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Natural starter cultures are undefined multiple-strains culture communities of mostly thermophilic lactic acid bacteria (LAB), in association with minor amounts of mesophilic bacteria, which structure could be affected by small changes in the parameters of the cheese/whey-making process. This study aims to investigate the complex microbiota of natural whey starter (NWS) used in Parmigiano Reggiano (PR) cheese-making, focusing on both the absolute and relative abundance of bacterial species and on the modification of the bacterial community under environmental and technological pressures. To reach this purpose a combined approach, using quantitative PCR (qPCR) and High-Throughput Sequencing (HTS), was used to investigate the bacterial dynamics of 91 whey samples collected during different steps of PR cheese-making, in one dairy, through two different lines of production, one Conventional and one Organic, over a 10 weeks period. Our results highlighted that NWS used for the production of PR cheese is a dynamic microbial community, which adapts to the different technological parameters encountered in the cheese/NWS manufacturing process, while retaining a high level of resilience of the thermophilic LAB species mainly involved in the steps of curd acidification and the early maturation process. Differences were also observed in bacterial species diversity between samples from Conventional and Organic line but, in conclusion, NWS resulted to be shaped by technological treatments, regardless of its initial different composition., Competing Interests: Declaration of Competing Interest The authors declare they have no conflicts of interest., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Over the last decade, several methods based on genomic DNA have been developed for the identification and genotyping of prokaryotic and eukaryotic organisms. These genomic methods differ regarding taxonomic range, discriminatory power, reproducibility, and ease of interpretation and standardization. The amplified fragment length polymorphism (AFLP) technique is a very powerful DNA fingerprinting technique for DNA of any source or complexity, varying in both size and base composition. In addition, this method shows high discriminatory power and good reproducibility allowing it to be efficient in discriminating at both the species and strain levels. The development and application of AFLP have allowed significant progress in the study of biodiversity and taxonomy of microorganisms. In the last years, the Applied Biosystems AFLP Microbial Fingerprinting Kit, now out of production, was widely used in various studies to perform AFLP characterization of selected bacteria strains (described by Vos et al. (Nucleic Acids Res 23(21):4407-4414, 1995)). Its replacement gives the possibility for laboratories to continue the use of the previous AFLP data as a reference for bacteria genetic fingerprinting analysis in biodiversity studies. To overcome this issue a result comparison, by using an improved AFLP protocol and the AFLP commercial kit, was performed. In particular, previous results on different species (Listeria monocytogenes, Lactobacillus plantarum, and Streptococcus thermophilus) obtained with the commercial kit were compared with the improved AFLP procedure to validate the protocol. When compared with the AFLP Microbial Fingerprinting Kit, the improved protocol shows high reproducibility, resolution, and overall, is a faster method with lower costs.