiltrate (UF) with different concentrations of lactate and casein hydrolysate. Milk or UF supplemented with 50 mM lactate and 5 g L(-1) casein hydrolysate allowed growth of all dairy propionibacteria studied. In these new fermented dairy products, dairy propionibacteria remained viable and stress-tolerant in vitro during minimum 15 days at 4 °C. The efficiency of milk fermented by the most tolerant Propionibacterium freudenreichii strain was evaluated in piglets. Viability and SCFA content in the colon evidenced survival and metabolic activity of P. freudenreichii. This work results in the design of a new food grade vector, which will allow preclinical and clinical trials.

Contribution of Surface β-Glucan Polysaccharide to Physicochemical and Immunomodulatory Properties of Propionibacterium freudenreichii

ABSTRACT Propionibacterium freudenreichii is a bacterial species found in Swiss-type cheeses and is also considered for its health properties. The main claimed effect is the bifidogenic property. Some strains were shown recently to display other interesting probiotic potentialities such as anti-inflammatory properties. About 30% of strains were shown to produce a surface exopolysaccharide (EPS) composed of (1→3,1→2)-β-d-glucan due to a single gene named gtfF. We hypothesized that functional properties of P. freudenreichii strains, including their anti-inflammatory properties, could be linked to the presence of β-glucan. To evaluate this hypothesis, gtfF genes of three β-glucan-producing strains were disrupted. These knockout (KO) mutants were complemented with a plasmid harboring gtfF (KO-C mutants). The absence of β-glucan in KO mutants was verified by immunological detection and transmission electron microscopy. We observed by atomic force microscopy that the absence of β-glucan in the KO mutant dramatically changed the cells topography. The capacity to adhere to polystyrene surface was increased for the KO mutants compared to wild-type (WT) strains. Anti-inflammatory properties of WT strains and mutants were analyzed by stimulation of human peripheral blood mononuclear cells (PBMCs). A significant increase of the anti-inflammatory interleukin-10 cytokine production by PBMCs was measured in the KO mutants compared to WT strains. For one strain, the role of β-glucan in mice gut persistence was assessed, and no significant difference was observed between the WT strain and its KO mutant. Thus, β-glucan appears to partly hide the anti-inflammatory properties of P. freudenreichii; which is an important result for the selection of probiotic strains.

Identification of a Secreted Lipolytic Esterase in Propionibacterium freudenreichii, a Ripening Process Bacterium Involved in Emmental Cheese Lipolysis

ABSTRACT Lipolysis plays an important role in the formation of cheese flavor. In Emmental cheese, the main part of lipolysis has been associated with the presence of Propionibacterium freudenreichii, a species used as a ripening culture. Our aim was to identify the most probable lipolytic esterase(s) involved in cheese lipolysis by P. freudenreichii. Since cheese lipolysis mainly occurs during P. freudenreichii growth, we hypothesized that P. freudenreichii possesses secreted lipolytic esterase(s). For 12 putative esterase genes previously identified from the genome of P. freudenreichii CIRM1, the level of expression was quantified by real-time reverse transcriptase (RT)-PCR, and the subcellular localization of esterases was predicted in silico. The esterase activity in extracellular and intracellular extracts of P. freudenreichii was characterized by zymography, and the extracellular esterases were identified by mass spectrometry. Finally, the best candidate was overexpressed in the same strain. All of the 12 genes encoding putative esterases were expressed. Esterase PF#279 was predicted to be secreted in the medium, PF#774 to be surface exposed, and the 10 remaining putative esterases to be intracellular. Zymography revealed that esterase activities in culture supernatant differed from the ones detected in intracellular extracts. PF#279 was identified as the sole esterase present in culture supernatant. Transformed P. freudenreichii CIRM1 clones overexpressing PF#279 showed 5 to 8 times more lipolytic activity on milk fat than the wild-type strain. Combining in silico, biochemical, and genetic approaches, we showed that PF#279 is the sole secreted esterase in P. freudenreichii and is active on milk fat. Therefore, it is likely a key component in cheese lipolysis by P. freudenreichii.