Development of an S-layer gene-based PCR-DGGE assay for monitoring dominant Lactobacillus helveticus strains in natural whey starters of Grana Padano cheese

Abstract

Monitoring L. helveticus strain dynamics in natural whey starters is of great interest at the industrial level due to the key role that this bacterial population plays in Grana Padano cheese production. In this study, we aimed to develop a PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) assay based on the slpH locus, in parallel with performing culture-dependent analysis of whey samples using optimized media to maximize the number of isolated strains. We designed new primers targeting the slpH locus to amplify a gene region that would be suitable for PCR-DGGE analysis and discriminating strains. Our results confirmed that the developed PCR-DGGE method was rapid and reliable for monitoring the L. helveticus population in whey starter cultures. All sequences of bands detected in the PCR-DGGE profiles from whey samples showed high similarity to S-layer genes of L. helveticus, and perfectly matched with the slpH locus sequences of dominant strains. Overall, our findings indicated that the target region of the slpH locus was sufficiently heterologous to discriminate L. helveticus strains, and that our PCR-DGGE analysis provided a more accurate picture of the population composition of whey starters compared to culture-dependent techniques that often fail to isolate the most abundant strains.