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Документ Influence of biological inductors on the synthesis and biological activity of microbial metabolites(2023) Pirog, Tatiana; Ivanov, MykytaThe increasing antibiotic resistance is a severe concern for humanity. Co-cultivation of microorganisms is a promising method for obtaining new secondary antimicrobial metabolites. An effective strategy for co-cultivation of microorganisms involves the usage of certain biological inductors. The aim of this review is to summarize existing scientific research in the literature related to the influence of physiologically different types of biological inductors on the synthesis and biological activity of microbial secondary metabolites. An analysis of the literature has shown that in such studies, either live or inactivated cells of the inductor are added to the culture medium at significantly lower concentrations compared to the producer cells of the final metabolites, or the supernatant (filtrate) after cultivation of a competitive microorganism is used as an inductor. According to the literature and our own experimental studies, the using inductors is an effective approach not only for intensifying the synthesis of bacteriocins, surfactants, and antibiotics, but also for increasing their biological activity. Additionally, it often leads to the production of novel antimicrobial compounds that are not typical for the producer. However, the mechanisms of effect of inductors on the synthesis of biologically active secondary metabolites require further research, as the literature suggests that their introduction into the cultivation medium of producer does not always lead to an intensification of the synthesis of the final product. Moreover, the biological activity of secondary metabolites depends on the cultivation conditions of the producer, including the presence of biological inductors in the culture medium. Therefore, it is essential to conduct further research on the interaction between producers and competitive microorganisms to regulate the biological activity of the synthesised metabolites. In addition, there is a necessity to search for more cost-effective substrates for the biosynthesis of secondary metabolites, optimize the composition of the culture medium and expand the range of both pro- and eukaryotic inductorsДокумент Biological activity of Acinetobacter calcoaceticus IMV B-7241 surfactants synthesized in the presence of competitive bacteria Bacillus subtilis BT-2(2023) Pirog, Tatiana; Ivanov, Mykyta; Shevchuk, TetianaCurrently, the effectiveness of technologies of microbial surfactant, which are characterized by a complex of practically valuable physicochemical and biological properties, is lower than that of synthetic analogues. To reduce the cost of these products of microbial synthesis, industrial waste is used as substrates for their biosynthesis. In previous studies, it was established that surfactants synthesized by Acinetobacter calcoaceticus IMV B-7241 on crude glycerol had lower antimicrobial activity compared to those obtained on purified glycerol. The main approaches to the regulation of the biological activity of microbial surfactants are their post-fermentation chemical modification, as well as the improvement of producer strains by methods of metabolic and genetic engineering. In recent years, the great amount of studies have appeared on the co-cultivation of producers of antimicrobial compounds with competitive microorganisms (biological inductors), in response to the presence of which the antimicrobial activity of the final product increases. Aim. To study the effect of live and inactivated cells of Bacillus subtilis BT-2, as well as the corresponding supernatant, on the antimicrobial, anti-adhesive activity and the ability to destroy biofilms of A. calcoaceticus IМV B-7241 surfactants, synthesized in a medium with glycerol of different degrees of purification. Methods. The IMV B-7241 strain was grown in the liquid mineral medium with purified and crude glycerol, into which live and inactivated B. subtilis BT-2 cells, as well as the supernatant after growing the BT-2 strain (2.5−10%, v/v) were added. Surfactants were extracted from the supernatant of the culture liquid with Folch's mixture. Anti-adhesive activity and the degree of destruction of biofilms were determined by the spectrophotometric method, antimicrobial activity − by the indicator of the minimum inhibitory concentration. The activity of enzymes of surface-active aminolipids biosynthesis (NADP+-dependent glutamate dehydrogenase) and glycolipids (phosphoenolpyruvate (PEP)-carboxylase, PEP-synthetase, PEP-carboxykinase, trehalose-phosphate synthase) was analyzed in cell-free extracts obtained after сells sonication. Results. It was established that the introduction of inactivated B. subtilis BT-2 cells and supernatant into the medium with both substrates did not affect the indicators of the surfactants synthesis, while in the presence of live cells of the BT-2 strain in the medium with purified glycerol, a decrease in the concentration of the final product by 1.5 times, and in the culture medium with crude glycerol - an increase of 1.4 times were observed compared to the indicators without the inductor. The study of the antimicrobial activity of surfactants showed that the most effective of the used inductors (live, inactivated cells, supernatant) were live cells of B. subtilis BT-2. The introduction of BT-2 strain live cells into the culture medium with both substrates was accompanied by the formation of surfactants, the minimum inhibitory concentrations of which in relation to bacterial (Bacillus subtilis BT-2, Staphylococcus aureus BMS-1, Proteus vulgaris PA-12, Enterobacter cloacae С-8 ) and yeast (Candida albicans D-6, Candida tropicalis PE-2) test-cultures were 3-23 times lower than established for those synthesized on the medium without this inductor. Anti-adhesive activity of surfactants obtained on purified and crude glycerol in the presence of all types of inductors was higher than those synthesized in the culture medium without inductors (cells adhesion of bacterial and yeast test-cultures on polyvinyl chloride was 13−70 and 33−96%, respectively). Introduction into A. calcoaceticus IMV B-7241 medium cultivation of both live and inactivated B. subtilis BT-2 cells, as well as the supernatant, was accompanied by the synthesis of surfactants in the presence of which the disruption of bacterial biofilms was on average 10-20 % higher compared to using surfactants synthesized without an inductor. In the presence of B. subtilis BT-2 in the medium, in the cells of the IMV B-7241 strain the activity of NADP+-dependent glutamate dehydrogenase (a key enzyme of aminolipids biosynthesis) increased by 1.5-2 times, while the activity of glycolipids enzymes biosynthesis remained practically at the same level as without an inductor. Such data indicated that the higher biological activity of surfactants obtained by A. calcoaceticus IMV B-7241 in the presence of biological inductors might be due to an increase in the content of aminolipids in their composition. Conclusions. As a result of research, it was established the possibility of regulating the antimicrobial and anti-adhesive activity, as well as the ability to disrupt biofilms of A. calcoaceticus IМV B-7241 surfactants by introducing into the culture medium of competitive bacteria B. subtilis BT-2. It is important that under such cultivation conditions the antimicrobial activity of surfactants synthesized on toxic crude glycerol significantly increased.Документ Destruction of biofilms under the influence of Acinetobacter calcoaceticus IMV B-7241 surfactants, synthesized in the presence of competitive microorganisms(2022) Pirog, Tatiana; Ivanov, MykytaIntroduction. The aim of this study was to investigate the role of surfactants synthesized by Acinetobacter calcoaceticus IMV B-7241 in media with glycerol in the presence of biological inductors in destruction of biofilms. Materials and methods. Cultivation of A. calcoaceticus IMV B-7241 was carried out in a mineral medium using refined glycerol or crude glycerol, the waste of biodiesel production, as carbon sources. Biological inductors were introduced as live or inactivated cells of Bacillus subtilis BT-2, as well as the supernatant after strain BT-2 cultivation. Surfactants were extracted from the supernatant of the culture liquid with a modified mixture of Folch (chloroform and methanol, 2:1). The degree of biofilm destruction in the presence of surfactants was determined by spectrophotometric method. Results and discussion. Regardless of the substrate used, the introduction of both live and inactivated cells of B. subtilis BT-2 into medium used for cultivation of A. calcoaceticus IMV B-7241 was accompanied by the synthesis of surfactants, the degree of biofilm destruction of which was higher than those obtained in the medium without an inductor. The degree of destruction of bacterial and yeast biofilms achieved by the action of A. calcoaceticus IMV B-7241 surfactants obtained on refined glycerol in the presence of inductor cells was 36.5–85% and was 1.5-3 times higher compared to using surfactants synthesized in medium without inductors. Note that, surfactants synthesized in the presence of biological inductors destroyed biofilms of the test cultures at fairly low (7.5–960 μg/ml) concentrations. Similar results were observed for the usage of surfactants obtained on the waste of biodiesel production. Therefore, introduction of live cells of B. subtilis BT-2 into the medium with the crude glycerol was accompanied by synthesis of surfactants, which at concentration 1.8-960 μg/ml caused destruction of B. subtilis BT-2, Proteus vulgaris PA-12 and Enterobacter cloacae C-8 biofilms at 30.1–80.7% and was higher than using similar surfactant concentrations obtained during cultivation without inductors (24.1–75%). The destruction of biofilms of Staphylococcus aureus BMS-1, Candida albicans D-6 and Candida tropicalis PE-2 under the action of surfactants (1.8-960 μg/ml) synthesized on crude glycerol in the presence of both live or inactivated cells of B. subtilis BT-2 was 1.5–8 times higher than surfactants synthesized in medium without inductor. Conclusion. The possibility to regulate the ability to destroy bacterial and yeast biofilms of surfactants synthesized by A. calcoaceticus IMV B-7241 by introducing into the medium competitive bacteria B. subtilis BT-2 was found.Документ Microbial co-cultivation: discovery of novel secondary metabolites with different biological activities(2023) Pirog, Tatiana; Ivanov, MykytaIn recent decades overuse and misuse of antibiotics as well as social and economic factors have accelerated the spread of antibiotic-resistant bacteria, making them a major problem for humanity. One of the most effective approaches to the discovery of new secondary antimicrobial metabolites is co-cultivation of microorganisms, in which the producer of the target products is grown together with competitive microorganisms (inductors), in response to the presence of which silent biosynthetic genes of the producer strain are activated and an increase in the biological activity of the synthesized secondary metabolites and/or even the synthesis of new metabolites is observed. The review summarizes the current literature data on the co-cultivation of antimicrobial substances producers with competitive microorganisms, which results in the synthesis of new metabolites with antimicrobial and cytotoxic activity, not typical for monocultures. During the co-cultivation of fungi, bacteria, and fungi with bacteria, the synthesis of new antimicrobial and anticancer metabolites, which are classified as alkaloids, phenylpropanoids, macrolides, polyketides, cyclopeptides, terpenoids, anthraquinones, and steroids, is observed. These data indicate that the mixed fermentation of microorganisms is a simple, cheap, and quite effective way to obtain new metabolites that are promising for use in medicine. В останні десятиліття надмірне та неправильне використання антибіотиків, а також соціальні та економічні чинники прискорили поширення стійких до антибіотиків бактерій, зробивши їх основною проблемою для людства. Одним із найефективніших підходів до відкриття нових вторинних антимікробних метаболітів є кокультивування мікроорганізмів, при якому продуцент цільових продуктів вирощують разом із конкурентними мікроорганізмами (індукторами), у відповідь на присутність яких мовчать біосинтетичні гени штам-продуцент активується і спостерігається підвищення біологічної активності синтезованих вторинних метаболітів і/або навіть синтез нових метаболітів. В огляді узагальнено сучасні літературні дані щодо спільного культивування продуцентів антимікробних речовин із конкурентними мікроорганізмами, що призводить до синтезу нових метаболітів з антимікробною та цитотоксичною активністю, нехарактерною для монокультур. При спільному культивуванні грибів, бактерій і грибів з бактеріями спостерігається синтез нових антимікробних і протипухлинних метаболітів, які класифікуються як алкалоїди, фенілпропаноїди, макроліди, полікетиди, циклопептиди, терпеноїди, антрахінони, стероїди. Ці дані свідчать про те, що змішана ферментація мікроорганізмів є простим, дешевим і досить ефективним способом отримання нових метаболітів, перспективних для використання в медицині.