The Bacterial Microbiota of Artisanal Cheeses from the Northern Caucasus

Abstract

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.

Metagenomic Insights into the Taxonomic and Functional Features of Traditional Fermented Milk Products from Russia

“…plantarum and Lcb. paracasei dominated in some dairy products analyzed here and are also frequently detected in cheeses [ 5 , 10 , 12 , 28 , 29 ], yogurts, kefir and other fermented dairy products [ 8 , 30 , 31 , 32 ]. They appear to be one of the crucial LAB in milk fermentation due to their high potential for producing important metabolites and for improving the quality characteristics of the fermented products, for example, synthesizing thickening hydrocolloids (exopolysaccharides, EPS) or lipolysis-derivative aroma compounds [ 33 , 34 ].…”

“…Together with the canonical LAB listed above, Macrococcus_B caseolyticus ( Staphylococcales order within the Firmictutes phylum) was present in some studied metagenomes (bryndza, cottage cheese (14TG only) and khurunga). Earlier it was detected in some kind of cheeses [ 12 , 42 ] and other fermented food [ 43 , 44 ] and can potentially enhance the release of flavor compounds in FMPs due to a high esterase activity [ 42 ]. Among Proteobacteria (currently Pseudomonadota ), MAGs affiliated to novel species of the Acetobacter genus were only detected in khurunga and shubat samples.…”

“…However, metabarcoding does not allow a reliable prediction of the functional features of the microbiomes inhabiting these products. Only recently, we published a metagenomics of traditional Caucasian cheeses’ microbiomes produced in Northern Ossetia-Alania [ 12 ]. According to the analysis of metagenome-assembled genomes (MAGs), it was revealed that the majority of dominating bacteria did not contain the genes of antibiotic resistance (ABR).…”

A Metabolomics Study by 1H HRMAS NMR: From Sheep Milk to a Pressed-Curd Cheese: A Proof of Concept

“…Differences between the elaboration processes are highly related to the development of the texture and flavor of each type of cheese [3,39]. In fact, the proteolysis pathways for each bacterium and the enzymatic processes and their repercussion on the changes during RT and the interaction with the elaboration procedure are continuously studied [58][59][60][61][62][63][64][65][66][67][68].…”

Gas chromatography-mass spectrometry characterization of the aroma of Ossetian cheeses

“…To date there has been only one such study on Ossetian cheese, where polar water-soluble organic acids (lactic, acetic, etc.) in Ossetian cheese samples were determined and quantified by HPLC [1]. On the other hand, no studies of Ossetian cheese have yet been conducted using gas chromatography-mass spectrometry, despite the fact that this method allows characterization of a much wider range of volatile metabolites and therefore is routinely used in the literature, for example, for reliable qualitative and quantitative characterization of volatile composition of semi-hard Spanish goat cheeses, Gouda and Cheddar cheeses [2][3][4][5].…”