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Документ Industrial wastes as substrates for synthesis of surfactants with antiadhesive activity by Rhodococcus erythropolis IMV Ac-5017(2022) Pirog, Tatiana; Kliuchka, Igor; Kliuchka (Nykytyuk), LiliaMicrobial surfactants can affect the degree of microorganism’s adhesion to different surfaces and lead to biodegradation of the already formed biofilms. They are also non-toxic to the environment. These properties allow to use the microbial surfactants in medicine and food industry.Документ Synergistic effect of surfactants of Nocardia vaccinii IMV B-7405 and essential oils on Candida genus yeast(2022) Pirog, Tatiana; Kliuchka (Nykytyuk), Lilia; Shevchuk, Tetiana; Muchnyk, FainaThe increase in the number of resistant strains of Candida genus representatives, capable of forming biofi lms on various surfaces, stimulates the search for new, alternative methods of combating them, one of which is the use of compounds of natural origin, such as essential oils. However, at the same time, their concentration should be minimal, which is due to the ability of essential oils to cause severe damage of the human’s central nervous system and aspiration pneumonia. This leads to the necessity of searching for new methods to reduce the concentration of essential oils and at the same time to preserve their properties, in particular, by their use in a mixture with other antimicrobial agents, which can be microbial surfactants. Previously, it was found that the degree of yeast biofi lm destruction under the action of Nocardia vaccinii IMV B-7405 surfactants dependson the nature of the growth substrate and is the highest in the presence of preparations synthesized on purified glycerol. Aim. To study the synergism of antifungal activity and the role in the destruction of biofilms of a mixture of Nocardia vaccinii IMV B-7405 surfactants synthesized on glycerol of different quality and essential oils. Methods. N. vaccinii IMV B-7405 was grown in a medium containing purified glycerol or waste from biodiesel production at a concentration of 2% (v/v) as carbon sources. The surfactants were extracted from the supernatant of cultural liquid by a modified Folch mixture. The antimicrobial activity of essential oils, surfactants, and their mixtures was determined by the index of the minimum inhibitory concentration. To assess the synergistic effect of a mixture of surfactants with essential oils, the fractional inhibitory concentration index was used. The degree of biofi lm destruction (%) was determined as the difference between the cell adhesion in untreated and treated with surfactants, essential oil, or their mixture wells of the polystyrene microplates. Results. It was found that the surfactants synthesized by N. vaccinii IMV B-7405 on both purifi ed glycerol and waste from biodiesel production showed synergistic antifungal activity in mixtures with cinnamon and lemongrass essential oils. Thus, the minimum inhibitory concentrations against Candida albicans D-6, Candida utilis BVS-65, and Candida tropicalis RE-2 of a mixture of surfactants synthesized on purified glycerol with cinnamon and lemongrass essential oils were 1.8—7.5 and 3.7— 15 μg/mL, respectively, and were lower than in the case of using surfactants (30-60 μg/mL), cinnamon or lemongrass essential oil (156—312 μg/mL) alone. The use of a mixture of surfactants obtained on waste from biodiesel production and cinnamon or lemongrass essential oils made it possible to reduce the minimum inhibitory concentrations of the latter against studied yeast test cultures by 14—56 times. At the same time, the index of fractional inhibitory concentration did not exceed 0.5, which indicates the synergism of the antifungal activity of the mixture of these compounds. The destruction of Candida yeast biofi lms under the action of surfactants synthesized on both purified glycerol and waste from biodiesel production in a mixture with cinnamon or lemongrass essential oils reached 60—67 and 67—77%, respectively, which is an average of 25—35% higher compared to the use of each monopreparation separately. Conclusions. The results presented in this paper confirm the previously obtained data that N. vaccinii IMV B-7405 surfactants, synthesized on both traditional substrates and toxic industrial wastes, have antimicrobial and antiadhesive synergistic action with essential oils, which allows us to consider them as potential components of the so-called «antifungal locks» in the fight against of Candida genus representatives.Документ Destruction of biofilms on silicone tubes under the action of a mixture of Nocardia vaccinii IMV B-7405 surfactants with other biocides(2021) Pirog, Tatiana; Kliuchka (Nykytyuk), Lilia; Shevchuk, Tetiana; Iutynska, GalynaThe formation of pathogenic microorganisms biofilms on the central venous catheter is the cause of catheter-associated infections. An alternative method of combating biofilms is the use of “antibacterial” and “antifungal” locks, which are solutions of antibiotics or antifungal drugs in a mixture with other natural compounds, which can be microbial surface-active substances (surfactants) or essential oils. Aim. To investigate the role of Nocardia vaccinii IMV B-7405 surfactants mixture with other antimicrobial compounds in the destruction of biofilm on silicone tubes. Methods. N. vaccinii IMV B-7405 was grown in medium containing as carbon source purified glycerol and waste from biodiesel production, refined sunflower oil, oil after frying French-fried potatoes, Potato wedges and meat. The surfactants were extracted from supernatant of cultural liquid by modified Folch mixture. 2 mL of surfactant solutions, antifungal drugs (nystatin, fluconazole) or tea tree essential oil of the same concentration (5–640 μg/mL) were added to test tubes with silicone tubes (with pre-formed biofilm from test culture). To study the synergistic effect of the biofilms destruction, a mixture of surfactant solutions and antifungal substances (or essential oil) of the same concentration in a ratio of 1:1 (1 mL of each solution) was added to the test tubes. Sterile tap water (2 mL) was added to control test tubes instead of surfactants preparations, antifungal substances or essential oil. The degree of biofilm destruction (%) was determined as the difference between the adhesion of cells on the inner side of silicone tubes, untreated and treated with surfactants, antifungal drugs, essential oil, or their mixture. Results. It was found that surfactants synthesized by N. vaccinii IMV B-7405 on all substrates showed synergism of yeast and bacterial biofilms destruction on silicone tubes in a mixture with nystatin, fluconazole and tea essential oil in the whole range of investigated concentrations (5–640 μg/mL), but the highest effect was achieved at a concentration of 20–40 μg/mL. Thus, the degree of Candida albicans D-6, Candida utilis BVS-65 and Candida tropicalis PE-2 biofilms destruction under the action of a mixture of surfactants synthesized on waste from the biodiesel production and waste oil, with antifungal drugs was 45.8–71.8 % and was higher than with only surfactants (21.2–41.6 %), nystatin (22.4–24.1 %) or fluconazole (28.1–31.3 %). The destruction of Candida genus yeast biofilms under the action of surfactants synthesized on oil-containing substrates in a mixture with both nystatin and fluconazole reached 50.1–71.2 %, which is 10–30 % higher compared to the use of surfactants alone or only antifungal agents. The degree of Pseudomonas sp. MI-2, Escherichia coli IEM-1, Staphylococcus aureus BMS-1, Bacillus subtilis BT-2 (spores) biofilms destruction on silicone tubes treated with a mixture of tea tree essential oil and surfactants synthesized on all oil-containing substrates was 10–29 % higher than in the case of using only solutions of surfactants (11.5–45.4 %) or essential oil (21.4–34.5 %) for the tubes treatment. Conclusions. The data obtained make it possible to consider surfactants synthesized by N. vaccinii IMV B-7405 on a wide range of cheap and accessible substrates as promising components of “antibacterial” and “antifungal” locks in combination with essential oils and antifungal agents.Документ Synergism of antimicrobial and anti-adhesive activity of Nocardia vaccinii IMV B-7405 surfactants in a mixture with essential oils(2020) Pirog, Tatiana; Kliuchka (Nykytyuk), Lilia; Kliuchka, Igor; Shevchuk, Tetiana; Iutynska, GalynaAn increase in the antibiotic resistance of pathogenic microorganisms has stimulated the search for alternatives to antibiotics substances of natural origin, which are essential oils (EO) and non-toxic biodegradable microbial surfactants. Aim. To investigate the antimicrobial and anti-adhesive activity of a mixture of EO and surfactants of Nocardia vaccinii IMV B-7405 synthesized on various oil-containing media. Methods. N. vaccinii IMV B-7405 was grown in medium containing as carbon source refined sunflower oil, oil after frying french fried potatoes, potato wedges and meat. The surfactants were extracted from supernatant of cultural liquid by modified Folch mixture. The antimicrobial action of tea tree, cinnamon and lemongrass EO, surfactants and their mixtures was determined by index of the minimum inhibitory concentration (MIC). Synergistic effect of surfactants and EO was evaluated by indicator of fractional inhibitory concentration. The degree of bacteria and fungi biofilms destruction under the action of surfactants, EO and their mixtures was determined by spectrophotometric method. Results. It was found that N. vaccinii IMV B-7405 surfactants synthesized on all oil-containing substrates showed a synergistic antimicrobial and anti-adhesive activity with the investigated EO. MIC of a surfactants and EO mixture against bacteria (Bacillus subtilis BT-2 (spores), Escherichia coli IEM-1, Staphylococcus aureus BMS-1) and yeast (Candida albicans D-6, Candida utilis BVS-65 and Candida tropicalis RE-2) were 2–20 μg/ml and were significantly lower than each compound separately (156–625 and 8–80 μg/ml for EO and surfactants, respectively). The destruction of bacterial and yeast biofilms under the action of a mixture of surfactants (20–40 μg/ml) and EO (20–40 μg/ml) was 1.3–2.9 times higher compared with using of each component separately at similar concentrations. Conclusions. The data presented the possibility of using a mixture of EO and surfactants not only to reduce their MIC, but also as effective antimicrobial and anti-adhesive agents.Документ Antimicrobial activity of a mixture of surfactants produced by Acinetobacter calcoaceticus IMV B-7241 with antifungal drugs and essential oils(2022) Pirog, Tatiana; Kliuchka, Igor; Kliuchka (Nykytyuk), LiliaIntroduction. The aim of the work was to study the effect of a mixture of surfactants synthesized by Acinetobacter calcoaceticus IMV B-7241 under various cultivation conditions with antifungal drugs (clotrimazole and fluconazole) and essential oils (cinnamon and lemongrass) on yeast of genus Candida. Material and methods. The cultivation of A. calcoaceticus IMV B-7241 was carried out in a basic medium that did not contain NaCl (medium 1), contained NaCl, 2.0 g/l (medium 2), contained NaCl, 2.0 g/l, and KCl, 1.0 g/l (medium 3). The surfactants were extracted from supernatant of cultural liquid by modified Folch mixture. Antimicrobial properties of the surfactants, antifungal drugs and essential oils were determined by index of the minimum inhibitory concentration (MIC). To assess the synergistic effect of a mixture of surfactants with antifungal drugs or essential oils the fractional inhibitory concentration index was used. Results and discussion. Surfactants synthesized by A. calcoaceticus IMV B-7241 on the basic medium were the most effective antimicrobial agents against the yeasts strains Candida albicans D-6, C. tropicalis RE-2 and C. utilis BVS-65 with MIC 22.5–45 μg/ml that were 2.6–17 times lower than the values determined for surfactants synthesized on modified media. At the same time, regardless of the strain cultivation in different media, all surfactants showed synergism of antifungal activity with clotrimazole, fluconazole, cinnamon or lemongrass essential oils. Thus, in the presence of surfactants synthesized on basic and modified media in a mixture with antifungal drugs, MIC of clotrimazole and fluconazole against the studied yeast test cultures decreased by 4–32 times. The use of a mixture of essential oils with surfactants synthesized by A. calcoaceticus IMV B-7241 growing in different media made it possible to reduce MIC of cinnamon and lemongrass oils against yeasts of Candida genus 4–18 and 8–32 times, respectively. At the same time, the index of fractional inhibitory concentration did not exceed 0.5, which indicates the synergism of anifungal activity between the studied compounds. Conclusion. The results confirm the possibility to reduce the minimum inhibitory concentrations of antifungal drugs or essential oils against members of genus Candida by their mixture with microbial surfactants.Документ Effect of cations on the activity of NADP+-dependent glutamate dehydrogenase in Acinetobacter calcoaceticus IMV B-7241, Rhodococcus erythropolis IMV Ac-5017 and Nocardia vaccinii IMV B-7405 grown on industrial waste(2021) Pirog, Tatiana; Paliichuk, Olesia; Lutsay, Dariya; Kliuchka (Nykytyuk), Lilia; Shevchuk, TetianaIntroduction. It is studied the activity of NADP+-dependent glutamate dehydrogenase in the presence of mono- and divalent cations (potential activators of this key enzyme of surface-active aminolipids biosynthesis) in A. calcoaceticus IMV B-7241, R. erythropolis IMV Ac-5017 and N. vaccinii IMV B-7405 during cultivation on waste of biodiesel production and sunflower oil waste. Materials and methods. Cultivation of strains was performed in liquid mineral media using as substrates: refined and waste (after frying potato) sunflower oil, refined glycerol and waste of biodiesel production. NADP+-dependent (EC 1.4.1.4) glutamate dehydrogenase activity in cell-free extracts was analyzed for glutamate formation during oxidation of NADPH at 340 nm. Monovalent (Na+, K+) and divalent (Mg2+, Ca2+, Zn2+) cations in the form of salts of NaCl, KCl, MgSO4 × 7H2O, CaCl2 and ZnSO4 × 7H2O were added to the reaction mixture, as well as into the medium for strains cultivation. Results and discussion. Calcium cations were found to be activators of NADP+-dependent glutamate dehydrogenase activity in R. erythropolis IMV Aс-5017 and N. vaccinii IMV B-7405 grown on refined and waste sunflower oil: in the presence of 1–5 mmol Ca2+ in the mixture, the activity of the enzyme increased 1.3–2 times compared with that without these cations. The increase in the concentration of CaCl2 to 0.2−0.4 g/l in oil-containing medium of strains IMV Ac-5017 and IMV B-7405 cultivation was accompanied by an increase in NADP+-dependent glutamate dehydrogenase activity by 1.3–1.5 times compared with that on basic medium. When additional quantity of CaCl2 (0.1−0.2 g/l) was introduced into the medium with purified glycerol for the cultivation of A. calcoaceticus IMV B-7241, an increase in NADP+-dependent glutamate dehydrogenase activity was observed by almost 2.5−3 times compared with those for strain IMV B-7241 on the basic medium. There was no impact of activating cations magnesium, zinc, potassium and sodium on NADP+-dependent glutamate dehydrogenase activity of all strains grown on oil-containing substrates and glycerol of different degrees of purification. Conclusion. The results demonstrate the possibility to increase activity of key enzymes of the biosynthesis of the desired product: the composition of the medium should be modified by changing the content of enzymes’ activators.Документ Factors affecting antibiofilm properties of microbial surfactants(2021) Pirog, Tatiana; Kliuchka (Nykytyuk), Lilia; Lutsay, Dariya; Stabnikov, VictorAntibiofilm activity of surface-active substances (SAS) synthesized by Rhodococcus erythropolis, Acinetobacter calcoaceticus, or Nocardia vaccinia was studied. The strains were cultivated using carbon ethanol, glycerol, hexadecane, sunflower oil, and carbon-containing wastes from biodiesel production as carbon sources. Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, and yeasts Candida albicans and Candida utilis were used as testing cultures. The antibiofilm activity of biosurfactants depended on the source and concentration of carbon source and duration of cultivation. The addition of calcium cations to media significantly increased antibiofilm activity. Replacement of pure sources of carbon for wastes provided the opportunity to receive biosurfactants with high antibiofilm properties on inexpensive substrates. Destruction of biofilms was the same in cases when supernatant or purified SAS was used. Вивчено антибіоплівкову активність поверхнево-активних речовин (ПАР), синтезованих Rhodococcus erythropolis, Acinetobacter calcoaceticus або Nocardia vaccinia. Штами культивували з використанням в якості джерел вуглецю вуглецевого етанолу, гліцерину, гексадекану, соняшникової олії та вуглецевмісних відходів виробництва біодизеля. Як тест-культури використовували Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, дріжджі Candida albicans і Candida utilis. Антибіоплівкова активність біосурфактантів залежала від джерела та концентрації джерела вуглецю та тривалості культивування. Додавання до середовищ катіонів кальцію значно підвищувало активність антибіоплівки. Заміна чистих джерел вуглецю на відходи дала можливість отримувати на недорогих субстратах біоПАР з високими антибіоплівковими властивостями. Деструкція біоплівок була однаковою у випадках використання супернатанту або очищеного ПАР.Документ Influence of cultivation conditions on antimicrobial properties of Nocardia vaccinii ІMV B-7405 surfactants(2016) Pirog, Tatiana; Panasiuk, Catherine; Kliuchka (Nykytyuk), Lilia; Iutinska, GalinaThe aim of the work was to study of antimicrobial effect of Nocardia vaccinii ІMV B-7405 surfactants, synthesized in various cultivation conditions, against phytopathogenic bacteria of genera Pseudomonas, Xanthomonas, and Pectobacterium. The antimicrobial properties of surfactants were determined in suspension culture by Koch method and also by index of the minimum inhibitory concentration. Surfactants were extracted from supernatant of cultural liquid using mixture of chloroform and methanol (2: 1). It has been established that antimicrobial properties of surfactants depend on the nature of the carbon source in the medium (refined sunflower oil, as well as waste oil after frying potatoes and meat, glycerol), the duration of the cultivation (5 and 7 days), the degree of surfactants purification (the supernatant of cultural liquid, purified surfactants solution) and the test culture type. The highest antimicrobial activity was exhibited by purified surfactants solutions synthesized by ІMV B-7405 strain on the waste oil after potato frying (decreased survival of pathogenic bacteria by 50–95%), and surfactants formed within 7 days of N. vaccinii ІMV B-7405 cultivation on all test substrates (minimum inhibitory concentration 7–40 μg/ml, which is several times lower than the surfactant, synthesized for 5 days). These data are promising for the development of ecologically friendly biopreparations to control the number of phytopathogenic bacteria.