Study of the Lactic Acid Bacteria during ripening in the traditional cheese of Lesvos island “Ladotyriâ€

Our research has been focused on the development and application of molecular biology techniques for the rapid and precise detection and quantification of lactic acid bacteria (LAB) in Ladotyri a traditionally prepared Protected Designation Origin cheese, preserved in extra virgin oil. A novel multiplex PCR has been developed for the identification of three genera and one species: Lactococcus spp., Lactobacillus spp., Leuconostoc spp and Streptococcus thermophilus . The primers sets, LactuF/R, LeucF/R, LachrcAF/R, LacBallF/R and StrThermF/R were designed based on the sequences of tuf, hrcA and LacZ genes. Three different PDO cheeses from raw milk and one from pasteurized milk were sampled and analyzed  at three days, one, three and six months after cheese making. The genus quantification was performed by a Real-Time qPCR assay using the same primers used at the multiplex PCR. The standard curves used for the quantification of LAB were generated from decimal dilutions of cultured reference strains. Quantification of all genera was linear providing precise results. At the beginning of the ripening Lactococcus spp., Lactobacillus spp. and Leuconostoc spp were identified and quantified in the three cheeses from raw milk, while in cheese from pasteurized milk Streptococcus thermophilus was also identified. The sampling from the first and third month cheeses showed that the population of Lactobacillus spp. increased in all cases. At the six-month sampling the three genera and Streptococcus thermophilus were still present in the cheese from pasteurized milk while Lactobacillus spp. was absent and Lactococcus spp. was decreased in the majority of the cheeses from raw milk. Â