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.

Toxic ingredients in food can lead to serious food-related diseases. Such compounds are bacterial toxins (Shiga-toxin, listeriolysin, Botulinum toxin), mycotoxins (aflatoxin, ochratoxin, zearalenone, fumonisin), pesticides of different classes (organochlorine, organophosphate, synthetic pyrethroids), heavy metals, and natural antinutrients such as phytates, oxalates, and cyanide-generating glycosides. The generally regarded safe (GRAS) status and long history of lactic acid bacteria (LAB) as essential ingredients of fermented foods and probiotics make them a major biological tool against a great variety of food-related toxins. This state-of-the-art review aims to summarize and discuss the data revealing the involvement of LAB in the detoxification of foods from hazardous agents of microbial and chemical nature. It is focused on the specific properties that allow LAB to counteract toxins and destroy them, as well as on the mechanisms of microbial antagonism toward toxigenic producers. Toxins of microbial origin are either adsorbed or degraded, toxic chemicals are hydrolyzed and then used as a carbon source, while heavy metals are bound and accumulated. Based on these comprehensive data, the prospects for developing new combinations of probiotic starters for food detoxification are considered.

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.

2,3-Butanediol (2,3-BD) is a reagent with remarkable commercial use as a platform chemical in numerous industries. The present study aims to determine the capabilities of non-pathogenic and cellulolytic Bacillus licheniformis 24 as a 2,3-BD producer. By applying the Plackett–Burman design and response surface methodology through central composite design (CCD), a complex optimization of medium and process parameters was conducted. Thus, among ten studied factors of medium content, four components were evaluated with a significant positive effect on 2,3-BD formation. Their optimal values for 2,3-BD production (yeast extract, 13.38 g/L; tryptone, 6.41 g/L; K2HPO4, 4.2 g/L; MgSO4, 0.32 g/L), as well as the optimal temperature (37.8 °C), pH (6.23) and aeration rate (3.68 vvm) were predicted by CCD experiments and validated in a series of batch processes. In optimized batch fermentation of 200 g/L of glucose 91.23 g/L of 2,3-BD was obtained, with the overall productivity of 1.94 g/L/h and yield of 0.488 g/g. To reveal the maximum 2,3-BD tolerance of B. licheniformis 24, fed-batch fermentation was carried out. The obtained 138.8 g/L of 2,3-BD with a yield of 0.479 g/g and productivity of 1.16 g/L/h ranks the strain among the best 2,3-BD producers.

The treatment of agricultural areas with pesticides is an indispensable approach to improve crop yields and cannot be avoided in the coming decades. At the same time, significant amounts of pesticides remain in food and their ingestion causes serious damage such as neurological, gastrointestinal, and allergic reactions; cancer; and even death. However, during the fermentation processing of foods, residual amounts of pesticides are significantly reduced thanks to enzymatic degradation by the starter and accompanying microflora. This review concentrates on foods with the highest levels of pesticide residues, such as milk, yogurt, fermented vegetables (pickles, kimchi, and olives), fruit juices, grains, sourdough, and wines. The focus is on the molecular mechanisms of pesticide degradation due to the presence of specific microbial species. They contain a unique genetic pool that confers an appropriate enzymological profile to act as pesticide detoxifiers. The prospects of developing more effective biodetoxification strategies by engaging probiotic lactic acid bacteria are also discussed.

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.

Inulin is a renewable and cheap carbon source used in microbial fermentations. Bacillus licheniformis 24 is known as an excellent 2,3-butanediol (2,3-BD) producer from fructose; therefore, the cloning and expression of a robust heterologous inulinase could enhance its 2,3-BD production from inulin. The inu gene of Lacticaseibacillus paracasei DSM 23505 encoding fructan-β-fructosidase (EC 3.2.1.80) was chosen for the purpose. PCR fragments containing the complete inu (3.6 kb) and its truncated variant inu-tr (2.2 kb, lacking Big3 cell wall attachment domains) were cloned into Escherichia coli StellarTM and B. licheniformis 24. The high quality of the recombinant constructs was confirmed by restriction analysis, PCR, sequencing, and phenotypic tests. The results showed that the inulinase activity of B. licheniformis cells harboring the full-length inu variant (T26) was eightfold higher compared to the wild type, retaining cell wall attachment in the B. licheniformis host. In contrast, the truncated variant inu-tr (T14) showed mostly extracellular but weak activity, thus suggesting that the Big3 domains are also important for the enzyme’s function. During flask-batch fermentation of 100 g/L raw chicory flour (containing 90% inulin), T26 produced acetoin and 2,3-BD from inulin. Contrariwise, T14 and the wild type formed products only from the mono- and disaccharides naturally found in the chicory flour. In the fermenter, from 200 g/L of raw chicory flour, the recombinant T26 degraded approximately 140 g/L of the inulin. However, the final concentrations of the produced 2,3-BD and acetoin were 18.5 g/L and 8.2 g/L, respectively, because of the accumulation of unconverted sucrose. To conclude, further strain improvement is necessary to make the process efficient for obtaining 2,3-BD from inulin by simultaneous saccharification and fermentation (SSF).

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