Vol 54, No 6 (2018)

Natural polyhydroxyalkanoates (PHAs) and their synthetic analogs are widely used in medicine, including the production of biodegradable medical devices (prostheses, patches, stents, and plugs) intended for regenerative intestinal surgery. The possibility of biosynthesis and biodegradation of PHAs, primarily, their most common representative, poly(3-hydroxybutyrate) (PHB), by different symbiotic and infectious human and animal bacteria, particularly, multiple bacteria of intestinal microbiota, as well as the physiological role of biopolymer in bacterial cells, is discussed in detail in the review. The review also focuses on the problem of endogenous PHB in humans and animals. The assumption that microbiota bacteria can be a source of endogenous PHB is also discussed. In addition, the use of PHAs in regenerative intestine surgery is considered.

A primary response of the innate immune system to a pathogen invasion or tissue damage is the synthesis of tissue cytokines. Interleukins (IL) 25 and 33 or thymic stromal lymphopoietin (TSLP) are produced by epithelial or muscle cells in response to pathogen fragments, i.e., pathogen associated molecular patterns (PAMPs) or endogenous stress factors (danger associated molecular patterns, DAMPs). The goal of this work is to compare the ability of the bacterial peptide N-formyl-met-leu-phe (fMLP) and an endogenous pruritus inducer, amino acid β-alanine, to stimulate the expression of tissue cytokines. It has been shown by quantitative PCR that fMLP stimulates the ex vivo gene expression of TSLP and IL-25 in murine trachea explants, and β-alanine also stimulates IL-33 gene expression. The in vivo level of TSLP and IL-33 has been evaluated by ELISA in lung homogenates 1 and 6 h after intratracheal injection of the stimulants. Both β‑alanine and fMLP induce the release of IL-33 and TSLP from the intracellular depot. It is shown for the first time that β-alanine and fMLP are activators of tissue cytokine synthesis.

Two halo-tolerant and alkaliphilic actinomycetes, Nocardiopsis alba OM-4 (GeneBank Number, KC119568) and Nocardiopsis alba TATA-13 (GeneBank Number, KC119569) isolated from the salt-enriched soil of the Coastal Gujarat (India) were studied for their proteases. The low molecular weight (19–20 kDa) alkaline proteases were purified by the hydrophobic interaction chromatography on Phenyl Sepharose 6 FF column. The enzymes were optimally active at 60–70°C and pH 10.0. NaCl enhanced the catalysis and enzyme stability at different temperatures and in the presence of up to 50% concentrations of various solvents. The purified enzymes were resistant against various surfactants and inhibitors, suggesting their potential applications in the detergent industry. The changes in the secondary structures were probed by the circular dichroism spectroscopy at various temperatures and solvents, followed by the K2D analysis. With increasing temperatures, the contents of the α- helices and β-sheets increased in the N. alba OM-4 protease, while a reverse trend was evident for the N. alba TATA-13 protease. On the other hand, the α-helix contents increased accompanied with decreased β-sheets in both proteases in the presence of different solvents.

Daptomycin, produced by Streptomyces roseosporus is a novel calcium-dependent cyclic lipopeptide used as antibiotic in treatment of human infections caused by Gram-positive bacteria. However, the yield of daptomycin needs to be further enhanced to reduce the high production cost, which has seriously restricted its clinical application. Here we obtained PRGN21 mutant of S. roseosporus producing the daptomycin with high-yield and having multiple antibiotic resistance to paromomycin, rifampicin, neomycin and gentamicin using the method of combing genome shuffling and ribosome engineering. The daptomycin yield produced by this strain approached 324 mg/L, which was nearly 4-fold higher of that for the parent strain of W2. In period from 48 to 120 h, the dptE transcriptional levels in the PRGN21 mutant of S. roseosporus were significantly enhanced compared to those in S. roseosporus W2 strain, which can explain the ability of PRGN21 mutant to improve the daptomycin biosynthesis.

Bacillus subtilis biomass was encapsulated in 2 biopolymeric membranes to evaluate the enzyme hydrolysis of whey protein and the functional characteristics of the obtained products. The encapsulates of membranes contained composite gel of sodium alginate and chitosan supplied with polyethylene glycol or glutaraldehyde for improving the mechanical properties of the composite. Morphology, porosity, water retention and biomass proliferation of membranes were analyzed. Protease activity from encapsulated B. subtilis was evaluated by the kinetic of protein hydrolysis for 8 h. Quality of hydrolysates was analyzed according to their functional properties. Conventional hydrolysis by commercial neutrase and free biomass of B. subtilis were used as control. Alginate-chitosan-polyethylene glycol composite gave the best results of bacterial encapsulation. Functional properties of the whey protein hydrolysates were comparable with those for products obtained after the action of neutrase and free microbial biomass. In addition, the enzyme activity remained after 5 cycles of hydrolysis. Thus, encapsulating microbial biomass could be an economic alternative of biocatalytic process to produce functional whey products.

The molecular genetic, morphometric, and physiological-biochemical characteristics of three isolates of the carotenogenic microalga Haematococcus sp. obtained in different regions of the Caucasus and Crimea were studied. According to the results of light microscopy and PCR analysis of 18s rDNA gene fragments, all isolates were identified as Haematococcus pluvialis (clade Chlorogonia, order Volvocales, class Chlorophyceae). It is shown that the strains isolated in the mountainous and valley areas of the Black Sea region differ in important characteristics relevant for mass cultivation: cell size, growth rate, stress tolerance, mass fraction of total carotenoids and astaxanthin in dry matter, and their yield per liter of the start culture. The H. pluvialis isolates most promising for further research and the development of a cultivation technology for the Black Sea regions of the Caucasus and Crimea were strains IMBR-3 (Sevastopol) and IMBR-1 (Adler), which were characterized by the highest astaxanthin production (16.77 ± 1.32 and 17.12 ± 0.72 mg/(L day), respectively).

In Saccharomyces cerevisiae, flocculation is mainly regulated by the expression of genes belonging to the FLO family. In this study, the flocculating protein Flo1p, which is encoded by FLO1 gene was isolated and purified from S. cerevisiae W303-1A F4. The protein was found to have a molecular weight of about 200 kDa and belongs to Flo1-type. In addition, the recombinant yeasts including S. cerevisiae ZWA46 (Δ196-240), S. cerevisiae ZWA46 (Δ196-209) and S. cerevisiae ZWA46 (Δ210-240), were constructed. The results showed that amino acid residues from 196 to 209 of Flo1p were the active mannose-binding region, while residues 210 to 240 were not. The latter, however, had a certain influence on mannose-binding ability of Flo1p.

In this study, highly sensitive immunochromatographic analyses (ICAs) of ciprofloxacin (CIP) are developed. CIP is an antibiotic of the fluoroquinolone group that is widely used in veterinary practice and contaminates agricultural products. The analyses are based on various techniques that introduce gold nanoparticles as markers. It has been shown that the schemes allow for the detection of CIP within 15 min with an instrumental detection limit of 10 pg/mL for both schemes and visual detection limits of 10 and 2 ng/mL for direct and indirect schemes, respectively. The developed systems have been tested to detect the antibiotic in milk samples. It has been shown that ICA can determine CIP in milk using a simple procedure of preliminary sample preparation with preservation of high analytical characteristics.

A rapid method for the identification of the osmotolerant yeast Zygosaccharomyces rouxii was developed based on Taqman RT-PCR. Osmotolerant yeasts cause spoilage of food with a high sugar content (honey, syrups, candies, jams, concentrated fruit juices, etc.). In the course of bioinformatics analysis, a DNA segment containing the 5.8S, 18S and 28S genes of ribosomal RNA and ITS1 and ITS2 intergenic regions specific for Z. rouxii was detected, and primers and Taqman probes were developed. The PCR was optimized both for the concentration of its components and for temperature cycles. It was shown that PCR is specific only for Z. rouxii, and the developed analysis allowed the detection of 10 femtograms of its DNA. The method was validated for the collection microorganisms of different taxonomic groups. As a result of the screening of 22 commercially available sugars and dried fruits, it was established that three types of sugar and six dried fruits were contaminated with these yeasts.

A method for the estimation of antioxidant properties of natural and synthetic preparations was developed based on inhibition of the autoxidation of paprika carotenoids. Carotenoids in the form of paprika extract were coated on a porous inert polysaccharide. Autoxidation of paprika carotenoids occurs more rapidly in such system than with direct methods for the determination of antioxidant inhibitory properties. It was shown that the developed method had good reproducibility. The behavior of paprika carotenoids, which was studied in systems with various types of starches, showed that the developed method could be used to evaluate the protective properties of food biopolymers in relation to lipid autoxidation. Moreover, the method is effective for comparison of the activity of both synthetic and natural antioxidants. The most important advantage of the method is that paprika carotenoids are almost identical to unsaturated fatty acids in their ability to interact with oxygen radicals of oxidants. Therefore, the results reflect the behavior of antioxidants in both real lipid-containing model systems and food products.