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.

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.

Correlation of the capsular phenotype in Propionibacterium freudenreichii with the level of expression of gtf, a unique polysaccharide synthase-encoding gene.

Many food-grade bacteria produce exopolysaccharides (EPS) that affect the texture of fermented food products and that may be involved in probiotic properties. Propionibacterium freudenreichii is a Gram-positive food-grade bacterium with reported probiotic capabilities that is widely used as starter in Swiss-type cheese. In this study, 68 strains of P. freudenreichii were screened for the beta-glucan capsular phenotype by immunoagglutination with a specific antibody and for the presence of the gtf gene coding for polysaccharide synthase. All strains were positive for PCR amplification with gtf gene-specific primers, but the presence of beta-glucan capsular EPS was detected for only 35% of the strains studied. Disruption of gtf in P. freudenreichii revealed that gtf is a unique gene involved in beta-glucan capsular EPS production in P. freudenreichii. The gtf gene was transferred into and expressed in Lactococcus lactis, in which it conferred an agglutination-positive phenotype. Expression of the gtf gene was measured by performing quantitative reverse transcription-PCR assays with RNA from four capsular and three noncapsular strains. A positive correlation was found between the beta-glucan capsular phenotype and gtf gene expression. Sequencing of the region upstream of the gtf open reading frame revealed the presence of an insertion element (IS element) in this upstream region in the four strains with the beta-glucan capsular phenotype. The role of the IS element in the expression of neighboring genes and its impact on interstrain variability of the P. freudenreichii capsule phenotype remain to be elucidated.