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We developed a polymerase chain reaction (PCR)-based method for the identification of lactobacilli at the genus level. One specific primer, LbLMA1-rev, was designed by analysing similarities between the nucleotide sequence of the spacer between the 16S and 23S rRNA genes in a number of Lactobacillus strains. Amplification with LbLMA1-rev and R16-1, a universal primer, generated a PCR product for 23 Lactobacillus species. Electrophoresis did not reveal any discrete bands when Escherichia coli, Lactococcus lactis, Leuconostoc mesenteroides, Streptococcus thermophilus, Carnobacterium pissicola, Pediococcus pentosaceus, Bifidobacterium bifidum, Weissella confusa, Enterococcus hirae, Staphylococcus aureus or Listeria monocytogenes DNA were used as template.

The diversity and dynamics of yeast populations in four batches of Livarot cheese at three points of ripening were determined. Nine different species were identified by Fourier transform infrared spectroscopy and/or sequencing, and each batch had its own unique yeast community. A real-time PCR method was developed to quantify the four main yeast species: Debaryomyces hansenii, Geotrichum candidum, Kluyveromyces sp. and Yarrowia lipolytica. Culture and molecular approaches showed that G. candidum was the dominant yeast in Livarot cheese. When D. hansenii was added as a commercial strain, it codominated with G. candidum. Kluyveromyces lactis was present only at the start of ripening. Yarrowia lipolytica appeared primarily at the end of ripening. We propose a scheme for the roles and dynamics of the principal Livarot yeasts.

Microscopic conformation, growth behaviour and freezing sensitivity of seven strains of Geotrichum candidum, a ripening starter, were studied and compared according to their macroscopic morphotypes. It has been shown that the thallus forming units (TFU)×ml−1/OD600nm ratio as a function of time is an interesting parameter to follow G. candidum sporulation through the growth behaviour. Microscopic conformation, growth behaviour and freezing sensitivity are clearly strain specific and mostly related to their corresponding morphotypes “yeast”, “mould” or “intermediate”. The two “mould” strains that sporulate weakly (UCMA103, UCMA499) showed a low survival rate to freezing stress whereas the “yeast” strains expressed a significant resistance owing to the arthrospore abundance. Interestingly, one strain (UCMA96) which appeared on solid medium in accord with the “mould” morphotype respond similarly to freezing stress.

We developed a polymerase chain reaction (PCR)-based method for the identification of lactobacilli at the genus level. One specific primer, LbLMA1-rev, was designed by analysing similarities between the nucleotide sequence of the spacer between the 16S and 23S rRNA genes in a number of Lactobacillus strains. Amplification with LbLMA1-rev and R16-1, a universal primer, generated a PCR product for 23 Lactobacillus species. Electrophoresis did not reveal any discrete bands when Escherichia coli, Lactococcus lactis, Leuconostoc mesenteroides, Streptococcus thermophilus, Carnobacterium pissicola, Pediococcus pentosaceus, Bifidobacterium bifidum, Weissella confusa, Enterococcus hirae, Staphylococcus aureus or Listeria monocytogenes DNA were used as template.

PCR primers were developed for the specific detection of Clavispora lusitaniae, Debaryomyces hansenii var hansenii, Geotrichum candidum, Kluyveromyces lactis and K. marxianus and Yarrowia lipolytica, yeast species commonly found on the surface of smear cheese. Forty eight representative strains frequently found in smear cheeses or taxonomically related to the target yeasts were used as templates, to validate the designed primers. The specific and selective detection of these yeasts was effective in situ, in Livarot smear, without yeast isolation and culture and was comparable with data obtained with a conventional method. The primers described here have thus potential for PCR studies applied to cheese. It should also be possible to use some of these primers with other substrates.

The aim was to select potentially probiotic lactobacilli from 88 strains isolated from unpasteurized milk and cheese products, and to incorporate these bacteria in a viable state into a soft cheese, without changing its quality. The survival of these bacteria was assessed in acidic and bile conditions, after freezing at −80 °C. Four strains from unpasteurized Camembert – two Lactobacillus plantarum strains and two Lb. paracasei/casei strains – were identified and typed by PCR and PFGE and were found to display potentially probiotic characteristics in addition to resistance to low pH and bile. These characteristics were resistance to lysozyme, adhesion to CACO-2 cells, antimicrobial effects against common foodborne pathogens (Listeria monocytogenes, Staphylococcus aureus, Salmonella spp., Escherichia coli), innocuity following the ingestion of high doses by mice and appropriate antibiotic susceptibility profiles. The potential of Lb. plantarum strain UCMA 3037 for incorporation into a soft cheese (Pont-l'Eveque registered designation of origin (RDO)) was investigated. This strain grew well and survived in sufficient numbers (more than 107 cfu/g throughout the shelf-life of the product) in the cheese. This strain did not change the quality score of the product until the best before date (75 days after manufacture). Thus, unpasteurized Camembert is a natural source of potentially probiotic lactobacilli, which could be used as an additive in the development of potentially probiotic soft cheeses. Further work is required to demonstrate the persistence and efficacy of these strains in the human host upon ingestion.

A collection of 32 lactococcal strains isolated from raw milk in the Camembert RDO (registered designation of origin) area were phenotypically and genotypically characterized. As expected for environmental isolates, all strains had a Lactococcus lactis subsp. lactis phenotype. The strains were then genotypically identified by the randomly amplified polymorphic DNA (RAPD) technique, using reference strains of lactococci. Two major clusters were identified containing the two subspecies lactis and cremoris. The subspecies lactis cluster could be divided into five subgroups whereas there was a high coefficient of similarity between all strains in the subspecies cremoris cluster. This RAPD classification was then compared with that of a traditional PCR assay using L. lactis species-specific primers corresponding to part of the histidine biosynthesis operon. The two subspecies were differentiated by the size of the fragment amplified (about 200 bp longer for subspecies cremoris). Unlike preliminary phenotypic assignments, the results of PCR experiments corroborated the genotypic identification of the lactococcal strains by RAPD allowing the technique to be reconsidered on the basis of its taxonomic efficiency.