The reported health effects of fermented dairy foods, which are traditionally manufactured in Bulgaria, are connected with their microbial biodiversity. The screening and development of probiotic starters for dairy products with unique properties are based exclusively on the isolation and characterization of lactic acid bacterial (LAB) strains. This study aims to systematically describe the LAB microbial content of artisanal products such as Bulgarian-type yoghurt, white brined cheese, kashkaval, koumiss, kefir, katak, and the Rhodope’s brano mliako. The original technologies for their preparation preserve the valuable microbial content and improve their nutritional and probiotic qualities. This review emphasises the features of LAB starters and the autochthonous microflora, the biochemistry of dairy food production, and the approaches for achieving the fortification of the foods with prebiotics, bioactive peptides (ACE2-inhibitors, bacteriocins, cyclic peptides with antimicrobial activity), immunomodulatory exopolysaccharides, and other metabolites (indol-3-propionic acid, free amino acids, antioxidants, prebiotics) with reported beneficial effects on human health. The link between the microbial content of dairy foods and the healthy human microbiome is highlighted.

Bacillus licheniformis is a soil bacterium with many industrial applications. In addition to enzymes, platform chemicals, antibiotics and phytohormones, the species produces exopolysaccharides (EPSs) of various biological activities. This study revealed that Bulgarian isolate B. licheniformis 24 produced EPSs consisting of galactose, glucose and mannose with substrate-dependent ratio. From glucose, B. licheniformis 24 secreted EPS1, consisting of 54% galactose, 39% glucose and 7% mannose. From fructose, the strain formed EPS2, containing 51% glucose, 30% mannose and 19% galactose. Batch cultivation in flasks yielded 2.2–2.6 g/L EPS1 and 1.90–2.11 g/L EPS2. Four to five times higher yields of EPS were obtained from both substrates during batch and fed-batch processes in a fermenter at 37.8 °C, pH 6.2 and aeration 3.68 vvm. The batch process with 200 g/L of starting substrates received 9.64 g/L EPS1 and 6.29 g/L EPS2, reaching maximum values at the 33rd and 24th h, respectively. Fed-batch fermentation resulted in the highest yields, 12.61 g/L EPS1 and 7.03 g/L EPS2. In all processes, EPSs were produced only in the exponential growth phase. Both EPSs exhibited antioxidant activity, but EPS2 was much more potent in this regard, reaching 811 μM Vitamin C Equivalent Antioxidant Capacity (versus 135 μM for EPS1). EPS1 displayed antibacterial activity against a non-O1 strain of Vibrio cholerae.

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 Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus were predominant in all samples. In addition, yoghurts contained accompanying lactic acid bacteria (LAB) including Lacticaseibacillus paracasei, Lb. helveticus, Limosilactobacillus fermentum, Lb. rhamnosus, Lactococcus lactis, Pediococcus acidilactici, Leuconostoc mesenteroides, and Leuc. pseudomesenteroides. A negligible amount of pollutant strains was found. Twenty-four LAB strains were isolated from the yoghurts and identified. Lb. delbrueckii subsp. bulgaricus 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.

β-galactosidase is an enzyme with dual activity and important industrial application. As a hydrolase, the enzyme eliminates lactose in milk, while as a trans-galactosidase it produces prebiotic galactooligosaccharides (GOS) with various degrees of polymerization (DP). The aim of the present study is the molecular characterization of β-galactosidase from a Bulgarian isolate, Lactobacillus delbrueckii subsp. bulgaricus 43. The sequencing of the β-gal gene showed that it encodes a new enzyme with 21 amino acid replacements compared to all other β-galactosidases of this species. The molecular model revealed that the new β-galactosidase acts as a tetramer. The amino acids D207, H386, N464, E465, Y510, E532, H535, W562, N593, and W980 form the catalytic center and interact with Mg²⁺ ions and substrate. The β-gal gene was cloned into a vector allowing heterologous expression of E. coli BL21(DE3) with high efficiency, as the crude enzyme reached 3015 U/mL of the culture or 2011 U/mg of protein. The enzyme’s temperature optimum at 55 °C, a pH optimum of 6.5, and a positive influence of Mg²⁺, Mn²⁺, and Ca²⁺ on its activity were observed. From lactose, β-Gal produced a large amount of GOS with DP3 containing β-(1→3) and β-(1→4) linkages, as the latter bond is particularly atypical for the L. bulgaricus enzymes. DP3-GOS formation was positively affected by high lactose concentrations. The process of lactose conversion was rapid, with a 34% yield of DP3-GOS in 6 h, and complete degradation of 200 g/L of lactose for 12 h. On the other hand, the enzyme was quite stable at 55 °C and retained about 20% of its activity after 24 h of incubation at this temperature. These properties expand our horizons as regards the use of β-galactosidases in industrial processes for the production of lactose-free milk and GOS-enriched foods.

The translational enhancement of the 5′-leader sequence of the tobacco mosaic virus (TMV) and tobacco etch viral 5′-leader sequence (TEV) was compared in relation to the expression of the human calcitonin (hCT) gene in transgenic potato plants. The synthetic human calcitonin tetrameric genes were cloned under the control of the 35S Cauliflower mosaic virus (CaMV)promoter linked to the TMV or TEV leader sequences. The resulting constructs were cloned into the binary vector Bin19 and potato minituber discs transformed using an agrobacterium strain. The expression of the hCT gene was studied by mRNA-DNA hybridization, a RT-PCR, ELISA and RIA. The results showed that the TMV omega element increased the yield of the expressed recombinant protein two to three-fold in comparison with the TEV leader sequence.