Endothelial cells in vivo are constantly exposed to mechanical forces such as cyclic strain. In endothelial cells, Nox4-containing NAD(P)H oxidase complexes have been identified as major sources of superoxide anion (.O(2)(-)) formation. In this study, we analyzed the effect of cyclic strain on endothelial ROS formation by electron paramagnetic resonance spectroscopy, cytochrome c assay, and dihydroethidium fluorescence, on NO formation by Griess reaction and on gene expression by RT-PCR and Western blot. Primary cultures of human umbilical vein endothelial cells were exposed to 2-18% cyclic strain for up to 24 h using the Flexercell system. Long-term application of 5-12% cyclic strain downregulated Nox4 expression and ROS formation in a time-dependent manner. Downregulation of Nox4 was further confirmed by promoter analysis using dual-luciferase assay. Cu/Zn SOD, MnSOD, and catalase expression was decreased after application of chronic 12% cyclic strain. In contrast, endothelial NO formation and eNOS were increased by cyclic strain. Strain-dependent Nox4 downregulation was abolished by eNOS inhibition with L-NAME. In conclusion, physiological levels of cyclic strain downregulate Nox4 expression and superoxide anion formation. This novel mechanism might contribute to a vasoprotective balance between NO and superoxide anions in response to physiological mechanical stimulation of endothelial cells.

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.

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Biobutanol is a promising alternative fuel with impaired microbial production thanks to its toxicity. Lactiplantibacillus plantarum (L. plantarum) is among the few bacterial species that can naturally tolerate 3% (v/v) butanol. This study aims to identify the genetic factors involved in the butanol stress response of L. plantarum by comparing the differential gene expression in two strains with very different butanol tolerance: the highly resistant Ym1, and the relatively sensitive 8-1. During butanol stress, a total of 319 differentially expressed genes (DEGs) were found in Ym1, and 516 in 8-1. Fifty genes were upregulated and 54 were downregulated in both strains, revealing the common species-specific effects of butanol stress: upregulation of multidrug efflux transporters (SMR, MSF), toxin-antitoxin system, transcriptional regulators (TetR/AcrR, Crp/Fnr, and DeoR/GlpR), Hsp20, and genes involved in polysaccharide biosynthesis. Strong inhibition of the pyrimidine biosynthesis occurred in both strains. However, the strains differed greatly in DEGs responsible for the membrane transport, tryptophan synthesis, glycerol metabolism, tRNAs, and some important transcriptional regulators (Spx, LacI). Uniquely upregulated in the butanol-resistant strain Ym1 were the genes encoding GntR, GroEL, GroES, and foldase PrsA. The phosphoenolpyruvate flux and the phosphotransferase system (PTS) also appear to be major factors in butanol tolerance.

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.

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.

To adapt to various ecological niches, the members of genus Bacillus display a wide spectrum of glycoside hydrolases (GH) responsible for the hydrolysis of cellulose and lignocellulose. Being abundant and renewable, cellulose-containing plant biomass may be applied as a substrate in second-generation biotechnologies for the production of platform chemicals. The present study aims to enhance the natural cellulase activity of two promising 2,3-butanediol (2,3-BD) producers, Bacillus licheniformis 24 and B. velezensis 5RB, by cloning and heterologous expression of cel8A and cel48S genes of Acetivibrio thermocellus. In B. licheniformis, the endocellulase Cel8A (GH8) was cloned to supplement the action of CelA (GH9), while in B. velezensis, the cellobiohydrolase Cel48S (GH48) successfully complemented the activity of endo-cellulase EglS (GH5). The expression of the natural and heterologous cellulase genes in both hosts was demonstrated by reverse-transcription PCR. The secretion of clostridial cellulases was additionally enhanced by enzyme fusion to the subtilisin-like signal peptide, reaching a significant increase in the cellulase activity of the cell-free supernatants. The results presented are the first to reveal the possibility of genetic complementation for enhancement of cellulase activity in bacilli, thus opening the prospect for genetic improvement of strains with an important biotechnological application.