The Bacterial Microbiota of Artisanal Cheeses from the Northern Caucasus

In this study, we used culture-independent analysis based on 16S rRNA gene amplicons and metagenomics to explore in depth the microbial communities and their metabolic capabilities of artisanal brine cheeses made in the North Caucasus. Additionally, analysis of organic acid profiles was carried out for cheese characterization. Twelve cheese samples (designated as 05SR-16SR) from various artisanal producers were taken from five different villages located in Northern Ossetia-Alania (Russia). These cheeses were made using methods based on cultural traditions inherited from previous generations and prepared using a relatively uncontrolled fermentation process. The microbial diversity of Caucasus artisanal cheeses was studied for the first time. The results showed a diverse composition in all cheeses, with Bacillota (synonym Firmicutes) (9.1-99.3%) or Pseudomonadota (synonym Proteobacteria) (0.2-89.2%) prevalence. The microbial communities of the majority of the studied cheeses were dominated by lactic acid bacteria (LAB) genera, like Lactococcus (10.3-77.1% in 07SR, 09SR, 10SR, 11SR, 13SR, 15SR, 16SR), Lactobacillus (54.6% in 09SR), Streptococcus (13.9-93.9% in 11SR, 13SR, 14SR, 15SR), Lactiplantibacillus (13.4-30.6% in 16SR and 07SR) and Lentilactobacillus (5.9-14.2% in 09SR, 10SR and 13SR). Halophilic lactic acid bacteria belonging to the Tetragenococcus genus accounted for 7.9-18.6% in 05SR and 06SR microbiomes. A distinctive feature of Ossetia cheese microbiomes was the large variety of halophilic proteobacteria, and in some cheeses they prevailed, e.g., Chromohalobacter (63-76.5% in 05SR and 06SR), Psychrobacter (10-47.1% in 08SR, 11SR, 12SR), Halomonas (2.9-33.5% in 06SR, 08SR, 11SR and 12SR), Marinobacter (41.9% in 12SR) or Idiomarina (2.9-14.4% in 06SR, 08SR and 11SR samples). Analysis of the genomes assembled from metagenomes of three cheeses with different bacterial composition revealed the presence of genes encoding a variety of enzymes, involved in milk sugar, proteins and lipid metabolism in genomes affiliated with LAB, as well as genes responsible for beneficial bioamine and bacteriocin synthesis. Also, most of the LAB did not contain antibiotic resistance genes, which makes them potential probiotics, so highly demanded nowadays. Analysis of the genomes related to halophilic proteobacteria revealed that they are not involved in milk fermentation; however, the search for "useful" genes responsible for the synthesis of beneficial products/metabolites was partially positive. In addition, it has been shown that some halophiles may be involved in the synthesis of inappropriate bioactive components. The results obtained by culture-independent analyses confirm the importance of using such techniques both to clarify the quality and health-promoting properties of the product, and to look for probiotic strains with specified unique properties. This study has shown that traditional dairy foods may be a source of such beneficial strains.