Application of denaturing high-performance liquid chromatography (DHPLC) for yeasts identification in red smear cheese surfaces

Aims: To evaluate and optimize the use of denaturing high-performance liquid chromatography (DHPLC) for yeasts identification in red smear cheese surfaces. Methods and Results: The resolution of DHPLC was first evaluated and optimized using a mixture of PCR amplicons of the internal transcribed spacer 2 (ITS2) region of 19 yeast reference strains representing 18 species that are common in the cheese microbiota. Sixteen of the 18 yeast species could be resolved by combining runs at temperatures of 57 center dot 5 and 59 degrees C. Then, DHPLC was used to investigate the yeast microbiota of pasteurized Maroilles, Munster and Livarot cheese surfaces by comparing their peak profiles with our reference yeast database and by collecting/sequencing of peak fractions. Debaryomyces hansenii and Geotrichum candidum for Munster and Maroilles cheeses, and Candida catenulata, Candida intermedia and G. candidum for Livarot cheese were identified using the reference database and collecting/sequencing of peak fractions. Conclusions: DHPLC technique was found to have good resolution properties and to be useful for investigating the yeast microbiota of red smear cheese surfaces. Significance and Impact of the Study: This is the first time that DHPLC is applied to study the yeast microbiota of red smear cheese surfaces.