Diversity and evolution of the microbial populations during manufacture and ripening of Casيn, a traditional Spanish, starter-free cheese made from cowʹs milk

Classical culturing and denaturing gradient gel electrophoresis (DGGE) techniques have been used for studying the microbial diversity and dynamics of the traditional Spanish Casيn cheese during manufacturing and ripening. As with other starter-free cheeses made from raw milk, the microbial diversity of Casيn was shown to be high by both culturing and DGGE analyses. The culture technique showed that lactic acid bacteria (LAB) species constituted the majority of the microbial populations. Of the 14 bacterial species identified, Lactococcus garvieae was predominant in the three-day-old cheese sample, although it was replaced by Lactococcus lactis subsp. lactis at day 30. As expected, the DGGE profiles obtained were complex, consisting, depending on the sample, in five to ten different amplification bands. Among these, a band corresponding to Streptococcus thermophilus was observed throughout the whole manufacturing process. This species had never been identified from traditional Spanish cheeses previously. Culturing and molecular methods showed high populations of undesirable microorganisms, arguing for a required improvement in the hygiene of Casيn manufacture. Random amplification of polymorphic DNA (RAPD) profiling suggested that the L. garvieae and L. lactis populations were composed of one and five strains, respectively. In addition, only a single L. lactis RAPD pattern was stably maintained from day three to day 30, indicating high succession of strains along ripening. After a thoroughly characterisation, strains of the two Lactococcus species could be used in designing specific starter cultures for Casيn. Additional species (such as Lactobacillus plantarum and Corynebacterium variabile) might be included as adjunct cultures.