Naukovi visti NTUU “КPІ”. – 2016. – N 3

INDOLEQUINOXALINES DERIVATIVES – PROMISING COMPOUNDS WITH ANTIVIRAL AND INTERFERON INDUCING ACTIONS /T.A. Bogun, L.G. Zholner, T.I. Bykova, N.M. Zholobak, M.Ya. Spivak - K. Naukovi visti NTUU “КPІ”. – 2016. – N 3
Background. The development of new drugs with antiviral activity and the ability to induce interferon is an urgent task, taking into account the rapid spread of viral infections, development of resistance the virus strains to existing antiviral drugs and the overall decline of the immune response.

Objective. Determine the antiviral activity and interferon inducer actions of indolequinoxalines derivatives in terms of in vitro.

Methods. The work on PST cell cultures in conditions of in vitro antiviral and interferon inducer of the 18 new indolequinoxalines derivatives was studied.

Results. The ability of indolequinoxalines derivatives to inhibit the development of viral cytopathic effect on the model PST-BBC only in therapeutic regimens was shown. In the model of inoculated cell cultures PST is shown that 12 compounds are capable of inducing interferon in low concentrations.

Conclusions. Among the indolequinoxalines derivatives RG-61 compound as a promising for further studies was identified, which has a low toxicity (as compared to a number of compounds derived), has antiviral activity in infected cells and is capable of inducing IFN.

Keywords: interferon inducers; antiviral activity; vesicular stomatitis virus; interferon-inducing action; amiksin; derivatives of indolohinoksalin.

HYDROGEN PRODUCTION DURING АNAEROBIC FERMENTATION OF CORN AND SUNFLOWER WASTE /N.B. Golub, D.I. Drapoi - K. Naukovi visti NTUU “КPІ”. – 2016. – N 3
Background. Obtaining hydrogen – an alternative energy carrier – from renewable resources (agricultural waste).

Objective. Establish rational parameters for alkaline pretreatment of sunflower and corn waste by an association of microorganisms to increase the speed of hydrogen production.

Methods. We used gas chromatography method to establish the qualitative and quantitative biogas content that was produced during the process of fermentation. We used light microscopy methods to determine lysis zones and to monitor species content of microorganisms association.

Results. We’ve investigated the effect of 5, 10, 20, 30, 40 % alkali concentration during pretreatment of corn and sunflower waste on hydrogen yield in the process of their anaerobic fermentation for 1, 2 and 3 hours. The highest hydrogen yield has been achieved by pretreatment with 20 % NaOH solution for 3 hours. We’ve studied the hydrogen yield during fermentation process with inoculum to culture medium ratio as: 1:6; 1:3; 1:2; 2:3; 1:1 by volume. The content of microorganisms in inoculum was 0.0011 g/cm3 and the dry residue of culture medium was 0.0267 g/cm3. It’s been shown that hydrogen yield in the gas phase reaches 87.5 ± 4.2 % for the association of microorganisms enriched by Clostridium and Bacillus genus with the ratio of inoculum to culture medium 2:3. Fermentation process has been studied in two temperature modes: 22 ± 2 and 35 ± 2 °C. It’s been determined that temperature increase from 22 ± 2 to 35 ± 2 °C increases hydrogen yield by 2 times. It’s been shown that the highest hydrogen yield is observed by holding enzymatic process at pH level near 6–6.5 pH decrease switches metabolic pathways from acetate fermentation type to the formation of butyrate, thus reducing hydrogen yield.

Conclusions. The most effective method of substrate pretreatment for the process of obtaining hydrogen by anaerobic fermentation of corn and sunflower waste (1:1) was the pretreatment with 20 % NaOH solution for 3 hours. The highest hydrogen yield has been observed in case of inoculum to culture medium ratio set at 2:3 with pH 6–6.5 and the temperature at 35 ± 2 °C.

Keywords: anaerobic fermentation; hydrogen; waste; microorganisms; inoculum; substrate; cellulosic materials.

BIOSAFETY IN THE CONTEXT OF LABOUR PROTECTION. BIOTECHNOLOGICAL AND REGULATORY ASPECTS /V.V. Danilovа, N.V. Dekhtiarenko, Yu.V. Gorshunov, A.Yu. Galkin - K. Naukovi visti NTUU “КPІ”. – 2016. – N 3
Вackground. The rapid spread of modern biotechnologies causes public concern about their potential harmful effects on biodiversity, taking into account risks to human health. One of the important aspects of the implementation of the biosafety requirements is the observance of rules of labor protection.

Оbjective. The aim of this work is to analyze the current status of biosafety through the implementation of requirements for labor protection in enterprises and research institutions of biotechnological (pharmaceutical) profile.

Methods. The analysis and synthesis of information on biotechnological and regulatory aspects of biosafety and occupational safety are conducted.

Results. The biological safety principle is to limit the spread or prevent spillage of infectious material from the lab environment, where various manipulations or maintaining in culture of the material are carried out. The main legal documents regulating the sphere of labor protection when working with BO in Ukraine: Law of Ukraine “On ensuring sanitary and epidemic wellbeing of population”, Law of Ukraine “On state Biosafety system while creating, testing, transportation and use of genetically modified organisms”, the State sanitary norms and rules on “Organization of work of laboratories in the material study, which contains pathogenic biological agents of I–IV groups of pathogenicity by molecular-genetic methods”, State sanitary rules on “Safety of work with microorganisms of I–II groups of pathogenicity”.

Conclusions. An important element of access for the products of the biotechnology industry of Ukraine to the markets of developed countries is compliance with international recommendations on biosafety, which is not possible without the implementation of relevant international instruments in domestic legislation. The formation of the system approach to biosafety and occupational safety in enterprises and research institutions of biotechnological profile will contribute to the development and implementation of regulations (licensing conditions) for the implementation of the activities with genetically modified organisms.

Keywords: biosafety; occupational safety; biological objects; risks.

SELECTION OF ‎METHODS FOR CAROTENOID EXTRACTION FROM SUBMERGED LAETIPORUS SULPHUREUS MYCELIUM /L.P. Dzyhun, O.A. Palyushok, O.M. Chudnivets - K. Naukovi visti NTUU “КPІ”. – 2016. – N 3
Background. To obtain natural carotenoids for their practical application is the actual problem. Xanthоcarotene obtained through biotechnology can be used in light industry as dye, as an alternative to artificial pigments. The need of dedication of natural carotenoids is also caused by their suitability for medical use. It is explained by diverse biological activity (antitumor, radioprotective, antimicrobial, antiviral, antioxidant) of these substances.

Objective. Objective of the study is selection of effective methods and conditions of carotenoid extraction from Laetiporus sulphureus biomass.

Methods. After the submerged producer’s cultivation was performed different methods of pigments extraction were carried out. Carotenoid content was determined.

Results. The most effective extraction was chosen. Carotenoid content in producer’s biomass was calculated.

Conclusions. Еthanol-hexane method was selected as the most effective extraction circuit. Also was found that the change of exposure’s temperature causes changes in the composition of the key faction.

Keywords: carotenoids; хanthоcarotene; extraction; optical density; submerged cultivation; Laetiporus sulphureus.

BIOGENIC RHAMNOLIPID SURFACTANTS IN COMPLEX PLANT GROWTH REGULATORS /I.V. Karpenko, G.G. Midyana, O.V. Karpenko - K. Naukovi visti NTUU “КPІ”. – 2016. – N 3
Background. The topical problem of biotechnology is the creation of ecologically safe agents for the crop production. Considering the environmental threat of synthetic agents, biological products are priority ones for plants. Among them biogenic surfactants deserve attention due to their physico-chemical and biological properties.

Objective.Influence of surface-active products of microbial synthesis of the strainPseudomonas sp. PS-17 (rhamnolipid biosurfactants) on the activity of phytohormones for the creation of complex plant growth regulators.

Methods. The effect of culture liquid supernatant (CLS) and rhamnolipid biocomplex (RBC) on phytohormone activity was determined in bioassays for auxin, cytokinins, gibberellins.

Results. It was shown that in the compositions with RBC the activity of phytohormones has increased: auxins – indolyl-3-acetic acid on 28 %, indolyl-3-butyric acid – 63 %; gibberellin – on 30 %, cytokinin – on 24–30 % compared to the options without biosurfactants. Such stimulating effect on phytohormones was also determined for CLS, in addition it manifested phytohormone-like action.

Conclusions. The obtained results prove the prospects of rhamnolipid surfactants for the creation of ecologically safe complex plant growth regulators.

Keywords: rhamnolipid surfactants; coleoptile; hypocotyl; cytokinin; gibberellic acid; auxin.

O.V. Kravchenko

OXIDIZING AGENTS TESTING FOR THE INTENSIFICATION OF THE BIOLOGICAL COMPONENTS OF THE IRON AND MANGANESE REMOVAL FROM WATER

Background. Iron and manganese groundwater are found in almost all regions of Ukraine. The concentration of these elements varies within a very wide range, and in some regions it is 20–30 mg/dm3 for iron and 2–3 mg/dm3 for manganese. The high content of these compounds in water impairs its organoleptic characteristics, leads to the formation of color films and precipitation and, therefore, to overgrowing water supply systems, water intake valves, sanitary products. That is why the improvement of existing or development of new iron and manganese removal technology remains an important issue of our time.

Objective. The aim is to test the effect of oxidizing – ozone and hydrogen peroxide – on the efficiency of iron and manganese removal from water.

Methods. The studies were conducted in laboratory columns filled with zeolite from Sokirnitskyi deposit fraction 1–4 mm. Columns are settled with pure cultures of microorganisms Leptothrix, as well as mixed cultures Leptothrix and Sphaerotillus. Model solutions of different compositions were passed through the columns, before treated with ozone (1 mg/dm3) and hydrogen peroxide solution (1 mg/dm3).

Results. The addition of low concentrations of oxidizing agents intensifies the process of iron and manganese removal by pure and mixed cultures of bacteria which can remove iron and manganese (up to 98 %). These results confirm the fact that the removal of iron and manganese from water is intensified biological component of the process, rather than chemical-physical.

Conclusions. Conclusions regarding that the ozonation is efficiency for iron and manganese removal from water. Considerable increase in removal efficiency of iron and manganese from solutions treated with ozone. Technologically the process is convenient to implement ozonation before filter loading settled with bacteria capable of oxidizing iron and manganese.

Keywords: iron removal; manganese removal; ozone; hydrogen peroxide.

METHODS OF ASSESSING BIOLOGICAL ACTIVITY OF RECOMBINANT HUMAN INTERLEUKIN-7 AND STABILITY STUDY OF THE PREPARATION ON ITS BASIS /T.N. Lutsenko, D.B. Starosyla, S.L. Rybalko, A.Yu. Galkin - K. Naukovi visti NTUU “КPІ”. – 2016. – N 3
Background. Interleukin-7 is one of the most important immune regulatory cytokines. Recombinant human interleukin-7 (rIL-7) in aqueous solution is subjected to chemical degradation mechanisms such as proteolysis, oxidation, disulfide exchange, oligomerisation etc. Such changes affect the shelf life of the preparation on the basis of rIL-7. Evaluation of the biological activity of rIL-7 can be carried out by assessing its antiviral activity.

Objective. Comparison of methods for inhibiting reproduction of the influenza virus, herpes simplex virus and hepatitis C virus with recombinant human interleukin-7 and research stability of the preparation on the basis of rIL-7.

Methods. We used immortalized cells: the kidneys of dogs, the bovine kidneys and kidneys of African green monkey Vero. The following viruses were used: hepatitis C virus surrogate (bovine viral diarrhea virus, BVDV), influenza virus (strain A/FM/1/47 (H1N1)), herpes simplex virus type 2 (HSV-2) (BH strain). To determine the antiviral activity of rIL-7 in vitro conditions using daily, immortalized cells. Cells were grown in RPMI-1640 medium.

Results. It was shown that rIL-7 in buffer stabilizing solutions and the culture medium after 1 week of storage was active at a dilution of 0.003 g/ml against BVDV. The study results of antiviral activity of rIL-7 drug in buffer solutions and culture medium against influenza virus A/FM/H1N1 can conclude that storage of the drug in buffer solution and in the intact state at 4 °C for one week has not affected its antiviral activity. It was shown that antiherpetic activity of preparations after 1 week of storage at 4 °C in buffer stabilizing solutions and intact state remained effective. The antiviral activity of drugs in a stabilizing solution has been persisted for 3 months at 4 °C, and in the intact state of rIL-7 lost its antiviral activity after 1 week against the herpes virus, and after 1 month regarding BVDV.

Conclusions. Methods for assessing the antiviral activity of rIL-7 towards BVDV, influenza virus, and HSV-2 were developed. It has been proven that the stabilizing buffer solutions proposed by us provide a high level of biological activity of rIL-7 preparations during storage at 4 °C for 3 months, which is a prerequisite for the development of liquid dosage forms of pharmaceutical preparations on their basis.

Keywords: interleukin-7; human recombinant; antiviral activity; biological standardization.

THE PULLULANASE ENZYME: ANALYSIS OF INDUSTRY USE OF PRODUCERS, A PROCESS FOR PREPARING /M.V. Molochko, N.V. Dekhtiarenko - K. Naukovi visti NTUU “КPІ”. – 2016. – N 3
Вackground. The work is dedicated to critical analysis of literature covering the main aspects of pullulanasе enzymes.

Оbjective. The aim is to determine promising areas of research in the field of pullulanasе enzymes.

Methods. The analysis and synthesis of information about the use, sources, mechanism of action, pullulanase enzyme properties and methods of cultivation and methods of isolation and purification of enzyme, immobilization techniques and methods of determining the activity of the enzyme were made.

Results. The analysis of the literature identified the existence of a large number of laboratory developments receiving pullulanase enzyme and lack of industrial production, both in our country and in neighboring countries.

Conclusions. A list of areas in which there is the successful use of pullulanase enzyme is outlined. A patent search of the contemporary producers of pullulanase was made, and characteristics of their cultivation were identified. The most common ways to obtain purified pullulanase and modern methods of immobilization have been revealed. Methods of determining the activity of pullulanase enzyme relatively pullulan substrate were established. Pullulanase enzymes word producers range is specified.

Keywords: pullulanase; enzyme; industry; mechanism of action; producer; property; activity; purification; manufacturers; strain.

THE EFFECT OF IRRADIATION BY BLUE RANGE OF THE SPECTRUM LIGHT ON INCREASING OF TRANSIENT ACCUMULATION OF RECOMBINANT REPORTER PROTEIN GFP IN PLANTS NICOTIANA BENTHAMIANA /A.A. Peterson, M.Y. Vasylenko, Y.S. Pryshchepa, L.B. Oriabinska, M.V. Kuchuk - K. Naukovi visti NTUU “КPІ”. – 2016. – N 3
Background. Plant expression systems are being increasingly used in scientific and industrial biotechnology. Research of the influence of different factors on efficiency of plant transient expression systems expands scientific basis for large-scale and economically efficient application of these systems in industrial biotechnology.

Objective. The object of this research was to determine the influence of Irradiation by blue light of plants Nicotiana benthamiana immediately before agroinfiltration on transient accumulation of reporter recombinant protein GFP.

Methods. Irradiation of plants was performed using LED light sources (λ = 440 nm). Quantity of accumulated reporter protein in plant leaves was measured by fluorimetric method.

Results. It was demonstrated that GFP accumulation in irradiated plant biomass was higher than in not irradiated biomass. Increase of level of accumulation was between 60–87 % depending on other agroinfiltration conditions.

Conclusions. Shown effect seems to be promising as a methodological procedure either in labs or in industrial practices. That is because the apparatus for irradiation is constructively simple, cheap, energy efficient and a period of irradiation time is short.

Keywords: plant expression system; transient expression; Nicotiana benthamiana; recombinant protein; green fluorescent protein; GFP.

CURRENT POSSIBILITIES OF OBTAINING ROSE FRAGRANCE ESSENTIAL OIL /V.Yu. Polishchuk, O.M. Dugan - K. Naukovi visti NTUU “КPІ”. – 2016. – N 3
Вackground. Rose essential oil is one of the most precious essential oils, which is in great demand and is widely used in perfumery and cosmetics, food and pharmaceutical industries. However, traditional methods of production have certain disadvantages; therefore, the biotechnological methods of obtaining rose fragrance essential oil become topical.

Оbjective. The aim is the analysis of current literary sources regarding the possibility of obtaining rose fragrance essential oil by traditional and biotechnological methods, search of the most promising microorganisms for further research.

Methods. In this paper monitoring and analysis of domestic and foreign literature are carried out.

Results. The traditional methods of obtaining rose essential oil, its properties and use are considered. The genera of microorganisms which are able to synthesize fragrant compounds being constituent parts of rose essential oil are identified. Representatives of genus Eremothecium relate to prospective producers that synthesize riboflavin as well as components of rose essential oil (β-phenylethanol, geraniol, citronellol and nerol).

Conclusions. The economic reasonability of production on the basis of Eremothecium can be increased at the expense of application of complex obtaining riboflavin and essential oil for satisfaction of medical, pharmaceutical, cosmetic and food industry requirements.

Keywords: rose essential oil; fungi; synthesis de novo; producer; Eremothecium.

METHANEPRODUCING OF MESOPHILOUS BACTERIA COMMUNITIES OBTAINED FROM LIVESTOCK WASTES /L.A. Khrokalo, A.I. Doloman, V.Yu. Chernenko - K. Naukovi visti NTUU “КPІ”. – 2016. – N 3
Background. The perspective substrates for elaboration of biopreparations in biogas technologies were considered.

Objective. The aim of the study is selection from farm wastes (pig manure, chicken excrements and fermented farm wastes) storage cultures of mesophilous bacteria communities, comparative analysis of methane producing, selection and identification of methane-producing bacteria cultures.

Methods. Bacteria cultures were grown in anaerobic conditions on liquid and solid Zhylina nutrient media with addition of sodium acetate under temperature +33 ± 2 °C. Content of gaseous metabolites was estimated by using gas chromatograph LHM-8MD. Culture patterns were taken in argon flow, were fixed, Gram stained and viewed in light microscope.

Results. Allselectedstorage cultures efficiently produced methane, about 10–15 vol. % of methane has been accumulated in bottles since the 12th day of cultivation. Сultures selected from fermented wastes had high yields of methane emission is about 28 vol. %. Cultures of methane-producing bacteria were received by passages on liquid and solid nutrient media with addition of amoxicillin. Bacteria of Methanosarcina and Methanosaeta genus were identified.

Conclutions. Obtained cultures are perspective ones for elaboration of bacterial preparations and their further using in biogas technologies.

кeywords: anaerobicmesophilous bacteria communities; methane-producing bacteria; Methanosarcina; Methanosaeta.

OBTAINING OF OXYCELLULOSE FROM COTTON FIBERS BY ACTION OF VARIOUS OXIDANTS /V.A. Barbash, U.Y. Nikitiuk, I.N. Deykun - K. Naukovi visti NTUU “КPІ”. – 2016. – N 3
Background. Development of production technologies of materials for various purposes of alternatives to crude oil and natural gas sources of raw material, in particular cellulose.

Objective.To determine the optimal values of technological parameters of cotton fiber oxidation by action of various oxidants – hydrogen peroxide, Oxone, PINO – on quality of oxycellulose.

Methods. Oxidation of mechanically chopped cotton fibers by solution of hydrogen peroxide, Oxone and PINO was performed with air bubbling by the compressor through solution of cellulose in round-bottomed flask immersed in a water bath of the required temperature. The methods of experiment planning and optimization were used to determine the optimum process parameters.

Results. Studied oxidants for oxycellulose of cotton fibers to increase efficiency are in the following order: hydrogen peroxide – Oxone – PINO. The method of full factorial experiment was used to determine the adequate regression equations that describe the process of oxycellulose obtaining by action of Oxone and PINO, and the Gauss-Seidel method was used to determine the optimal values of technological parameters of oxycellulose obtaining. It is shown that oxidation of cotton fibers for optimal values of technological parameters allows getting oxycellulose with the content of carboxyl groups to 3 %.

Conclusions. The obtained oxycellulose can be used in medicine as a component of non-toxic, hemostatic, antimicrobial materials.

Keywords: cotton fiber; hydrogen peroxide; Oxone; PINO; oxycellulose; carboxyl group.

SOLID PHASE EXTRACTION OF TRACE AMOUNTS OF CHLORAMPHENICOL ON THE SILICA GEL SURFACE MODIFIED WITH OCTADECYL GROUPS /O.M. Ivanova, T.V. Sikolenko - K. Naukovi visti NTUU “КPІ”. – 2016. – N 3
Background. Chloramphenicol (CAP) is a broad spectrum antibiotic. It’s widely used in various fields of veterinary and can be accumulated in food of animal origin. A minimum required performance limit (MRPL) for CAP in food is significantly restricted in the EU and the USA. Most modern methods make it difficult to determine the trace amounts of the drug from various matrices without concentration. The method of liquid-liquid extraction (LLE) may be accompanied by loss of trust component. It is therefore advisable to use solid phase analyte extraction to increase the determination sensitivity by instrumental methods and CL withdrawal stability.

Objective. Development of the solid phase extraction method of chloramphenicol from honey, followed by enzyme linked immunoassay.

Methods. Quantitative determination of chloramphenicol was determined using enzyme linked assay (ELISA) test-kit for determination of chloramphenicol in foods, provided by LLC “XEMA”. Solid phase extraction of chloramphenicol from honey samples was studied on silica gel surface modified with octadecyl groups (SiO2-C18), “Agilent”. This method of solid phase extraction was carried out on samples of honey, that previously checked by ELISA using LLE for sample preparation.

Results. Our results showed the possibility of solid phase extraction of CAP on the surface of the silica gel with oktadecyl groups. The optimal conditions for extraction the analyte on the surface of sorbent were found and the eluation condition for CAP was studied. We tested the method of solid phase extraction of CAP from honey matrics on control materials – honey samples with known concentrations of CAP, that were bought by LLC “XEMA”.

Conclusions. Solid phase extraction of CAP could be used for CAP extraction form honey. The proposed method of sample preparation reduces the time of preparation for honey samples. This method is perspective for developing the method of simultaneous concentration of antibiotics from different groups followed by ELISA determination.

Keywords: Chloramphenicol; solid phase extraction; honey; sorption concentration; ELISA.

RADICAL CHAIN OXIDATION OF CUMENE IN THE PRESENCE OF DIFFERENT SUPRAMOLECULAR SYSTEMS CONTAINING OXYGEN RADICAL ANION /O.V. Smirnova, I.V. Efimova - K. Naukovi visti NTUU “КPІ”. – 2016. – N 3
Background. In most publications on the problems associated with reactive oxygen species, highlights their destructive effect on the membranes, nucleic acids and proteins. But many researchers overlooked the vast array of data showing the absolute necessity of reactive oxygen to vital processes. So many empirical data are in conflict with established in classical biochemistry scheme in which reactive oxygen species are seen only as hyperactive chemical particles that can disrupt normal harmonious progress of biochemical processes. In this regard, the study of superoxide anion role in the radical-chain oxidation processes is important, where its function as mediator remains virtually unexplored.

Objective. The aim is to research the process of initiated cumene oxidation in the presence of different supramolecular systems containing anion radical oxygen.

Methods. The paper used the equilibrium solutions of supramolecular complexes KO2*crown-ether, for which balance achieving time was estimated by measuring the electrical conductivity. The cumene oxidation kinetics in the presence of oxygen radical anion was monitored by gas volumetric method. To study the influence of the cation nature on the experimental mixture oxidation, we used various crown ethers to form supramolecular systems KO2*crown in DMSO.

Results. It was stated that effectiveness of the complex action depends on the crown-ether nature, which allows choosing the most optimal system for the inhibiting mixture preparation.

Conclusions. Our studies allow choosing the best mode and the complex structure – a source of oxygen anion radical, depending on the objectives and goals of the experiment. The received data can have significant value in studying the role of reactive oxygen species in biochemical processes.

Keywords: radical-chain oxidation; inhibitor; superoxide anion.