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Документ Synthesis and biological activity of Аcinetobacter calcoaceticus IMV B-7241 surfactants depending on monovalent cations content in cultivation medium(2021) Pirog, Tatiana; Lutsay, Dariya; Shevchuk, Tetiana; Iutynska, GalynaMicrobial surfactants (biosurfactants) are multifunctional preparations due to a combination of physicochemical (reduction of surface and interfacial tension, emulsifying activity) and biological (antimicrobial and antiadhesive activity, the ability to destroy biofilms) properties. However, the disadvantage of biosurfactants synthesized as a complex of compounds is the possibility of changing the biological activity depending on the conditions of producer cultivation. Aim. To study the effect of potassium and sodium cations on the NADP+-dependent glutamate dehydrogenase activity of cell-free extract of Acinetobacter calcoaceticus ІМV B-7241 with subsequent appropriate modification of the nutrient medium composition and determination of antimicrobial and anti-adhesive activity of surfactant synthesized. Methods. A. calcoaceticus ІМV B-7241 strain was grown in media containing 2% of sunflower oil waste as a carbon source, as well as various concentrations of potassium and sodium chloride (basal – 1.0 g/l NaCl, medium # 1 that did not contains NaCl, medium # 2 in which the concentration of NaCl was 2.0 g/l, medium # 3 in which the concentration of NaCl and KCl was 1.0 g/l each). The surfactants were extracted from the supernatant liquid culture with a modified Folch mixture. Anti-adhesive activity and the degree of biofilms degradation were determined by spectrophotometric method, antimicrobial activity − by the indicator of the minimum inhibitory concentration (MIC). Activity of enzymes of surface-active aminolipids biosynthesis (NADP+-dependent glutamate dehydrogenase) and glycolipids (phosphoenolpyruvate (PEP) carboxylase, PEP-synthetase, PEP-carboxykinase, trehalose phosphate synthase) were analyzed in cell-free extracts obtained after the destruction of cells by ultrasound. Results. It was found that potassium and sodium cations in concentrations of 50 and 100 mM are inhibitors of NADP+-dependent glutamate dehydrogenase, and in lower concentrations (5–20 mM) – activators of this enzyme, as well as PEP-carboxykinase and PEP-synthetase. The increase in the biosurfactant concentration to 6.1−7.7 g/l during cultivation of A. calcoaceticus ІМV B-7241 in medium # 1 and # 3 was due to the predominant synthesis of glycolipids under such conditions, which was evidenced by the increase in 1.8−6.5 times in the activity of PEP-carboxylase, PEP-carboxykinase, PEP-synthetase and trehalose phosphate synthetase compared to the indicators on the basal medium. The concentration of surfactants synthesized in the basal medium was 3.6 g/l, but such surfactants were characterized by the highest antimicrobial and anti-adhesive activity. Their MIC against the test-cultures of studied bacteria (Pseudomonas sp. MI-2, Bacillus subtilis BT-2, Escherichia coli IEM-1, Staphylococcus aureus BMS-1, Enterobacter cloaceae C-8) and fungi (Candida albicans D-6, Rhizopus nigricans P1, Aspergillus niger P-3, Fusarium culmorum T-7) were 0.88−56 μg/ml and were by 2−3 orders of magnitude lower compared to established for surfactants synthesized in modified media # 1–3. In the case of treatment of abiotic materials with surfactant solutions obtained on the basal medium, the adhesion of bacteria and fungi was on average 10–20 % lower than after surface treatment by the surfactant synthesized in modified media. In the presence of 148−296 μg/ml of surfactants obtained in the basal medium, destruction of S. aureus BMS-1 and B. subtilis BT-2 biofilms was 45−66 %, and C. albicans D-6 yeast – 39−44 %. Under the action of similar concentrations of surfactants synthesized in modified media, the destruction of bacterial and yeast biofilms was lower: 6-52 and 20–46 %, respectively. Conclusions. The obtained results are consistent with the data of our previous studies on the possibility of regulating the antimicrobial and antiadhesive activity of surfactants in the process of producer cultivation by changing the content of cations in the medium, which are inhibitors/activators of enzymes responsible for the synthesis of components of the surfactants complex, which have certain biological properties.Документ Regulation of biological activity of surfactants under cultivation of Acinetobacter calcoaceticus IMB B-7241 on glycerol(2021) Pirog, Tatiana; Lutsay, Dariya; Yarova, HannaIntroduction. The aim of this research was to study the biological activity of surfactants synthesized by Acinetobacter calcoaceticus IMV B-7241 in the medium with glycerol of various degrees of purification and high content of calcium cations (activators of NADH+-dependent glutamate dehydrogenase, which is a key enzyme of biosynthesis of surface-active aminolipids responsible for antimicrobial activity of surfactant complex). Materials and methods. Cultivation of A. calcoaceticus was carried out in liquid mineral medium using as substrates refined glycerol and the waste from biodiesel production. The base medium did not contain calcium chloride, the content of CaCl2 in the modified medium was 0.1 and 0.2 g/l. Surfactants were extracted from the supernatant of the culture luquid with a modified mixture of Folch. The number of adhered cells and the degree of biofilm destruction in the presence of surfactants was determined by spectrophotometric method, antimicrobial activity of surfactants - by the minimum inhibitory concentration (MIC). Results and discussion. It was found that the additional introduction of 0.1−0.2 g/l of CaCl2 into cultivation medium with refined glycerol was accompanied by the synthesis of surfactants, the MIC of which against bacteria (Bacillus subtilis BТ-2, Enterobacter cloacae C-8, Staphylococcus aureus BМS-1) and yeast (Candida albicans D-6) were 1.01–21.3 μg/ml and were 1.4–29 times lower compared to the MIC of surfactants obtained in base medium (1.83−58.8 μg/ml). The adhesion of test cultures on abiotic materials treated with such surfactants was 8–13% lower, and the degree of biofilms destruction was 5–19% higher compared to the values, established for surfactants obtained on base medium. The increasіng antimicrobial and anti-adhesive activity of surfactants synthesized on waste of biodiesel production was observed only when CaCl2 was introduced into the medium at a concentration of 0.2 g/l. Surfactants synthesized in the presence of calcium cations in the medium with the waste of biodiesel production proved to be more effective destructors of bacterial biofilms in comparison with those obtained in the base medium only at low concentrations (0.7–5.5 μg/ml). Conclusion. The results demonstrate the possibility of regulating the biological activity of A. calcoaceticus IMV B-7241 surfactants by changing in the composition of medium content of calcium cations - activators of NADH+-dependent glutamate dehydrogenase (key enzyme of surface-active aminolipids biosynthesis). Surfactants synthesized under different cultivation conditions of A. calcoaceticus IMB B-7241 on glycerol are more effective biofilm destructors and antimicrobial and anti-adhesive agents compared to the known lipopeptides and rhamnolipids formed on glycerol.Документ Calcium and magnesium cations influence on antimicrobial and antiadhesive activity of Acinetobacter сalcoaceticus ІMV B-7241 surfactants(2016) Pirog, Tatiana; Sidor, Inga; Lutsay, DariyaThe aim of the work was to study the effect of calcium and magnesium cations on NADP+-dependent glutamate dehydrogenase activity (key enzyme of biosynthesis of Acinetobacter calcoaceticus ІMV B-7241 surface-active aminolipids) followed by modification of medium composition and determining antimicrobial and antiadhesive activity of synthesized surfactants. The strain IMV B-7241 was grown in medium with ethanol. NADP+-dependent glutamate dehydrogenase activity of the cell-free extract was analyzed using the formation of glutamate in the oxidation of NADPH. Surfactants were extracted from supernatant of cultural liquid by mixture of chloroform and methanol (2:1). Antimicrobial against bacteria properties of the surfactants were determined by index of the minimal inhibitory concentration. The number of attached cells and the degree of biofilm destruction were analyzed spectrophotometrically. It was established that in the presence of 10 mM Cа2+ and Mg2+ NADP+-dependent glutamate dehydrogenase activity in the cell-free extract increased to 1.5 times in comparison with that without cations. Increasing concentration of magnesium sulfate to 0.2 g/l, or adding CaCl2 (0.1 g/l) into cultivation medium of IMV B-7241 strain was accompanied by rise of NADP+-dependent glutamate dehydrogenase activity in 2.4 and 3.0 times respectively, as well as increasing antimicrobial and antiadhesive activity of synthesized surfactants. Minimal inhibitory concentration of surfactants synthesized in modified media against some bacteria was in 1.3–3.5 times, adhesion on abiotic surfaces treated with such surfactants in an average of 5–17% lower, and the degree of biofilm destruction in 7–13% higher as compared to indicators for the surfactant produced in the base medium. The obtained results indicate the possibility of regulating antimicrobial and anti-adhesive activity of surfactants under producer cultivation.