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

• Published in: International journal of food microbiology Vol. 144; no. 1; pp. 10 - 19
• Main Authors: Falentin, Hélène, Postollec, Florence, Parayre, Sandrine, Henaff, Nadine, Le Bivic, Pierre, Richoux, Romain, Thierry, Anne, Sohier, Danièle
• Format: Journal Article Conference Proceeding
• Language: English
• Published: Amsterdam Elsevier B.V 15.11.2010; [Amsterdam; New York, NY]: Elsevier Science; Elsevier
• Subjects: accuracy; Acidification; Agar; Bacteria; Bacterial Load; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biological and medical sciences; Carbon; Cell culture; Cheese; Cheese - microbiology; cheese ripening; cheesemaking; Emmental cheese; Fermented food; food analysis; food composition; Food Handling; Food industries; Food Microbiology; Fundamental and applied biological sciences. Psychology; Gene expression; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Gram-Positive Bacteria - genetics; Gram-Positive Bacteria - growth & development; Gram-Positive Bacteria - metabolism; Lactic acid; Lactic acid bacteria; Lactobacillus - genetics; Lactobacillus - growth & development; Lactobacillus - metabolism; Lactobacillus helveticus; Lactobacillus paracasei; Life Sciences; Limit of Detection; metabolism; Microbiology and Parasitology; Milk and cheese industries. Ice creams; plate count; Polymerase chain reaction; Propionibacterium - genetics; Propionibacterium - growth & development; Propionibacterium - metabolism; Propionibacterium freudenreichii; real time reverse transcriptase polymerase chain reaction; Reverse Transcriptase Polymerase Chain Reaction; Reverse transcription; Ripening; ripening bacteria; RT-qPCR; Sampling; stationary phase; Streptococcus thermophilus; Streptococcus thermophilus - genetics; Streptococcus thermophilus - growth & development; Streptococcus thermophilus - metabolism; Stress; stress tolerance; Transcription; transcription (genetics); Ripening; Bacteria; Lactic acid bacteria; Gene expression; Emmental cheese; RT-qPCR; Dairy product; Reverse transcription; Cheese; Metabolism; Real time; Polymerase chain reaction; Molecular biology; bacterium; ACTIVITE METABOLIQUE; EMMENTAL; fromage; bactérie; SPECIFIC METABOLIC ACTIVITY; RIPENING; lactic acid bacteria; EMMENTAL CHEESE; affinage; pcr; bactérie lactique
• ISSN: 0168-1605; 1879-3460
• DOI: 10.1016/j.ijfoodmicro.2010.06.003

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.