Biotechnologia Acta

5-23

Authors: O. M. Klyuchko, L. P. Buchatsky, O. V. Melezhyk

The aim of the work was to develop electronic database of biological organisms (fishes, viruses) on the base of the methods that have been used before in mathematics, engineering, physics. The methods of object-oriented system analysis, construction of models of subject spaces (problem space, solution space), ER-diagrams, of constructing databases and others were applied. The examples of prototypes of modern databases with information about fishes developed over the past years in Western European and American countries were presented. An overview of applied methods, concepts and terminology from the areas of object-oriented system analysis, database designing was disclosed. In conclusions the description of constructed database and practical recommendations for the development of databases with the information about domestic biological organisms for electronic information systems are given.

41-49

Authors: V. Sokolik, O. Berchenko, N. Levicheva, S. Shulga

The aim of the study was to determine the effect of miR-101 on the level of β-amyloid peptide and activation of the cytokine system in the brain regions of animals with an experimental model of Alzheimer’s disease. MiR-101 is the key deactivating operator of mRNA function for the amyloid-β protein precursor. Hence, miR-101 is capable to suppress its synthesis and amyloidogenic processing. Aged male rats were injected intrahippocampally with single-dose unilaterally of β-amyloid peptide 40 aggregates (15 nmol). After 10 days, nasal administration of the liposomal form of miR-101 or empty liposomes was started. After 10 days of therapy, the level of toxic endogenous form β-amyloid peptide 42 and the activity of the cytokine system were determined by the indicators of tumor necrosis factor α, interleukin-6, and interleukin-10 in neocortex, hippocampus and olfactory bulbs. It was found that in rats, aggregates of exogenous β-amyloid peptide 40 model the amyloidogenic and pro-inflammatory situation after 20 days in the neocortex and hippocampus (a significant increase in the concentrations of β-amyloid peptide 42 by 36% and cytokines by 16–18% in the neocortex, and β-amyloid peptide 42 by 27%, proinflammatory cytokines tumor necrosis factor α, interleukin-6 by 14% in the hippocampus), but not in olfactory bulbs. The ten-day course of nasal therapy of liposomal miR-101 normalized the level of β-amyloid peptide 42 and cytokines: in neocortex, the concentration of endogenous toxic β-amyloid peptide 42 decreased by 33%, in the hippocampus by 15%, and concentration of pro-inflammatory cytokines fell by 11–20%. Thus, nasal therapy of miR-101 in liposomes caused a significant anti-amyloidogenic effect in rats with the Alzheimer’s disease model, whereas its anti-inflammatory effect was primarily due to a decrease in β-amyloid peptide 42 concentration.

57-66

Authors: M. Kryvokhyzha1, Y. Libantova2, N. Rashydov

The aim of the work was to estimate the impact of the short wavelengths ultraviolet radiation (wavelength is 230 nm) on Arabidopsis thaliana. The stress response on some key flowering determination genes AP1, GI, LFY, FT, CO, and the repair gene RAD51 expression were investigated. The grown plants were applied by red (610–700 nm), violet (400–450 nm), neutral white (mixture wavelengths 380–750 nm), 20 V and high intensive white light (mixture wavelengths 380–750 nm) 40V LED. The experimental group of plants was irradiated by short wavelengths ultraviolet on ontogenesis stage 5.1 by Boyes classification. The leaf length as growth parameter mark also was analyzed. The short wavelengths ultraviolet influence caused differences in photoperiodic pathway genes expression in plants grown under different illumination. Acceleration flowering phases under influence white intensive illumination and delay ones in case of violet and common white illumination were observed comparing with control groups. It was revealed that cryptochrome and phytochrome formation play an important role in plant development and stress resistance. It enables to understand the best way of plant cultivation in stressful condition.

75-81

Authors: О. Hrytsev, J. Shevchenko, N. Vorobiova, L. Skivka

The aim of the study was to develop rapid and sensitive assay for the simultaneous detection of R. cerealis and R. solani. Pure cultures of fungi were grown on a potato dextrose agar for 5 days at 28 °C, and mycelium was harvested and used for DNA extraction. Total DNA was extracted using a commercial test-systems. Molecular identification of phytopathogenic fungi was performed using a multiplex-touchdown PCR with further electrophoretic separation of amplification products in agarose gel. The specific sequence characterized amplified region primers RtubR4/RtubF4 for R. cerealis and ITS1/GMRS–3 for R. solani were tested for their specificity and useability in PCR multiplex capacity. The specificity of the multiplex-touchdown PCR was tested using DNA from wide range of fungal species and non-target DNA from healthy wheat. The used primer pairs provided only specific fragments for R. cerealis and R.solani. No PCR products were obtained during amplification with the negative control or non-target DNA templates from other species. Coupled to this we have optimized the temperature regime for the multiplex PCR protocol. Taken together, our protocol convincingly demonstrated the simultaneous ability to detect Rhizoctonia cerealis and Rhizoctonia solani and can be used for the diagnosis of compound Rhizoctonia root rot.

5-26

Authors: Klyuchko O.M., Aralova A.A., Aralova N.I.

The purpose of the work was to analyze data about the construction of electronic automated work places for organization of more perfect ones for biotechnology. Methods of program and mathematic simulation, imitation modeling were used for these works. The information about some prototypes of electronic automated work places constructed for biology and linked sciences in Ukraine during the last 25–30 years was discussed. The results of some automated work places constructed by the authors were presented. In conclusion the observed experience was summarized and the set of recommendations for its practical implementation were done.

46-55

Authors: M. M. Marchenko, I. V. Dorosh, L. M. Cheban

The aim of the study was to develop the biotechnology approach for obtaining secondary carotenoids of green microalgae Desmodesmus armatus (Chod.) Hegew. under conditions of two-stage cultivation on waste water from recirculating aquaculture system in response to the action of inducers of different origin. By chemical nature, secondary carotenoids are C40-ketocarotinoids - intermediates of enzymatic oxidation of β-carotene to astaxanthin. The study presents the conditions for cultivation of D. armatus on the waste water from the recirculating aquaculture system by a two-stage accumulation process, where conditions for rapid growth of biomass were created at the first stage, and the biosynthesis of the target product was induced by the introduction of carotenoid biosynthesis precursors (C6H12O6, CH3COONa), promoters of free radical oxidation (FeSO4 / H2O2) or osmotic stress (NaCl) into the nutrient medium. It was shown that the first phase of cultivation was characterized by high growth and productive indices: the amount of biomass was up to 13 g/l, the content of total proteins was 37.9 %, lipids – 26 % and total carotenoids – 7.5 % per gram of dry biomass. Among carotenoids, the presence of zeaxanthin, lutein, β-carotene, insignificant amounts of astaxanthin, canthaxanthin, esters of adonixanthin and astaxanthin were detected. The features of the adaptive response of D. armatus to the influence of factors that induce secondary carotenogenesis are established. Among them is retention of the number of cells or doubling of their number during the use of chemical activators. Decrease in the activity of cytochrome oxidase as an indicator of the metabolic activity of the culture. Thus, the possibility of increasing the content of β-carotene and astaxanthin in D. armatus biomass, essential for fish and crustaceans, by introducing promoters of free radical oxidation and osmotic stress NaCl (200 mM) or Fe2+ (200 mM) and H2O2 (10–4 mM) into the waste water from RAS in the second phase of cultivation was established. Metabolic disbalance in D. armatus cells, which were observed under the influence of chemical factors, led to a redistribution of the main nutrients profile. Biosynthesis and accumulation of lipids were activated against the background of intensive carotenogenesis.

63-70

Authors: M. Gahramanova, 2, R. Dovhyi, M. Rudyk, O. Molozhava, V. Svyatetska, L. Skivka

The aim of the work was to explore phytochemical characteristics of water extract from polyherbal composition based on P. oleracea and it’s effect on oxidative metabolism of murine peritoneal macrophages. The qualitative phytochemical analysis was conducted by colorimetric method. Quantitative analysis of phenols was performed in the test with gallic acid as a standard. Murine peritoneal macrophages were isolated without previous sensitization. Leukotoxicity of the water extract from polyherbal composition leukotoxicity was evaluated in MTT test. Reactive oxygen species generation was assayed by the nitroblue tetrazolium reduction method. Phytochemical analysis revealed the presence of water-soluble and insoluble phenols, tannins, saponins, flavonoids, cardiac glycosides and coumarins in the studied plant mixture. The water extract from polyherbal composition used in a range of concentration 1–1000 μg/ml (lyophilisate in distilled H2O) didn’t exhibit any toxic effects on murine peritoneal macrophages. Water extract from polyherbal composition caused statistically significant dose-dependent increase in oxidative metabolism of murine peritoneal suggest modulatory effect of studied water extract from polyherbal composition on innate immunity cells.

79-87

Authors: O. V. Kyrychenko

The aim of the work was to estimate the action efficiency of pre-sowing soybean seed bacterization with complex inoculants based on Bradyrhizobium japonicum 634b and Azotobacter chroococcum Т79 under influence of phytolectins in vegetation conditions. It was shown, that during all vegetation period the soybean plants formed vegetative mass more actively: (in 1.2–1.5 times) above-ground part and in 1.2–1.7 times root system by the the complex seed bacterization as compared to the mono-inoculation. There is a direct dependence of soybean vegetative height on the functional (nitrogen-fixing) ability of the symbioses. Advantages of the application of complex compositions for intensification of beans formations (more early terms of reproductive organs forming, greater amount of beans on plants with their mass, exceeding control in 1.1–1.7 time) are shown. The middle increase of soybean harvest to control made from 13% (binary bacterial composition on basis of rhizobium and azotobacter) to 21% (polycomposition on basis of rhizobium and azotobacter activated by the wheat lectin). The compositions based on rhizobium activated by the soybean lectin provided 18% increased seed harvest. Polycomposition containing nitrogen-fixing bacteria activated by appropriate plants lectins led to the 19% increased harvest. It is shown that the harvest increased with higher values of almost all indexes of its structure. The compositions based on rhizobia and azotobacter activated by wheat lectin as well as the compositions based on rhizobia activated by soybean lectin are the most productive for practical use to increase the soybean yield.

5-28

Authors: O. M. Klyuchko

The newest biotechnical systems for environment ecological monitoring based on the use of modern information and computer technologies and existing databases of chemical substances have been analyzed. In particular, there were observed such modern biophysical research methods as imitation and program modeling, based on the author results obtained in the experiments with registration of chemo-sensitive transmembrane electric currents in neurons in voltage clamp mode, use of neuronal fluorescent markers and accounting of organisms-bioindicators. The developed systems and methods allow revealing and identification of substances hazardous to living organisms and to make conclusions about their possible biological effects. The functioning of biotechnical information systems for environmental monitoring was analyzed in a wide time ranges, using modern databases, expert subsystems and interfaces capable to identify different types of chemicals. It is shown that for such systematic environmental monitoring it is possible to study and predict the effects of substances influences for a long time ¾ from the first moments of their exposure to individual cells of organism to months and years after exposure to the whole organism.

30-57

Authors: T. P. Pirog, D. A. Lutsay, L. V. Kliuchka, K. A. Beregova

The recent literature data about the antibacterial and antifungal activity of microbial surfactants (lipopeptides synthesized by representatives of genera Bacillus, Paenibacillus, Pseudomonas, Brevibacillus, rhamnolipids of bacteria Pseudomonas, Burkholderia, Lysinibacillus sp., sophorolipids of yeasts Candida (Starmerella) and Rhodotorula), and our own experiments data concerning antimicrobial activity of surfactants synthesized by Acinetobacter calcoaceticus IMB B-7241, Rhodococcus erythropolis IMB Aс-5017 and Nocardia vaccini IMV B-7405 were presented. The analysis showed that lipopeptides were more effective antimicrobial agents compared to glycolipids. Thus, the minimum inhibitory concentrations (MIC) of lipopeptides, ramnolipids and sophorolipids are on average (μg/ml): 1–32, 50–500, and 10–200, respectively. The MIC of surfactants synthesized by the IMV B-7241, IMV Ac-5017 and IMV B-7405 strains are comparable to those of the known microbial lipopeptides and glycolipids. The advantages of glycolipids as antimicrobial agents compared with lipopeptides were the possibility of their synthesis on industrial waste and the high concentration of synthesized surfactants. The literature data and our own results indicate the need to study the influence of microbes’ cultivation conditions on the antimicrobial activity of the final product.

66-74

Authors: N. V. Dziubenko, H. M. Kuznietsova, O. V. Lynchak, V. K. Rybalchenko

Aim of the work was to investigate the suspended C60-fulleren effect on liver and pancreas state under intraperitoneal and intragastrial administration on rat experimental cholangitis model. Acute cholangitis was simulated by a single ingestion of ɑ-naphthyl isothiocyanate — ANIT. C60-fullerene aqueous colloid solution (C60FAS, 0.15 mg/ml) was administered to animals at a volume containing C60-fullerenes at a dose of 0.5 mg/kg body weight in 24 and 48 h after ANIT administration. After72 h of the experiment, the animals were euthanized. Blood serum ALT and AST activities were measured, the liver and pancreas states were analyzed by light-microscopy level. It was found that intragastrial and intraperitoneal administration of C60FAS contributes to the correction of negative effects in the liver and pancreas caused by the induction of acute cholangitis. This was proved by the normalization of ALT activity, reduction of pancreatic parenchymal edema and liver fibrosis, and increased blood flow in these organs. Application of C60FAS could improve the state of the liver and pancreas under acute cholangitis in rats.

81-85

Authors: A. V. Zlatska, I. M. Gordiienko, D. О. Zubov, R. G. Vasyliev, S. N. Novikova

The aim of the study was to investigate the level of estrogen (ESR1, ESR2) and progesterone (PGR) receptors expression in the primary culture of endometrial multipotent mesenchymal stromal/stem cells endometrial multipotent mesenchymal stem cells and during in vitro cultivation under different athmospheric oxygen content. The dynamics of changes in the level of expression of the sex hormones receptors in the primary culture and during cultivation under different oxygen content in the athmosphere was shown.

5-28

Authors: O. M. Klyuchko

The purpose of the present work was to alalyse the prototypes of information expert systems, their structure, functions and practical application, and to develop a new one for solving practical problems in biotechnology, laboratory practice and environmental protection. The observed prototypes were developed for the use in genetic studies, agricultural production, nature protection from pests and environmental pollutants, for works in medicine, and etc. During the work, following methods were used such as methods of comparative research of the samples of technical devices, imitation and program modeling, which were based on numerical results obtained in experiments with the recording of chemosensitive transmembrane electrical currents in neurons in voltage clamp mode. As a result, an original expert system was developed. It was coupled with a detector group, databases and interface. The developed expert system was able to distinguish automatically the certain types of chemicals at the input, to display their identification data and, if necessary, the reports about their harmfulness. Conclusions were done about the practical value of these data for the elaboration of new electronic expert systems for monitoring the presence of harmful substances in the environment. It was also discussed the possibility of developed expert system application for new methods of qualitative and quantitative analysis of some organic compounds.

39-46

Authors: A. V. Kotinskyi, A. I. Salyuk, S. A. Zhadan

The aim of the work was to study the impact of glycine on Spirulina growth and photosynthetic processes intensity. For research the culture of Spirulina platensis (Gom.) Geitl cyanobacterium strain LGU — 603 from collection of Kholodny Institute of Botany of the National Academy of Sciences of Ukraine was used. The cultivation process was carried out in Zaruk nutrient medium in a vertical tubular apparatus of 8 cm in diameter and of volume 2 dm3 in a constant mixing of the culture medium with air. Introduction of glycine in a cultural environment resulted in the increase of the Spirulina productivity, maintenance of basic photosynthetic pigments and protein. Glycine introduction allowed to increase the growth rate of spirulina and to content achieve the high density of the culture due to the increased length of log-phase. Intensification of Spirulina platensis growth and its productivity increase was due to the fragmentation of cyanobacteria trichomes, so the increase of trichomes number with a small number of cells, which grow quickly. The degree of fragmentation depended on the concentration of introduced glycine and on the culture development stadium and also on introduced glycine concentration.

55-66

Authors: I. A. Blaydа, N. Yu. Vasylieva, T. V. Vasylieva, L. I. Sliusarenko, O. I. Dzhambek

The aim of the work was to optimize the process of bacterial desulfurization of energy coal, namely, to determine the influence of the component composition of the nutrient medium and the conditions of the process, which ensure the maximal development and activity of the aboriginal association of acidophilic chemolithotrophic bacteria and, as a consequence, the maximal index of sulfur decrease in coal in minimal time. We used the method of mathematical planning of the experiment adapted to the plan in Greek-Latin squares. The calculations in this approach are based on the analysis of variance (ANOVA). The formal planning of experiments has been carried out with four operating factors (nutrient medium components) at four levels (concentrations). The calculations were performed in Excel. The selection of operating factors and their combinations was made with the usage of unifactor ANOVA, correlation analysis and the method of principal components PCA. Researches were carried out in R 3.4.0 program and were founded on data of the preliminary evaluating experiments. Acidithiobacillus ferrooxidans Coal 17 aboriginal strain was used to obtain the most significant desulfurization effect. This strain was isolated from the investigated coal, studied and identified. The significance of the factor level for each nutrient medium component was analyzed using the Duncan’s multiple range test, the uniformity of the variances was examined with the Cochran’s test, and the significance of the factors was tested by the Fisher’s criterion. As a result, for the optimal nutrient medium the next combination of factors and their levels, which corresponds to the composition, g/dm3, was recommended: (NH4)2SO4 — 0.15; K2HPO4 — 0.50; FeSO4.7H2O — 44.0; KCl — 0.10; MgSO4·7H2O — 0.10; Ca(NO3)2 — 0.10; yeast extract — 0.025% (vol.); strain A. ferrooxidans Coal 17 (titre 1·108 CFU/ml) — 1.60% (vol.). This makes it possible to reduce the sulfur content in coal by 66.31% in a short period (seven days). This result could not be got before.

73-81

Authors: O. B. Tashyrev, I. B. Sioma, G. O. Tashyreva, V. M. Hovorukha

The aim of the research was to develop theoretic principles of an efficient biotechnology for microbial purification of concentrated sewage filtrate from a wide range of organic and non-organic compounds, and experimentally confirmed these principles. The experiment combined conventional microbiological, physical and chemical methods in which ten different types of solid carriers, both natural and artificial, were used. A comparative analysis of these types of solid carriers used in the flow system and a structured evaluation of treatment parameters was provided. The obtaining results demonstrated the effectiveness of the use of approach sewage microbial purification in a flow system. Developed principles can be used as a basis for new efficient biotechnologies for sewage microbial purification.

5-25

Authors: O. M. Klyuchko

The purpose of the work was to analyze existing information systems (IS) of biological objects and to propose the methods for development of such IS for insects on the example of Noctuidae (Lepidoptera). A detailed analysis of technical information concerning the distributed networked systems, access to computer systems to the common data in electronic IS and the organization of biomedical databases in the Internet was done. The peculiarities of IS’ prototypes development for environmental monitoring of the fauna have been discussed, in particular changes in the number of butterflies’ populations throughout France (including western and southern departments), the Noctuidae (Lepidoptera) in the steppe zone of Ukraine (Striltzivskyi Steppe), and the development of such IS for all territory of Ukraine. The results could be used to develop electronic IS for other biological organisms.

35-41

Authors: N.V. Borzova, O. V., Gudzenko, L. D. Varbanets

The aim of the work was to investigate the possibility of practical use substrate specificity of α-Lrhamnosidase Cryptococcus albidus. p-Nitrophenyl derivatives of monosaccharides were used to determine the activity and specificity of the enzyme. The ability to hydrolyze of natural substrates was evaluated by Davis and high-performance liquid chromatography methods. It was shown that the enzyme exhibits narrow specificity towards the glycon of synthetic substrates and hydrolyzes only p-nitrophenyl-α-L-rhamnopyranoside (Km 4.5 mM) and p-nitrophenyl-α-D-glucopyranoside (Km 10.0 mM). C. albidus α-L-rhamnosidase the most active degrades naringin (Km 0.77 mM), releasing prunin and naringenin. Km for neohesperidin was 3.3 mM. The efficacy of the naringin hydrolysis in grapefruit and pomelo juice was 98 and 94% in 60 min (40 оC, 2 U/ml). As the result of treatment by α -L-rhamnosidase of green tea and orange juice, there was a decrease in the content of rutin, narirutin and hesperidin, indicating that the α -1,2- and α -1,6-linked rhamnose can be cleaved from natural flavonoids. Thus, the study shows the efficiency of treating citrus juices and green tea with C. albidus α -L-rhamnosidase for the purpose of improving their taste qualities and obtaining bioavailable flavonoids glucosides.

49-53

Authors: O. V. Kravchenko, O. S. Panchenko

The aim of the research was the isolation from drinking water the pure cultures of iron- and manganese-oxidizing microorganisms with further assessment of their efficacy to remove these contaminants on rapid filters. To assess the effectiveness selected strains were grown on the solid nutrient medium; the suspension was prepared and was treated to zeolite loading. Ten pure cultures of iron- and manganese-oxidizing bacteria were isolated and identified as 6 genuses: Siderocapsa, Leptothrix, Sphaerotillus, Galionella, Metallogenium, Hyphomicrobium. Comparison the efficiency of genuses Leptothrix, Sphaerotillus, Metallogenium has shown that under conditions of these experiments Leptothrix more effectively removed iron and manganese at low concentrations in model solution.

65-74

Authors: L. Oriabinska, O. Dziuba, O. Dugan

The aim of the work was to find strains of lactic acid bacteria capable to synthesize extracellular tannase enzyme − the key enzymehe hydrolyzing tannins which are plant food constituent. One of the main product of tannins hydrolyzis is gallic acid- the compound with proven antioxidant and onco-protective properties. As a result of lactobacteria screening, two biocompatible strains of lactic acid, namely L. rhamnosus LB3 and L. delbrueckii subsp. delbrueckii with a high level of enzyme productivity, were selected. The maximum accumulation of tannase, corresponding to 0.031 ± 0.002 U/ml for L. rhamnosus LB3 and 0.03 ± 0.002 U/ml for L. delbrueckii subsp. delbrueckii, was observed after 48 h of cultivation. Both strains showed rapid growth and performance of tannase in MRS medium in the presence of glucose or lactose as a carbon source. It was shown that gallic acid, which was a necessary component of the medium as a target enzyme inducer, did not affect the accumulation of lactobacilli biomass. The selected strains are of interest as producers of a bicomponent probiotic with antioxidant properties and require further investigation.