Austrian raw-milk hard-cheese ripening involves successional dynamics of non-inoculated bacteria and fungi

Cheese ripening is a complex process where the rind microbiota has a key impact on the development of organoleptic properties and on the quality and safety of the final products. In this study we analyzed the rind microbiota of Austrian Vorarlberger Bergkäse (VB), an artisanal surface-ripened cheese produced from raw cow milk without the addition of ripening cultures, along the ripening process. The organoleptic properties of VB can vary widely depending on the ripening time, which can last from three to 18 months. Therefore, the investigation of microbial events occurring at the different time points is crucial. A total of 200 VB rind samples, collected from two ripening facilities at five different time points (0, 14, 30, 90 and 160 days of ripening) were investigated. Real-time quantitative PCR results targeting 16S rRNA or 18S rRNA genes for bacteria or fungi, respectively, revealed that both kingdoms were abundant in VB rinds along the ripening process and that bacterial (BCE) and fungal (FCE) cell equivalents varied throughout ripening time. BCE were 3-4 logs higher than FCE at every time point investigated. Additionally, high-throughput gene-amplicon sequencing targeting the near full-length 16S rRNA gene (by using a PacBio Sequel platform) or the internal transcribed spacer 2 (ITS2) region (by using an Illumina MiSeq platform) was performed to characterize the bacterial or fungal communities on VB rinds, respectively. The results showed dynamic changes in VB rind microbiota throughout ripening. At early ripening times, VB rinds were dominated by Staphylococcus equorum and Candida. Further, S. equorum was highly abundant in every VB rind investigated, while the Candida abundance decreased over time. At 14 to 30 days during ripening, Psychrobacter and Debaryomyces flourished, although their high abundance was limited to these time points. At the latest ripening times investigated (90-160 days of ripening), VB rinds were dominated by Staphylococcus, Brevibacterium, Corynebacterium and Scopulariopsis, although their abundances differed between ripening facilities. Pearson correlation showed a strong relationship between diverse bacterial and fungal genera that might be linked to the proper development of VB ripening. This study deepens our understanding of the ripening process in this particular type of cheese and highlights the succession of several bacteria and fungi to be strongly linked to specific periods of the ripening process.