Metagenomic analysis of the microbial community in an Iranian local cheese

The consumption of local and traditional dairy products have increased in recent years and some local cheeses as functional foods with desirable organoleptic attributes have positive effects on human health. However, there is concern that consumption of these products may increase the risk of exposure to food born bacteria such as Enterobacteriaceae family, Staphylococcus aureus, and Listeria monocytogenes. The microbiome of fermented products such as cheese is one of the most powerful and important parameters in flavor development and ripening. Furthermore, cheese flavor formation as a dynamic biochemical process is related to environmental conditions including milk source, ripening time, and temperature of storage. These parameters affect the microbial community structures and metabolic pathways.

In this study, a local cheese made from cow milk was prepared based on a local recipe. The traditional cheese was manufactured using mesophilic starter culture. The cheese was ripened at 10°C for 3 months. The samples were collected from the surface of the cheese. The serial dilution was performed until 10-10 dilution in sterile ringer. For isolation and phenotypic identification of lactic acid bacteria, a 100 µl of diluted sample was cultured on MRS agar and M17 agar, followed by incubation at 37°C for 48 h under anaerobic conditions with Gas-Pak A. After purification of colonies, the Gram-positive and catalase-negative isolates were phenotypically identified at genus level using physiological tests including capacity of gas production, growth at different pHs (9.6 and 4.4), salt tolerance (%6.5 and 18%), and different growth temperatures (10°C and 45°C). DNA extraction was performed with DNeasy®Blood & Tissue Kit. The microbial population of the cheese and its functional potential for ripening were investigated by whole-metagenome sequencing. The prepared library using Nextera™ DNA approach was sequenced by using the Illumina HiSeq® 2000, 2×100 bp paired- end reads. The metagenomics data of cheese microbiome were analyzed for taxonomic profiling and functional potential by De Novo Assemble Metagenome and Bin Pangenomes. The metabolic pathways were extracted from the KEGGdatabase.

The results of phenotypic identification showed that most of the lactic acid bacteria strains belonged to Streptococcus, Lactococcus, and Lactobacillus. Also, the results of metagenomics analysis showed that there were various genera including Streptococcus, Lactococcus, Lactobacillus, Acinetobacter, Enterococcus, Glutamicibacter, and Weissella in cheese. Streptococcus thermophilus, Lactococcus lactis, and Lactobacillus helveticus were identified as dominant species. Pathogenic bacteria such as Enterobacter, Listeria, and Staphylococcuswere also slightly found and therefore there is nearly no concern for consumers and human health. The microbiome of this cheese showed the metabolic potential for the biosynthesis of a wide range of  aroma compounds and associated with flavor development that related with the metabolism and biosynthesis of methane, branched chain amino acids (isoleucine, valine, and leucine), aromatic amino acids (tyrosine, tryptophan, and phenylalanine), other amino acids (beta-alanine, L-lysine), fatty acids (arachidonate, palmitate, stearate), and monosaccharides. The enzymes related to biosynthesis and metabolism of amino acids were found during ripening of this cheese. These enzymes included 4-hydroxy-tetrahydrodipicolinate reductase, 2-isopropylmalate synthase, 3-dehydroquinate dehydratase, 3-hydroxyisobutyryl-CoA hydrolase, 5-carboxymethyl-2-hydroxymuconate delta-isomerase, and 3-hydroxyacyl-CoA dehydrogenase. Based on the results of KAAS (KEGG Automatic Annotation Server), proteins involved in metabolic pathways of microbial community on the surface of the traditional cheese included Cytochrome P450 Photosynthesis Proteins, Peptidases & Inhibitors, Glycosyltransferases, Lipopolysaccharide Biosynthesis Proteins, Peptidoglycan Biosynthesis and Degradation Proteins, Lipid Biosynthesis Proteins, Protein Kinases, Polyketide Biosynthesis Proteins Prenyltransferases, Protein Phosphatases & Associated Proteins, and Amino Acid Related Enzymes. The cheese under our study as a functional food showed health benefits for consumers due to the presence of probiotic bacteria and genes encoded for biosynthesis of valuable compounds including antibiotics, drugs, and antioxidants.