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

Abstract

Vorarlberger Bergkäse (VB) is an artisanal Austrian washed-rind hard cheese produced from alpine cows’ raw milk without the addition of ripening cultures. Ripening time is a key factor in VB, as it strongly influences the microbial communities present in the cheeses and the organoleptic properties of the product. In this study, the microbial and metabolic transcriptional profiles in VB rinds at different ripening times were investigated. VB products before (30 days of ripening) and after (90 days of ripening) selling were selected, RNA was extracted and subjected to shotgun metatranscriptomic sequencing. The analysis revealed some of the previously described abundant bacterial taxa of Brevibacterium, Corynebacterium, Halomonas, Psychrobacter, and Staphylococcus to be highly active in VB rinds. Additionally, the investigation of most important metabolic pathways in cheese ripening clearly showed differences in the gene transcription profiles and the active microbiota between the two ripening points investigated. At 30 days of ripening, metabolic events related with the degradation of residual lactose, lactate, citrate, proteolysis, and lipolysis were significantly more transcribed and mainly associated with Staphylococcus. On the other hand, genes involved in the degradation of smaller compounds derived from previous metabolism (i.e., metabolism of free amino acids and fatty acids) were significantly more expressed in VB rinds with 90 of ripening, and mainly associated with Brevibacterium and Corynebacterium. These latter metabolic activities are responsible of the generation of compounds, such as methanethiol and 2,3-butanediol, that are very important for the flavor and aroma characteristics of cheeses. This study shows the dynamic changes in the gene transcriptional profiles associated with energy substrates metabolism and the generation of organoleptic compounds during VB ripening and uncovers bacterial taxa as key drivers of the ripening process. These taxa might be the target for future studies toward an accelerated cheese ripening and the enhancement of its organoleptic properties.

Dynamics of Starter and Non-Starter Lactic Acid Bacteria Populations in Long-Ripened Cheddar Cheese Using Propidium Monoazide (PMA) Treatment

“…A more thorough description of genotypic variation within species using shotgun metagenomics [61] will help delineate the metabolic potential and diversity within species during long periods of ripening. Moreover, shotgun metagenomics will enable better characterization of microbial communities within cheese [18,62], inferring functional information [63], enriching taxonomic profiling with an unprecedented depth up to species and strain level variation that could affect the flavor of cheese, and discovering phage abundance [64]. However, a primary challenge facing this approach is the difficulty in assembling genomes from highly diverse sequences of cheese microbiota using as reference the available genomes deposited in widely used public databases [65].…”

Metatranscriptomic and metataxonomic insights into the ultra-small microbiome of the Korean fermented vegetable, kimchi

“…Metatranscriptome analysis has been used for the investigation of functional and taxonomic dynamics in fermented foods such as hard cheese ( Quijada et al, 2022 ), camembert cheese ( Lessard et al, 2014 ), liquor starter ( Huang et al, 2017 ), pao cai ( Xiao et al, 2021 ), and kimchi ( Jung et al, 2013 ). The transcriptomes of USM in the kimchi sample were compared with those of the NM using metatranscriptomic analysis with the SEED subsystem ( Figure 2 ; Supplementary Table 3 ).…”

“…The shotgun-based high throughput sequencing approach provides an opportunity to map the microbiome of foods to an unprecedented depth, highlighting the importance and key factors shaping the composition and activity of the resident microbiome, influencing food quality and safety ( De Filippis et al, 2021 ; Quijada et al, 2022 ). An example is metatranscriptome analysis, which interprets RNA transcripts by high-throughput sequencing and offers unprecedented opportunities to analyze the functional and taxonomic dynamics of a microbiome ( Bikel et al, 2015 ; Gallardo-Becerra et al, 2020 ).…”

Taxonomy, Sequence Variance and Functional Profiling of the Microbial Community of Long-Ripened Cheddar Cheese Using Shotgun Metagenomics

“…De Fillipis (2016) demonstrated temperature-driven changes in metabolic activities that were related to the maturation rate of Italian cheeses. Metatranscriptomics of Austrian washed-rind hard cheeses before (30 d) and after (90 d) ripening showed the transition to higher expression of amino acid and fatty acid metabolism by Brevibacterium and Corynebacterium, leading to the generation of compounds such as 2,3-butanediol and methanethiol [ 32 ]. The challenge at this point is the strain-dependent variation within species, for example in the case of L. casei in long-ripened cheeses [ 8 ].…”