Biotechnologia Acta

23-36

Authors: Pirhanov G.G., Zhernossekov D.D.

Sinorhizobium meliloti is a Gram-negative soil nitrogen-fixing bacterium that increases the yield of legumes. There is information in the literature about the complete genome sequence of this bacterium, in addition, the polysaccharide composition of the biofilm, which is actively involved in nitrogen fixation, has been studied. The well-known nucleotide sequence, as well as the genetic and biochemical features of S. meliloti make this organism an ideal model for biotechnological research. The purpose of this work was to analyze the current data provided in the literature on the symbiotic interaction of Sinorhizobium meliloti with the host plant, and to characterize the main directions of the use of this bacterium in agriculture, bioremediation and medicine.

44-52

Authors: O. K. Gulevskyy, Yu. S. Akhatova

One of the modern transfusiology tasks is to preserve the properties of erythrocytes after hypothermic storage. Worsening of their functional state due to the storage leads to deterioration of the transfusion medium quality and a number of clinical problems. Plausible protective effects of a human cord blood low-molecular fraction (CBF) and the Actovegin drug were evaluated in the present study in order to use them as components of recovery medium. Aim. The purpose of our study was to investigate the CBF and the Actovegin drug effect on erythrocytes morphology, energy balance, and oxygen transport function of erythrocytes after their hypothermic storage. Materials and methods. In the research erythrocyte mass from human donor blood was used, which was stored in hypothermia for 7–21 days. Every 7 days, the CBF or Actovegin (final concentration 0.6 mg/ml) were added to aliquots of erythrocyte mass and incubated for 1 h at 37 °C, followed by further studies. CBF was obtained from human cord blood by ultrafiltration and lyophilization. The erythrocytes morphology was assessed using light microscopy. The content of ATP and 2.3-DPG was determined by the amount of inorganic phosphorus and the photoelectrocalorimetric method. The indicators of erythrocytes oxygen transport function (saturation, oxygen and carbon dioxide tension) were assessed using an analyzer of blood gases and electrolytes IL GEM Premier - 3000. The ratio of hemoglobin forms was studied by photometry. Results. It was shown that both CBF and the Actovegin drug promoted to restore the morpho-functional characteristics of erythrocytes after 21 days of their storage at 24 °C. This was indicative as an increase in the normocytes number increase and restoration of oxygen tension, saturation, ATP and 2,3-DFG content, and normalization of the hemoglobin forms ratio. It was suggested that the mechanisms of the compared tested substances action were associated with ATP synthesis activation and 2,3-DFG formation. Conclusions. The use of media with containing CBF or the Actovegin drug enabled to restore effectively the properties of erythrocytes disturbed during their prolonged storage.

60-70

Authors: N. Kobylinska, T. Bohdanovych, V. Duplij, I. Pashchenko, N. Matvieieva

Aim. The profiles of polyphenolic phytochemicals in extracts of “hairy” roots of Artemisia tilesii Ledeb. and Artemisia annua L. were studied. Analytical separation and quantification of main components in extracts were evaluated. Methods. “hairy” roots were grown in vitro on Murashige and Skoog medium. High-performance chromatography coupled with different types of detection (photo diode array detection (DAD) and electrospray ionization with ultra-high resolution Qq-Time-of-Flight mass spectrometry) was used to identify and quantify the main biologically active components in ethanol extracts of “hairy” roots. Results. The amount of flavonoids was 94.71–144.33 mg RE/g DW and 33.52–78.00 mg RE/g DW in “hairy” roots of A. annua and A. tilesii, respectively. In most samples of “hairy” roots, the amount of flavonoids was higher than the content in the control plant roots. The presence of Apigenin (0.168 ± 0.003 mg/L and 0.178 ± 0.006 mg/L), Quercetin (0.282 ± 0.005 mg/L and 0.174 ± 0.005 mg/L) in the extracts of A. annua and A. tilesii was shown by reverse-phase HPLC-DAD method. Chlorogenic acid, Kaempferol, and other flavonoids were detected. Conclusions. The developed HPLC-DAD method demonstrated the high percentage of recovery, low limit of detection and quantification (9,11 ng/ml ≤ LOQ ≤16,51 ng/ml), accuracy and correctness. Thus, the method is suitable for the simultaneous quantification of phenolic acids and flavonoids in various plant extracts with short time and high efficiency.

21-37

Authors: Kanyuk M. I.

The use of multifunctional nanosystems in medicine and research is of contemporary interest. Aim. The purpose of the work was to summarize publications on the prospects of creating and using nanocontainers based on curcumin (Cur). Cur fluorescence in nanoparticles (NP) makes it possible to investigate the distribution of fluorescent and non-fluorescent components, significantly accelerating the study and implementation of drugs in practice. Particular attention is paid to the use of hydrophobic substances in NP, to penetrate into a living cell. Understanding the interaction of NP with living cells is extremely important when these particles are used to transport and deliver water-insoluble drugs to cells. Cur is one of the drugs with various and very promising pharmaceutical effects, it is poorly soluble in aqueous media, and the use of nanocarriers is an effective way to significantly increase its bioavailability. Cur has its own fluorescence, which enables to use it in multifunctional fluorescent nanosystems, for example, with Pluronic® micelles. The use of the fluorescence method makes it possible to trace the stages of interaction of Cur-loaded NP with cultured cells and their localization in cell organelles. With this approach, nanoscale dynamics of drug distribution and stability is observed over time. Conclusions. The main conclusion is that for unstable in the aquatic environment drugs such as Cur, it is necessary to use the most hydrophobic nanostructures without traces of water, which include the nuclei of Pluronic® micelles. This method makes it possible to use other poorly water-soluble drugs. A promising area of nanomedicine is the creation of complex bio-compatible nanomaterials based on several active drugs that reduce the toxicity of preparations to normal cells.

49-55

Authors: Tigunova O. O., Umansikiy M. O., Bratishko V. V., Balabak A. V., Shulga S. M.

Aim. The purpose of the study was to investigate the effect of ultrasonic disintegration on the lignocellulosic raw materials (biomass of the non-cereal part of rape) with its subsequent use as a substrate for the production of biobutanol. Methods. Butanol-producing strains and the biomass of the non-cereal part of rape Brassica napus were used in the present study. Ultrasonic disintegration of lignocellulosic raw materials was performed on the specially designed equipment. Results. The effect of ultrasonic disintegration on lignocellulosic raw materials was investigated for further application in biofuel production based on microbiological conversion. The possibility of using the obtained components after the pre-treatment of lignocellulose by ultrasonic disintegration as a substrate for the microbiological synthesis of butanol was shown. The highest accumulation of butanol (2.4 g/l) was obtained with the use of 5% dry matter content in the medium, 5 min treatment and the specific power of ultrasonic disintegration of 0.72 W/ml. Conclusions. The possibility of producer strains of the genus Clostridium to use cellulose in the fermentation process has been shown. When using ultrasonic disintegration for pretreatment of the noncereal part of the biomass of rape, the accumulation of butanol increased by 3 folds.

63-73

Authors: Korniienko I. M., Yastremska L. S, Polonchuk L. Y., Baranovsky M. M.

Lactic acid bacteria play a key role in human microecology and biotechnology — form organoleptic characteristics of products, increase the nutritional, including biological value of functional foods. Natural resistance to antibiotics is one of the important factors that determine the probiotic properties of lacto- and bifidobacteria. Aim. Study of the antibiotic resistance of functionally active probiotic cultures of «VIVO probioyogurt» leaven to determine the possibility of using a fermented milk product, which is prepared on its basis, during antibiotic therapy to maintain and restore normal intestinal microflora. Methods. Pure cultures of lactic acid bacteria (LAB) were selected for the study: (Lactobacillus delbrueckii ssp., L. acidophilus, L.casei, L. rhamnosus, L.paracasei, Streptococcus thermophilus, Bifidobacterium lactis (2 strains), B. infantis), which are part of leaven „VIVO probioyogurt“ the quality of which is confirmed by certificates of the International Organization for Standardization ISO 9001: 2008, as well as ISO 22000: 2005. The method of the experiment consisted of the following stages: preparation of nutrient media („Lactobacagar“, „Bifidoagar“, glucose-peptone medium), working solutions of antibiotics; working suspension of LAB; suspensions of cultures (lacto- and bifidobacteria), cultivation LAB on elective nutrient media with the addition of antibiotics and evaluation of research results. Determination of antibiotic resistance of LAB was performed by the method of double dilutions. Results. The use of this technique enabled to establish the minimum inhibitory concentration (MIC) of antibiotics of different groups relative to the LAB. The results of the research were processed using a licensed computer program Microsoft Excel. Conclusions. Evaluation of the results of studies to determine the MIC of antibiotics — benzylpenicillin, azithromycin, lincomycin, gentamicin sulfate, ceftriaxone, norfloxacin, amoxil, streptomycin, tetracycline, erythromycin in relation to IBD; fermented milk product, which was prepared on the basis of this starter culture, it was advisable to use during antibiotic therapy to restore and maintain normal intestinal microflora.

28-37

Authors: Krasnopolsky Yu. М., Pylypenko D. М.

Rabies is a neurological disease of a viral nature, leading to death. Rabies virus is an RNA virus that invades the central nervous system, leading to neuronal dysfunction. Timely vaccination can prevent the diseases development. Aim. The article is devoted to immunobiotechnological research aimed at creating antirabic vaccines. Results. The history of the antirabic vaccines creation from the first inactivated vaccines obtained from nervous tissue to the cultivation of the virus on animal cell cultures is considered. The article presents commercially available anti-rabies vaccines: their composition, the used rabies virus strains, cell cultures, the methods of inactivation and purification. The technology of producing an anti-rabies vaccine based on a Pitman Moore virus strain and a chicken fibroblast cell culture is presented. The advantages of different vaccine types are considered: live attenuated, peptide, liposomal, RNA vaccines, vaccines based on viral vectors, transgenic plants and reverse genetics methods. Conclusions. The development of biotechnology, immunology and virology makes it possible to improve constantly vaccine preparations, including those against rabies, increasing their effectiveness and safety.

53-63

Authors: O. M. Yaroshko, M. V. Kuchuk

Local cultivars of A. caudatus: Helios and Karmin were used as plant material. Amaranth is a new pseudocereal introduced in Ukraine. The plant biomass of amaranth is used in medicine, food industry and cosmetology industry. Aim. The purpose of the work was to identify the optimal conditions for the transient expression of reporter genes in Amaranthus caudatus cultivars. Methods. Biochemical and microscopy methods were used in the following work. Seedlings and adult plants of different age were infiltrated with agrobacterial suspensions separately (genetic vector pCBV19 with a uidA gene and genetic vector pNMD2501 with a gfp gene in Agrobacterium tumefaciens GV3101 strain). Results. Transient expression of the uidA and gfp genes was obtained in amaranth plants after conduction series of experiments. The most intensive transient expression of gfp and uidA genes was observed in seedlings infiltrated at the age of 1 day. The maximum fluorescence of the GFP protein was observed on 5th–6th days. Conclusions. It was shown that the cultivar Helios was more susceptible to agrobacterial infection than the cultivar Karmin. The effectiveness of Agrobacterium mediated transformation was from 16% to 95% for the Helios cultivar and from 12% to 93% for the Karmin cultivar. The obtained results indicate that the studied amaranth cultivars can potentially be used for obtaining transient expression of target genes and synthesizing target proteins in their tissues in the future.

70-79

Authors: V. M. Hovorukha, O. A. Havryliuk, I. O. Bida, Ya. P. Danko, O. V. Shabliy, G. V. Gladka, L. S. Yastremska, O. B. Tashyrev

The accumulation of solid and liquid organic waste requires their treatment to develop energy biotechnologies and prevent environment pollution. Aim. The goal of the work was to study the efficiency of the purification of the filtrate from dissolved organic compounds by aerobic oxidation and methane fermentation. Methods. The standard methods were used to determine рН and redox potential (Eh), the gas composition, the content of short-chain fatty acids, the concentration of dissolved organic compounds counting to the total сarbon. The efficiency of two types of microbial metabolism for the degradation of soluble organic compounds of filtrate was compared. Results. The aerobic oxidation was established to provide 1.9 times more efficient removal of dissolved organic compounds, compared with the anaerobic methane fermentation. However, it provided CH4 yield 1 L/dm3 of filtrate (сarbon concentration — 1071 mg/L). The necessity to optimize the methods for purifying filtrate to increase the efficiency of the process was determined. Conclusions. The obtained results will be the basis to develop complex biotechnology providing not only the production of environmentally friendly energy H2 via the fermentation of solid food waste, but also the purification of filtrate to solve the ecological and energy (CH4 production) problem of society.

22-29

Authors: Golub N.B., Shi Xinhua, Levtun I.I.

With the development of antibiotics application, their spread in the natural environment increases dramatically. The presence of antibiotics in the environment changes microorganism and other living beings ratio and composition, which causes a negative impact on biochemical processes that take place in the environment. The spread of antibiotic resistance genes in environmental microorganisms is a growing problem of environmental safety and human health. Aim. The objective of the work was to analyze the adaptation mechanisms of microorganisms to the influence of antibiotics and methods for antibiotics utilization. Results. The mechanisms of microorganisms’ adaptation to antibiotics are shown. The conditions for utilization of different antibiotics classes by microorganisms are provided. Conclusions. Methods of antibiotics destruction depend on its structure and physicochemical properties. Physico-chemical methods are used for local waste purification and are not suitable for antibiotics disposal in the natural environment. The decomposition products can also have a negative effect on the microorganisms’ cells. Depending on the class of antibiotics, their biodegradation occurs by different types of microorganisms. It has been shown that sulfonamides and amphinecoles are easily destroyed by many heterotrophic bacteria; biodegradation of aminoglycosides occurs by a strain of Pseudomonas spp.; tetracyclines - mushrooms; β-lactams - through the microorganisms’ association including: Burkholderiales, Pseudomonadales, Enterobacteriales, Actinomycetales, Rhizobiales, Sphingobacteriales. A consortium of destructors must be created to dispose of a specific classes of antibiotics.

39-45

Authors: L.A. Koldar, L.L. Dzhus, М.V. Nebykov

Development of effective technology of rare and endangered plant species mass reproduction in vitro is one of the urgent nowadays tasks. Diantus hypanicus Andrz. of the Caryophyllaceae Juss. family belongs to this group of plants. It is an endemic, narrow-localized species listed in the Red Book of Ukraine, the European Red List and the Annex to the Berne Convention. Prospects for the conservation of this species are associated with the study of its viability and require the development of effective reproduction methods. Aim. The purpose of the experiment was to determine the effect of different growth regulators concentrations on the D. hypanicus explants regenerative capacity during in vitro propagation. Methods. For the experiment the seed were used collected from plants of natural habitats and sown on a hormone-free nutrient medium according to the recipe of Murashige and Skoog. For 12–15 days, 4–6 cm long seedlings were obtained which were transferred to nutrient media modified with the content of growth-regulating substances: BAP 0.5–2.0 mg / l, IBA 0.01 mg / l and IAA 0.1 mg / l. Results. Initiation of adventitious buds with subsequent shoot formation was observed for 12–16 days. Each variant plants differed in number, growth activity and intensity, morphometric parameters. After 35–40 days from the explant introduction, conglomerates with well-developed leafy shoots were formed. It was found that, when modifying nutrient media with growth regulators BAP, IBA, IAA of different concentrations, D. hypanicus explants were actively undergoing regeneration processes of different intensity. Conclusions. It was proved that high ability to regenerate was the characteristic of explants cultured on a nutrient medium modified by the addition of BAP – 0.5 mg / l and IAA – 0.1 mg / l, where, on average, 18 ± 0.24 shoots of 2.42 ± 0.17 cm long were formed with a net reproduction of 19.06 ± 0.14. Shoots with 3.0–5.0 pairs of leaves that reached 3.5–6.0 cm were selected and transferred to nutrient media for rooting. Less developed shoots were planted on nutrient media for further reproduction. The next stage of the study is aimed at modifying nutrient media to achieve explants rhizogenesis and obtain a large number of plants necessary for the preservation of this endemic narrow-localized species in culture with subsequent repatriation to natural places of growth.

54-61

Authors: D. D. Gaiya, L. L. Ashafa, M. E. Entonu , S. K. Udu

Aim. Search of healthy and edible alternative oils from algae. Such oil provides many health benefits mainly because of docosahexaenoic acid (DHA) form of omega-3 fattyacids and some other micro nutrients in smaller amounts. Methods. Soxhlet extraction method was used to extract the oil with n-hexane as the solvent. The proximate composition was determined by AOAC methods, while the mineral contents were determined by AAS. FTIR and UV-Visible spectra of the oil were run using Agilent- FTIR Spectrometer and UV-Visible Spectrophotometer respectively. Results. The oil yield was very low (1.05%). The proximate composition reveals carbohydrate as the major nutrient in the residue (79.18%), others include lipid (8.03%), crude protein (5.00%), moisture (2.78%), crude fibre (3.01%) and ash (2.00%). The mineral composition reveals high amount of potassium (1602.5 mg/100g) and calcium (632.5 mg/100g) with low levels of phosphorous (14.9 mg/100g) and sodium (12.8 mg/100g). The FTIR spectrum of algae oil is similar to the normal vegetable oil. Stretching vibrations at 2922.2 cm-1 and 2855 cm-1 are attributed to methylene (-CH2--) and methyl (-CH3) groups while absorption bands at 1710 cm-1 and 1744 cm-1 showed carboxylic groups for algae oil and vegetable oil (control) which was attributed to C=O stretching vibrations (esters). The UV-Visible spectrum of algae oil showed two peaks at 408 nm and 660 nm for carotenoids and chlorophyll A respectively, which corroborate with previous studies. Conclusions. We conclude that the oil and cake characterized from Spirogyra porticallis has great potential for medicinal and nutritional usage.

19-27

Authors: P. R. Zubik, V. V. Motronenko, O.B. Besarab

In order to save money, some farms use the seed obtained in the process of cultivation not only for sale, but also for sowing, which has not found supporters among companies engaged in the production of genetically modified seed. To protect their rights, the latter have created technologies to limit the use of genetic material, which are intended to be used for protection the intellectual rights to reproduce plants with a changed genotype. However, these technologies contain also a commercial component and violate a number of moral principles and international acts. Aim. To describe the types of terminator technologies, their genetic and molecular basis and purpose. To assess a correspondence of their compliance with the international documents and norms. Method. Terminator technologies types, genetic bases and application and their analysis from the standpoint of international norms were studied. To achieve the goal, the methods of fact analysis, comparison and generalization were used. Results. There are two types of terminator technologies (variety- and trait-specific), which are based on the interaction of three genes, which leads to the implementation of certain phenotypic manifestations. It was found that the technologies for limiting the use of genetic material are both contradictory and consistent with a number of international legal acts, which did not make it possible to determine clearly the appropriateness of their use in agriculture. Conclusions. Terminator technologies application is still a controversial fact since they are based on the duality principle: to carry simultaneously a positive and a negative manifestation for people.

37-46

Authors: О. Yu. Bielikova, S. I. Тarasjuk, A. I. Mruk, Т. A. Nahorniuk, L. P. Buchatskyi

Rainbow trout is a valuable species of aquaculture, which is characterized by a high level of variability. Aim. The goal was to study the peculiarities of genetic variability of rainbow trout based on polymorphism of transferrin (TF), posttransferrin (PTF), esterase (EST) (EC 3.1.1.1.) and albumin (ALB) loci. Methods. Electrophoretic separation of plasma proteins of rainbow trout from three local stocks (Chernivtsi, Kharkiv and Transcarpathian) was carried out in the native polyacrylamide gel. Results. Peculiarities of the distribution and relative electrophoretic mobility of allelic variants of the studied loci of rainbow trout of Ukrainian local stocks were established.The results demonstrated the effectiveness of the analysis using methods of biochemical genetics to establish the characteristics of the genetic structure, the level of heterozygosity of local stocks and to carry out their differentiation. Conclusion. The use of selected markers would allow monitoring the dynamics of changes in the state of local stocks under the established conditions of cultivation in the future.

59-66

Authors: M. O. Twardovska, I. I. Konvalyuk, K. V. Lystvan, I. O. Andreev, V. A. Kunakh

Aim. The aim of the study was to determine the quantitative and qualitative content of phenolic compounds and flavonoids in Deschampsia antarctica E. Desv. tissue cultures obtained from plants originating from different islands of the maritime Antarctic. Methods. In vitro tissue culture, Folin-Ciocalteu method, spectrophotometry, HPLC analysis. Results. The quantitative content of phenolic compounds and flavonoids in D. antarctica tissue cultures obtained from plants of six genotypes (DAR12, DAR13, G/D12-2a, Y66, R30 and L57) was determined. The highest content of phenolic compounds (4.46 and 3.75 mg/g) was found in tissue cultures obtained from root and leaf explants of plant genotype L57. The highest amount of flavonoids (7.17 mg/g) was accumulated in G/D12-2a tissue culture of root origin. The content of the studied biologically active compounds (BACs) did not change with increasing number of subculture generations (from passage 10 to 19). HPLC analysis showed that in D. antarctica tissue cultures, a shift in the biosynthesis of BACs occurred towards the synthesis of more polar metabolites compared to explant donor plants. Conclusions. It was found that the transition of cells to undifferentiated growth affected the content of BACs, the amount of which decreased 2–5 times simultaneously with a significant change in their profile. This provided a basis for further biochemical studies, as well as for careful selection of tissue culture of D. antarctica to use it as a potential source of BACs.