Austrian Raw-Milk Hard-Cheese Ripening Involves Successional Dynamics of Non-Inoculated Bacteria and Fungi

Abstract

Cheese ripening involves successional changes of the rind microbial composition that harbors a key role on the quality and safety of the final products. In this study, we analyzed the evolution of the rind microbiota (bacteria and fungi) throughout the ripening of Austrian Vorarlberger Bergkäse (VB), an artisanal surface-ripened cheese, by using quantitative and qualitative approaches. The real-time quantitative PCR results revealed that bacteria were more abundant than fungi in VB rinds throughout ripening, although both kingdoms were abundant along the process. The qualitative investigation was performed by high-throughput gene-targeted (amplicon) sequencing. The results showed dynamic changes of the rind microbiota throughout ripening. In the fresh products, VB rinds were dominated by Staphylococcus equorum and Candida. At early ripening times (14–30 days) Psychrobacter and Debaryomyces flourished, although their high abundance was limited to these time points. At the latest ripening times (90–160 days), VB rinds were dominated by S. equorum, Brevibacterium, Corynebacterium, and Scopulariopsis. Strong correlations were shown for specific bacteria and fungi linked to specific ripening periods. This study deepens our understanding of VB ripening and highlights different bacteria and fungi associated to specific ripening periods which may influence the organoleptic properties of the final products.

Metatranscriptomic Analyses Unravel Dynamic Changes in the Microbial and Metabolic Transcriptional Profiles in Artisanal Austrian Hard-Cheeses During Ripening

“…In this study, we performed a metatranscriptomic analysis on VB rinds at two different ripening times. Our group has investigated the VB rind microbiota and the influence of the producing environment, highlighting several taxa to be very abundant in most of the products and producing surfaces regardless the facility or ripening time, such as Staphylococcus, Brevibacterium, Corynebacterium, Halomonas , and Psychrobacter , among others ( Schornsteiner et al, 2014 ; Quijada et al, 2018 , 2020b ; Schmitz-Esser et al, 2018 ; Anast et al, 2019 ). In this study, VB rind samples were taken at 30 and 90 days of ripening as they represent either young cheeses before they are able to be sold (day 30) or “early ripened” cheeses at the first time that these products are sold (day 90).…”

“…The shift in the transcription pattern is in agreement with our previous studies, as we found Staphylococcus (mainly assigned to Staphylococcus equorum according to 16S rRNA gene sequences) to be very abundant since the beginning of ripening (when the cheese wheel is extracted from the brine tank and placed in the ripening cellar), throughout the ripening and in the final products. On the other hand, Brevibacterium (mainly assigned to Brevibacterium linens and Brevibacterium aurantiacum ) and Corynebacterium (mainly assigned to Corynebacterium variabile ) followed different dynamics, as they appeared at latest ripening times and dominated in the final products ( Quijada et al, 2020b ). These bacterial groups are not inoculated to the VB during manufacture and were highly abundant in the production environment, that might act as a source of natural inoculation ( Quijada et al, 2018 ).…”

“…Additionally, the genomes of Advenella , Brevibacterium, Psychrobacter , and Psychroflexus strains isolated from the VB rinds were investigated by using whole genome sequencing and a putative metabolic pathway for histamine degradation was found in different Brevibacterium strains from VB ( Schmitz-Esser et al, 2018 ; Anast et al, 2019 ). More recently, the bacterial and fungal diversity of VB rinds was evaluated by high-throughput gene-targeted sequencing at different times throughout VB ripening, unraveling microbial dynamics and potential correlations of different bacteria and fungi during ripening ( Quijada et al, 2020b ). Such microbial correlations may be critical for the unique organoleptic properties of the VB products that are sold at different time points during ripening.…”

Environmental sources along natural cave ripening shape the microbiome and metabolome of artisanal blue-veined cheeses

“…The role of some members of the Cabrales cheese microbiome in the ripening process is clear based on previous studies for these sort of cheeses. Indeed, Lactococcus, Lactobacillus, Staphylococcus, Corynebacterium, Brevibacterium, Penicillium, Geotrichum or Debaryomyces have all previously been detected at high abundance in blue-veined rind washed cheeses, and in some cases also in cheese processing environments 5,6,12 , with very clear contributions to cheese fermentations. Moreover, some of these taxa, such as Corynebacterium or Brevibacterium, play a particularly important role in aroma development during cheese ripening 5,12,13 .…”

“…Historically, the microbiome of artisanal cheeses has been characterized using culture-based methods, which have several limitations 3,4 . High throughput sequencing methodologies (e.g., amplicon sequencing or shotgun whole metagenome sequencing) have transformed the way we study microbial ecology in complex ecosystems, such as those of artisanal cheeses 3,[5][6][7][8] , as they provide access to di cult-to-culture or viable but not culturable microorganisms 1,6 , and facilitate the mechanistic understanding of community assembly in such a way that allows studying temporal dynamics of microbial communities at an unprecedented level of detail 6 . Nevertheless, there is also a general consensus that metagenomic studies need to be complemented to, for example, investigate the microbial and chemo-sensory relationships from a multi-omics perspective 9 .…”