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Development of primers for the specific detection of Geotrichum candidum, Debaryomyces hansenii, Kluvyeromyces lactis/marxianus and Clavispora lusitaniae

The major yeasts present on the surface of five smear cheeses: Limburger (Germany), Reblochon (France), Livarot (France), Tilsit (Austria) and Gubbeen (Ireland) have been identified by FTIR spectroscopy during this project. It has been shown that the main yeast species occurring on the cheeses were Debaryomyces hansenii, Geotrichum candidum, Kluyveromyces lactis and/or Kluyveromyces marxianus, Yarrowia lipolytica and, for Gubbeen, Clavispora lusitaniae.

Our objectives were to construct specific PCR primers for identification of the yeasts listed above and to test when appropriate their ability for in situ detection of the yeasts from total DNA from Livarot cheese.

In order to assess the selectivity of PCR primers to be designed, thirty-six yeast strains representative of 17 yeast species were selected to be studied as belonging to the more frequently described yeasts in smear cheeses. They were obtained from different culture collections or were commercial cultures used in cheese processing. Livarot curd and cheeses were used for in situ validation of the primers. Alignments of nucleotide sequences were conducted using Basic Local Alignment Search Tool in order to localize nucleotide sequences suitable for the selective DNA amplification for all strains of a given yeast species. Primers designed in this study were compared to each other by aligning the published sequences from the DNA sequence libraries (DDBJ, EMBL, GenBank) and their specificity was evaluated using DNA extracted from the other yeast species.

A couple of primers were designed leading to DNA amplification from 7 reference and commercial strains of Kluyveromyces lactis and K. marxianus. An other couple of PCR primers were designed leading to specific DNA amplification from five Y. lipolytica reference strains. We designed specific primers leading to a single band of approximately 200bp, when used in PCR, for one out of two collection strains of Debaryomyces hansenii and for a commercial strain. Using two other primers, a 200 bp PCR product was obtained for Clavispora lusitaniae collection strains CBS 4413T and CBS 52992. We were also able to design two sequences specific for Geotrichum candidum and validated on reference strains and two commercial strains. Using these primers, Kluyveromyces lactis/marxianus, Debaryomyces hansenii, Geotrichum candidum and Yarrowia lipolytica were detected in situ as well in Livarot curd than in ripened cheese and in frozen smears. Primers designed for C. lusitaniae were also tested in situ although this species was not identified in Livarot cheese. As expected, no DNA amplification occurred.

To validate primers specificity from a taxonomic point of view, they should be tested with species whose phylogeny is closely related to the species we intended to detect. In accordance with the objectives of the SCM project, primers specificity was only checked in a context of smear cheese microbiology. However, this work constitutes a first stage towards a better knowledge of the principal yeast species on the surface of smear ripened cheese. Further application for those primers is their use in real time PCR in order to quantify yeast populations and to follow dynamics during ripening.

These results will be disseminated by the mean of a scientific paper which is currently in preparation. No benefits are expected. End-users of these results are the scientific community in particular for ecological studies, but also producers of commercial yeasts and sectors of the food industry where the yeasts listed above are useful or often encountered as contaminants. In particular Geotrichum candidum is widely used in the cheese industry as ripening agent, but it is also responsible for the degradation of fresh cheeses and fruit and vegetable juices. It proliferates in refrigerated cakes and in frozen branch vegetables. It attacks fresh fruit from the orchard as well as warehoused fruit, in which it produces aqueous, viscous or slimy rot. The specific detection of this species could be interesting in quality control. To our knowledge, one probe has already been described to detect G. candidum but the use of primers is easier. One couple of primers was described which efficiency was checked in the context of medical studies. This is the first time, to our knowledge, that specific primers were designed for studies in food microbiology.

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Laboratoire de Microbiologie Alimentaire - Université de Caen Basse-Normandie
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