| Summary: | Bulgarian yoghurt is associated with health benefits and longevity of consumers. The specific microflora producing bioactive metabolites is responsible for this effect. The present study examines the biodiversity in four homemade yoghurts from regions containing endemic microflora. Metagenome sequencing indicated <i>Lactobacillus delbrueckii </i>subsp.<i> bulgaricus </i>and <i>Streptococcus thermophilus</i> were predominant in all samples. In addition, yoghurts contained accompanying lactic acid bacteria (LAB) including <i>Lacticaseibacillus paracasei</i>,<i> Lb. helveticus</i>,<i> Limosilactobacillus fermentum</i>,<i> Lb. rhamnosus, Lactococcus lactis</i>,<i> Pediococcus acidilactici</i>,<i> Leuconostoc mesenteroides</i>, and <i>Leuc. pseudomesenteroides</i>. A negligible amount of pollutant strains was found. Twenty-four LAB strains were isolated from the yoghurts and identified. <i>Lb. delbrueckii </i>subsp.<i> bulgaricus</i> strains were genotyped by randomly amplified polymorphic DNA–PCR (RAPD), multi-locus sequence typing (MLST), and pulse field gel electrophoresis (PFGE), which demonstrated their uniqueness and non-commercial origin. To estimate the bioactive metabolites produced by the accompanying microflora, yoghurts fermented by single LAB strains were analyzed using liquid chromatography and mass spectrometry (LC-MS). The fermented samples contained large amounts of free essential amino acids (arginine, citrulline, tryptophan, lysine, and histidine), the neuroprotector indole-3-propionic acid (IPA), and significant quantities of the cyclic antimicrobial peptides cyclo(phenylalanyl-prolyl) and cyclo(leucyloprolyl). The disclosure of these special qualities draws attention to the accompanying microflora as a source of potential probiotic strains that can fortify the yoghurts’ content with bioactive compounds.
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