The molecular techniques used to investigate the microbial community composition in cheese can be divided into two different types, culture-dependent and culture-independent methods. Culture-dependent methods are based on the cultivation of microorganisms on selective culture media, followed by molecular identification and/or typing. In the culture-independent approach, DNA or RNA is extracted directly from cheese and then submitted to a variety of different analyses. DNA restriction analysis has been proven to be a powerful tool to identify and examine biodiversity. PCR-based techniques are also widely used to genotype bacteria isolated from cheese. DNA amplification using arbitrary primers allows simultaneous strain typing, species affiliation and individual strain differentiation and has been successfully used to monitor cheese microbiota during manufacturing and ripening. Among the culture-independent methods, PCR-DGGE/TGGE and PCR-SSCP have been widely employed to investigate biodiversity in cheese environments. The most interesting results are obtained using pyrosequencing, which allows minor bacterial populations in cheese to be detected and the relative abundances of the individual bacterial species to be estimated. Specific microbial activities during production and ripening can also be monitored using RNA targeting techniques.
