CC BY
19
UDC 759.873.088.5:661.185 https://doi.org/ 10.15407/biotech9.06.050
CALCIUM AND MAGNESIUM CATIONS INFLUENCE
ON ANTIMICROBIAL AND ANTIADHESIVE ACTIVITY OF Acinetobacter calcoaceticus IMV B-7241
SURFACTANTS
T. P. Pirog
I. V. Sidor National University of Food Technology, Kyiv, Ukraine
D. A. Lutsai
E-mail: [email protected]
Received 23.09.2016
The 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 IMV 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 Ca2+ 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.
Key words: Acinetobacter calcoaceticus IMV B-7405, surfactants, activity of NADP+-dependent glutamate dehydrogenase, calcium and magnesium cations.
Earlier research [1, 2] has established the dependence of antimicrobial and anti-adhesive activities of surface-active substances (surfactants) produced by Acinetobacter calcoaceticus IMV B-7241 on the presence of growth factors and certain microelements in the culture medium. Changing yeast autolysate and microelements mixture in the medium containing ethanol to copper sulfate and iron sulfate was followed by an increase of surfactants production [3], yet their antimicrobial and anti-adhesive activity decreased [1, 2]. We supposed that the phenomenon might be caused by microbial surfactants being secondary metabolites commonly synthesized as a complex of such
compounds [4] whose ratio (and therefore, the properties of the final product) may change depending on the different conditions of the producer cultivation.
By their chemical nature, surfactants of A. calcoaceticus IMV B-7241 are a complex of neutral, glyco- and aminolipids [4]. According to the data from literature [5, 6], aminolipids are more efficient antimicrobial agents than glycolipids, and neutral lipids are characterized by very low antimicrobial activity. Therefore, an increased fraction of aminolipids might be accompanied by a stronger antimicrobial activity of the surfactants. However, today the influence of cultivation conditions on surfactants'
antimicrobial and anti-adhesive activity and the possibility of regulating them remain outside the researchers' attention, although the first studies concerning the link between the chemical composition of microbial surfactants and their properties go back almost fifteen years [7]. Meanwhile, according to [8], the biosynthesis of aminolipids with preformed properties is impossible, and can be only achieved by post-fermentation chemical modification of the synthesized surfactants or using respective gene-modified producer strains [9].
In [1, 2] we suggested that in the presence of yeast extract and microelements mixture in A. calcoaceticus IMV B-7241 cultivation medium, the content of aminolipids in the produced surfactant complex is higher than in that obtained in the medium with copper sulfate and iron sulfate. Also, we assumed that among the microelements composing the cultivation medium for strain IMV B-7241 there can be cations activating NADP+-dependent glutamate dehydrogenase — a key enzyme of aminolipids biosynthesis in this strain [10]. Later this hypothesis was experimentally supported. Thus, later it was showed [11] that zinc cations are activators of NADP+-dependent glutamate dehydrogenase, and adding Zn2+ (38 pM) into the medium containing ethanol and sulfates of copper and iron was followed by the production of surfactants with higher antimicrobial and anti-adhesive activity.
According to literature [12, 13] the activators of NADP+-dependent glutamate dehydrogenase in microorganisms are the cations of calcium and magnesium. Thus, in the archaea Thermococcus sp. this enzyme's activity increased in the presence of 5 mM CaCl2, MgCl2 and MnCl2 [12]. Later [13] it was showed that cations of calcium and magnesium also activated NADP+-dependent glutamate dehydrogenase in the archaea Thermococcus waiotapuensis: under 10 mM CaCl2 and 10 mM MgSO4 the activity rose 1.3 times compared to the control without metal cations.
Hence, the purpose of this work is to study influence of calcium and magnesium cations on the NADP+-dependent glutamate dehydrogenase activity of A. calcoaceticus IMV B-724 cell-free extract with the following modification of cultivation medium composition and determining antimicrobial and antiadhesive activity of synthesized surfactants.
Materials and Methods
Object of study. The object of our study was strain Acinetobacter calcoaceticus K-4, registered in the Depository of Microorganism Strains of the Zabolotny Institute of Microbiology and Virology of the NAS of Ukraine under IMV B-7241.
Medium composition and cultivation conditions. Strain A. calcoaceticus IMV B-7241 was grown in a liquid mineral medium of the following composition (g/l): (NH2)2CO — 0.35, NaCl — 1.0, Na2HPO412H2O — 0.6, KH2PO4 — 0.14, MgSO47H2O — 0.1, distilled water — up to 1 l, pH 6.8-7.0. The medium was also augmented with yeast autolysate — 0.5 % (v/v) and microelements solution — 0.1 % (v/v) containing (g/100 ml): ZnSO47H2O — 1.1; MnSO4H2O — 0.6; FeSO47H2O — 0.1; CuSO45H2O — 0.004; CoSO47H2O — 0.03; H3BO3 — 0.006; KI — 0.0001; EDTA — 0.5.
As carbon and energy source we used 2% ethanol (v/v).
In one version of the experiment, the basic medium had MgSO47H2O content raised to 0.2 g/l (medium 1), in another the basic medium was augmented by additional dose of 0.1 g/l CaCl2 (medium 2).
Inoculum. Seeding material was a culture in the middle of the exponential growth phase grown in a basic medium with 0.5 % (v/v) of ethanol. The amount of inoculate was 5 % of the mediums volume (104-105 cells/ml). The culture was grown in 750 ml flasks with 100 ml of the medium on a shaker (320 rev/ min) at 30 °C for 120 hr.
Determining antimicrobial and antiadhe-sive activity of surfactants. In the research we used surface-active substances in the form of cultural liquid supernatant and solutions of surfactantas extracted from supernatant by the Folch mixture (chloroform and methanol, 2:1) as described in [1, 2, 3, 11].
The antimicrobial properties of surfactants were analyzed according to their minimum inhibitory concentration (MIC). MIC was determined by two-fold serial dilutions in meat-peptone broth (MPB) [2, 11]. In aseptic conditions, MPB was added to ten test tubes at 1 ml. In the first test tube, we added 1 ml of sterile solution of the surfactant a certain concentration, mixed it, 1 ml was taken and placed into the next tube. Similarly, we carried out the dilution in the next nine test tubes. From the last test tube, we pipetted out 1 ml. Therefore, the final volume in every test tube was 1 ml (MPB and the surfactant solution), but the concentration of the surfactant was
twice reduced at every step. As a control we used 1 ml of MPB without surfactants solution. Next, into every test tube 0.1 ml of the test culture suspension (105-106 CFU/ml) was added and the contents were mixed. The test tubes were incubated for 24 hours at 30 °C. The results were estimated visually by the cloudy or transparent medium: (+) flasks where the medium grew cloudy (indicating growth of test culture), (-) flasks without cloudiness (no growth). The MIC of the surfactant solution was determined as the surfactant concentrations in the first tube where the test culture failed to grow.
Antiadhesive properties were studied as follows [1, 11]: the purified plates of materials (Dutch tile, stainless steel, plastic, linoleum) of the same size (1 cm2) were sterilized at 112 °C for 30 min and then were added into the surfactants solution or supernatant and dried for 24 h in a thermostat at 30 °C. One day bacterial test cultures grown on meat peptone agar (MPA) were suspended in 100 mL of sterile tap water; the materials pretreated with surfactants and untreated (control) samples were placed into the suspension, incubated for 2 h in a thermostat at 30 °C, and rinsed with 10 mL of sterile tap water to remove non-adherent cells.
The plates of materials were treated with methanol (99%) for 15 min to fix the attached cells, dried at room temperature, placed for 5 min into 1% gentian violet solution, and rinsed with tap water. After drying, the materials were treated with 10 mL of 33% acetic acid solution and the optical density of the resultant suspension of desorbed cells was measured. The number (%) of attached cells (adhesion) was determined as a ratio of the optical density of the suspension obtained from surfactant-treated (supernatant, surfactant solution) materials to that of the controls (without surfactant treatment) and measured in %.
The study of surfactants' action on the biofilm destruction was carried out after [14]. To obtain biofilm, 180 pl of MPB or liquid wort and 20 pl of one-day test culture suspension were added into polystyrene microplates, incubated for 24 h at the optimal temperature. Then the cultural liquid was poured off and another 180 pl of fresh MPB (liquid wort) and 20 pl of the suspension of the test culture were added and again incubated for 24 h. In the study [14] it was established that such 48 h culture is sufficient for biofilm formation in the wells of microplate. After 48 h the cultural liquid was poured off, and into each of the wells of the microplate (pre-covered by the biofilm) 200 pl of preparations with
different surfactant concentrations (0.0051.28 mg/ml) were added. Into the control wells, surfactant preparations were replaced with distilled tap water (200 pl). After 24 h of exposition the wells were thrice washed by 200 pl of distilled water and the amount of adherent cells was determined spectrophotometrically just as it was done for the anti-adhesive research [1, 11]. The degree of biofilm destruction (%) was determined as the difference between cell adhesion in untreated and surfactant-treated wells of the polystyrene plate.
As test cultures to evaluate the biological properties of surfactants we used bacterial strains Escherichia coli 1ЕМ-1, Bacillus subtilis BT-2, Enterobacter cloaceae C-8, Staphylococcus aureus ВМС-1, Proteus vulgaris PA-12 from the living cultures collection of the Department of Biotechnology and Microbiology of the National University of Food Technologies.
Enzyme analysis. To obtain cell-free extracts the culture liquid was centrifuged (5000 g, 20 min, 4 °С). The remnants of the medium were twice washed out of the obtained cell pellet with 0.05 М K+-phosphate buffer (рН 7.0) using centrifugation (4000 g, 15 min, 4 °С). The washed cells were resuspended in 0.05 М K+-phosphate buffer (рН 7.0) and destroyed with ultrasound (22 kHz) thrice for 20 s at 4 °С on UZDN-1 ultrasonic disperser. The resulting mush was centrifuged again (12000 g, 30 min, 4 °С), the pellet was discarded, and the supernatant was used in further research as cell-free extract.
NADP+-dependent glutamate dehydro-genase activity of the cell-free extract (EC 1.4.1.4) was analyzed by measuring glutamate synthesis during NADPH oxidation at 340 nm [11]. To study the effect of cations on the enzyme activity we added to the reagents mixture 0.01-10 mM Ca2+ and Mg2+ as solution of CaCl2 and MgSO47H2O.
The enzyme activity was measured as the number of product nmol per 1 min of reaction per 1 mg protein. The protein content in cellfree extracts was determined after Bradford. The enzymatic activity was analyzed at 28-30 °С — the optimal temperature for A. calcoaceticus IMV В-7241 growth.
All experiments were carried out in triplicate, the number of parallel measurements in the experiments was 3-5. The statistical treatment of the experimental data was carried out as previously described [1-3]. The differences between the means were considered significant at P<0.05.
Results and Discussion
To begin with, we analyzed NADP+-dependent glutamate dehydrogenase activity of A. calcoaceticus IMV В-7241 cell-free extract depending on Ca2+ and Mg2+ content in the reaction mixture (Table 1). Experiments showed that at 10 mM Ca2+ and Mg2+ the activity increased 1.5 times compared to the cation-less control.
Next, NADP+-dependent glutamate de-hydrogenase activity of the cell-free extract obtaining from A. calcoaceticus IMV В-7241 cells grown in a liquid media with increased content of the enzyme activators was determined (Table 2).
We established that twice higher magnesium sulfate concentration or addition of CaCl2 into cultivation medium of IMV В-7241 strain was followed by 2.4- and 3 times rise in NADP+-dependent glutamate dehydrogenase activity, respectively.
The data on antimicrobial activity of the surfactants, synthesized by A. calcoaceticus IMV В-7241 in basic medium, and in medium with increased concentration of calcium and magnesium cations are presented in Table 3.
The research showed that for all studied bacteria (except for S. aureus BMC-1), the MIC of surfactants produced in the medium 2 was 1.3 3.5 times lower than that of surface-active substances produced in the basic medium. Previous research [11] showed that adding zinc cations into the cultivation medium of A. calcoaceticus IMV В-7241 (another activator of NADP+-dependent glutamate dehydrogenase in this strain) was followed by synthesis of surfactants, the MIC of which against E. coli IEM-1, E. cloaceae C-8, S. aureus BMC-1 and P. vulgaris PA-12 was 14, 28, 7 and 14 ug/ml, respectively, which is practically same as we obtained in our current work (Table 3).
Also, the antimicrobial activity of the A. calcoaceticus IMV В-7241 surfactants against some test cultures was higher than of well-known aminolipids of the bacteria genera Bacillus and Paenibacillus [5]. Thus, MIC of surfactin, iturin and polypeptin against different strains of E. coli IEM-1 was 15, 6, >300 and 3.1-12.5 ug/ml respectively; of polypeptin and octapeptin against P. vulgaris — 50-100 and 6.3 ug/ml; of iturin, polipeptin and octapeptin against S. aureus >400, 6.3 and 50 ug/ml, respectively. The minimum inhibitory concentration of the aminolipids produced by Streptomyces amritsarensis sp. nov. against B. subtilis MTCC 619 did not exceed 10 ug/ml, and against Staphylococcus epidermidis MTCC 435 it was 15 ug/ml [15].
Let us note here that the antimicrobial activity of surfactants produced by A. calcoaceticus IMV B-7241 in the basic medium and medium 1 was practically the same (Table 3). Therefore, adding more Ca2+ into the cultivation medium of strain IMV B-7241 influenced the antimicrobial activity of the produced surfactants more than raising the Mg2+ concentration.
Similar results were found when studying the anti-adhesive properties of the
A. calcoaceticus IMV B-7241 surface-active substances obtained in the media with different concentrations of Ca2+ and Mg2+ (Table 4). In these experiments we used surfactant solutions with base concentrations of 5 pg/ml, since earlier data [1] showed that this was the concentration with the maximal anti-adhesive effect of surface-active substances synthesized in the basic medium.
The data in Table 4 show that the adhesion of B. subtilis BT-2 spore cells on plastic, tile, steel and linoleum was minimal (14-26%) if the surfaces were pre-treated with the solution of surfactant produced by strain IMV B-7241 in the medium 2 with additional CaCl2. Also, unlike the practically equal antimicrobial activity of surfactants produced in the basic medium and medium 1 with increased magnesium content, their anti-adhesive properties turned out to be different (Tables 3 and 4). Treating all studied materials with the solution of surfactants produced in the medium 1, was followed by the decrease in the amount of B. subtilis BT-2 adherent cells by 70-84%, while the preparation obtained in the basic medium only lowered it by 52-77% compared to surfaces untreated with surfactants (Table 4). Notably, surfactants produced in the media 1 and 2 exhibited higher anti-adhesive activity at 1.25 pg/ml, while lowering the concentration from 5 to 1.25 pg/ml for surfactants obtained in the basic medium caused a 6-11% increase in
B. subtilis BT-2 adhesion on pre-treated surfaces (Table 4).
Analysis of anti-adhesive properties of the known microbial surfactants that we reviewed in [16] showed that aminolipids produced by bacteria of the genus Bacillus are more efficient anti-adhesive agents compared to rhamno- and sophorolipids. Thus, the efficient concentration of aminolipids is, on average, 2-50, and sophorolipids 12-200 pg/ml. However, the available literature did not contain information on the effect of aminolipids on the B. subtilis adhesion to different materials. Hence we compared our results (Table 4) to those in [17], where the authors studied the efficiency of Pseudomonas aeruginosa LCD12 rhamnolipids in preventing
Table 1. The effect of Ca2+ and Mg2+ cations on NADP+-dependent glutamate dehydrogenase activity
of A. calcoaceticus IMV B-7241 cell-free extract
Cations Concentration in the reagents mixture, mM Activity, nmolmin xmg 1 of protein
No cations (control) 0 345±17
Ca2+ 0.01 N. d.
5 431±21*
10 517±25*
Mg2+ 0.01 310±15*
5 345±17*
10 517±25*
Note. * — P < 0.05 compared to control (enzyme activity in the absence of Ca2+ and Mg2+ in the reaction mixture). N. d. — the parameter was not determined.
Table 2. NADP+-dependent glutamate dehydrogenase activity of A. calcoaceticus IMV B-7241 cell-free extract depending on Ca2+ and Mg + concentrations in the cultivation medium
Cultivation medium Concentration in the medium (g/l) Activity, nmolmin ^mg 1 of protein
MgS04-7H20 CaCl2
Basic 0.1 0 380±19
Medium 1 0.2 0 920±46*
Medium 2 0.1 0.1 1140±57*
Note. * — P < 0.05 compared to control (enzyme activity during cultivation of IMV B-7241strain in the basic medium).
Table 3. The minimum inhibitory concentration of surfactants produced by A. calcoaceticus IMV B-7241
in media with different Ca2+ and Mg2 concentrations
Test culture MIC (^g/ml) of surfactants, produced on
basic medium medium 1 medium 2
Bacillus subtilis BT-2 (spores) 14 12* 4*
Enterobacter cloaceae C-8 42 48* 32*
Staphylococcus aureus BMC-1 7 12* 8*
Proteus vulgaris PA-12 14 12* 8*
Escherichia coli IEM-1 28 24* 16*
Note. When measuring the minimum inhibitory concentration of surfactants, the error did not exceed 5%. * — P < 0.05 compared to control (MIC of surfactants produced by IMV B-7241 strain cultured in the basic medium).
the adhesion of B. subtilis RI6, E. coli PJ3, S. aureus FD5, and Staphylococcus epidermidis LK8 to polystyrene surface. It was found that the adhesion of test cultures was 50-80% if the wells of the plate were pre-treated with rhamnolipids at 8-64 pg/ml. The data in Table 4 show that surfactants produced in the process of A. calcoaceticus IMV B-7241 cultivation on all studied media exhibit higher
anti-adhesive activity at significantly lower concentrations.
According to the latest research, surfactants synthesized by bacteria (Pseudomonas, Lactobacillus, Bacillus) and yeasts (Saccharomyces) are able to not only prevent microorganisms adhesion on various materials, but to destroy established biofilms [16, 18-20].
In our previous study [16] we showed that A. calcoaceticus IMV B-7241 surfactants (0.04-1.28 mg/ml) synthesized on ethanol, glycerol and n-hexadecan are able to destroy more than 21-88% of the biofilm formed by S. aureus BMC-1, B. subtilis BT-2 and E. coli IEM-1, and the degree of bacterial biofilms destruction the grew with increasing surfactants' content.
Table 5 provides the data on the effect of A. calcoaceticus IMV B-7241 surfactant preparations, synthesized in the media with different concentrations of Ca2+ and Mg2+ on the destruction of B. subtilis BT-2 biofilm. In these researches, the biofilm destruction was studied under lower concentrations of strain IMV B-7241 surfactants, than in the previously discussed research [16].
It was found that regardless of the concentration and level of purification (supernatant, surfactant solution) the preparations of surfactants synthesized in the media 1 and 2 caused more efficient destruction of B. subtilis BT-2 biofilm than preparations obtained in the basic medium (19-58 and
12-45%, respectively). The highest degree of biofilm destruction (57-58%) was reached by preparations with surfactant concentration of 62-124 pg/ml.
Das et al. [17] showed that rhamnolipids of P. aeruginosa IMP67 at a concentration of 64 pg/ml were able to destroy 50% biofilm formed on polystyrene by B. subtilis RI6. Surface-active substances synthesized by Saccharomyces cerevisiae D3 at 100 pg/ml caused destruction of B. subtilis BT37 biofilm by 30% [21]. Therefore, surfactants produced by strain IMV B-7241 are more efficient destructors of biofilms than those of S. cerevisiae D3 and rhamnolipids of P. aeruginosa IMP67, which supports the possibility of using them as components of novel disinfectants to destroy bacterial biofilms.
Thus, our work showed that adding Ca2+ or increasing the concentration of Mg2+ in the cultivation medium of A. calcoaceticus IMV B-7241 was followed by synthesis of surfactants with higher antimicrobial and anti-adhesive activity than surfactants obtained in the basic medium.
Table 4. The effect of surfactants synthesized by A. calcoaceticus IMV B-7241 in media with different Ca2+ and Mg2+ concentrations on the adhesion of B. subtilis BT-2 spore cells to various surfaces
Cultivation medium Surfactant concentration, №/ml Materials,% adhesion
plastic tile steel linoleum
Base 5 23 37 24 30
1.25 29 48 30 36
Medium 1 5 21* 30* 15* 25*
1.25 16* 20* 16* 19*
Medium 2 5 19* 26* 14* 25*
1.25 16* 21* N.d. 14*
Note. When measuring the adhesion the error did not exceed 5%. * — P < 0.05 compared to control (adhesion after treatment with surfactants, produced by IMV B-7241 in the basic medium). N.d. — not determined.
Table 5. Destruction of B. subtilis BT-2 biofilm under the action of A. calcoaceticus IMV B-7241 surfactants
produced in the media of various composition
Cultivation medium Preparations Destruction (%) of the biofilm after treatment with surfactant (^g/ml)
3.9 7.8 15.5 31 62 124
Basic Supernatant 26 28 29 31 33 35
Surfactant solution 12 17 29 31 36 45
Medium 1 Supernatant 33* 43* 45* 45* 55* 57*
Surfactant solution 19* 24* 40* 42* 58* 58*
Medium 2 Supernatant 39* 43* 45* 50* 52* 57*
Surfactant solution 29* 33* 45* 48* 51* 58*
Note. During the measurement of biofilm destruction, the error did not exceed 5%. * — P < 0.05 compared to control (destruction of the biofilm after treatment with surfactant preparations, synthesized by strain IMV B-7241 in the basic medium).
The data are in agreement with our previous results [11] and support the
possibility of regulating antimicrobial and anti-adhesive activity of surfactants under producer cultivation.
REFERENCES
1. Pirog T. P., Savenko I. V., Shevchuk T. A. Effect of cultivation conditions of Acinetobacter calcoaceticus IMV B-7241 on surfactants antiadhesive properties. Microbiol. Zh. 2016, 78(1), 2-12. (In Russian).
2. Pirog T. P., Savenko I. V., Shevchuk T. A., Krutous N. V., Iutynska G. O. Antimicrobial properties surfactants synthesized under different cultivation conditions of Acinetobacter calcoaceticus IMV B-7241. Microbiol. Zh. 2016, 78(3), 2-12. (In Ukrainian).
3. Pirog T. P., Shevchuk T. A., Mashchenko O. Yu., Parfenyuk S. A., Iutinskaya G. A. Effect of growth factors and some microelements on biosurfactant synthesis of Acinetobacter calcoaceticus IMV B-7241. Microbiol. Zh. 2013, 75(5), 19-27. (In Russian).
4. Pirog T. Р., Konon A D. Microbial surfactants. I. Glycolipids. Biotechnol. acta. 2014, 7(1), 9-30. doi: 10.15407/biotech7.01.009. (In Ukrainian).
5. Cochrane S. A., Vederas J. C. Lipopeptides from Bacillus and Paenibacillus spp.: a gold mine of antibiotic candidates. Med. Res. Rev. 2016, 36(1), 4-31. doi 10.1002/med.21321.
6. Bernat P., Paraszkiewicz K., Siewiera P., Moryl M, Plaza G., Chojniak J. Lipid composition in a strain of Bacillus subtilis, a producer of iturin A lipopeptides that are active against uropathogenic bacteria. World J. Microbiol. Biotechnol. 2016, 32(10). doi: 10.1007/s11274-016-2126-0.
7. Abalos A., Pinazo A., Infante M. R., Casals M., Garcia F., Manresa A. Physicochemical and antimicrobial properties of new rhamnolipids produced by Pseudomonas aeruginosa AT10 from soybean oil refinery wastes. Langmuir, 2001, 17(5), 1367-1371.
8. Mandal S. M., Barbosa A. E., Franco O. L. Lipopeptides in microbial infection control: scope and reality for industry. Biotechnol. Adv., 2013, 31(5), 338-345. doi: 10.1016/j. biotechadv.2013.01.004.
9. Zhihui X., Jiahui S., Bing L., Xin Y., Qirong S. and Ruifu Z. Contribution of bacillomycin D in Bacillus amyloliquefaciens SQR9 to antifungal activity and biofilm formation. Appl. Environ. Microbiol. 2013, 79(3), 808-815. doi: 10.1128/ AEM.02645-12.
10. Pirog T. P., Shevchuk T. A., Antonyuk S. I., Kravchenko Ye. Yu., Iutiynska G. O. Effect of univalent cations on synthesis of surfactants by Acinetobacter calcoaceticus IMV B-7241. Microbiol. Zh. 2013, 75(2), 10-20. (In Russian).
11. Pirog T. P., Savenko I. V., Shevchuk T. A. Effect of Zn2+ on synthesis of Acinetobacter
calcoaceticus IMV B-7241 surfactants with antimicrobial and antiadhesive properties. Microbiol. Zh. 2016, 78(4), 49-58. (In Russian).
12. Hudson R. C., Ruttersmith L. D., Daniel R. M. Glutamate dehydrogenase from the extremely thermophilic archaebacterial isolate AN1. Biochim. Biophys. Acta. 1993, 1202(2), 244-250.
13. Lee M. K., González J. M., Robb F. T. Extremely thermostable glutamate dehydrogenase (GDH) from the freshwater archaeon Thermococcus waiotapuensis: cloning and comparison with two marine hyperthermophilic GDHs. Extremophiles. 2002, 6(2), 151-159.
14. Gomes M.-Z. V., Nitschke M. Evaluation of rhamnolipids surfactants as agents to reduce the adhesion of Staphylococcus aureus to polystyrene surfaces. Lett. Appl. Microbiol. 2012, 49(1), 960-965.
15. Sharma D., Mandal S. M., Manhas R. K. Purification and characterization of a novel lipopeptide from Streptomyces amritsarensis sp. nov. active against methicillin-resistant Staphylococcus aureus. AMB Express. 2014, N4. doi: 10.1186/s13568-014-0050-y.
16. Pirog T. P., Savenko I. V., Lutsay D. A. Microbial surface-active substances as antiadhesive agents. Biotechnol. acta, 2016, 9(3), 7-22. doi: org/10.15407/ biotech9.03.007.
17. Das P., Yang X.-P., Ma L.Z. Analysis of biosurfactants from industrially viable Pseudomonas strain isolated from crude oil suggests how rhamnolipids congeners affect emulsification property and antimicrobial activity. Front. Microbiol. 2014, N5. doi: 10.3389/fmicb.2014.00696.
18. Jolly M. J. Inhibitory effect of biosurfactant purified from probiotic yeast against biofilm producers. IOSR-JESTFT. 2013, 6(1), 51-55.
19. Turbhekar R., Malik N., Dey D., Thakare D. Disruption of Candida albicans biofilms by rhamnolipid obtained from Pseudomonas aeruginosa RT. IJRSB. 2015, 3(3), 73-78.
20. Vilela S. F., Barbosa J. O., Rossoni R. D., Santos J. D., Prata M. C., Anbinder A. L., Jorge A. O., Junqueira J. C. Lactobacillus acidophilus ATCC 4356 inhibits biofilm formation by C. albicans and attenuates the experimental candidiasis in Galleria mellonella. Virulence. 2015, 6(1), 29-39.
21. Jolly M. J. Inhibitory effect of biosurfactant purified from probiotic yeast against biofilm producers. IOSR-JESTFT. 2013, 6(1), 51-55.
ВПЛИВ КАТ1ОН1В КАЛЬЦ1Ю I МАГН1Ю НА АНТИМ1КРОБНУ ТА АНТИАДГЕЗИВНУ АКТИВН1СТЬ ПОВЕРХНЕВО-АКТИВНИХ РЕЧОВИН
Acinetobacter calcoaceticus 1МВ В-7241
Т. П. Пирог, I. В. Сидор, Д. А. Луцай
Нащональний ушверситет харчових технологш, Киïв, Укра1на
E-mail: [email protected]
Метою роботи було дослщити вплив Ca2+ i Mg2+ на НАДФ+-залежну глутаматдегщро-геназну актившсть — ключовий ензим б^-синтезу поверхнево-активних амшолшдав Acinetobacter calcoaceticus 1MB В-7241 — з наступною модифшащею складу середовища i визначенням антимiкробноï та антиадгезивно1 активностi поверхнево-активних речовин.
Штам 1MB В-7241 вирощували у середови-щi з етанолом. НАДФ+-залежну глутаматде-гiдрогеназну активнiсть екстракту аналiзува-ли за утворенням глутамату тд час окиснення НАДФН. Поверхнево-активнi речовини екстра-гували iз супернатанта культуральноï рiдини сумiшшю Фолча. Антиадгезивну актившсть визначали спектрофотометричним методом, антимшробну — за показником мiнiмальноï iнгiбуючоï концентраци.
Встановлено, що за наявностi у середови-щi 10 мМ Са2+ i Mg2+ НАДФ+-залежна глута-матдегiдрогеназна активнiсть екстракту шд-вищувалася в 1,5 раза порiвняно з такою без каиошв. Збiльшення концентрацiï сульфату магшю до 0,2 г/л або додавання CaCl2 (0,1 г/л) у середовище культивування супроводжувало-ся тдвищенням цiеï активностi у 2,4 i 3,0 рази вщповщно, а також посиленням антимшроб-ноï та антиадгезивноï активностi синтезованих поверхнево-активних речовин. Мшмальна ш-гiбуюча концентрацiя поверхнево-активних речовин, синтезованих на модифшованих середо-вищах, щодо деяких бактерш була в 1,3-3,5 раза, ïх адгезiя на абiотичних поверхнях, обро-блених такими речовинами, — у середньому на 5-17% нижчою, а ступiнь руйнування бiоплiв-ки на 7-13% вищим порiвняно з показниками, встановленими для поверхнево-активних речо-вин, утворюваних на базовому середовищi.
Наведенi даш свiдчать про можливiсть ре-гуляцп антимiкробноï та антиадгезивноï ак-тивносм поверхнево-активних речовин у про-цеи культивування продуцента.
Ключовi слова: Acinetobacter calcoaceticus 1MB В-7405, поверхнево-активнi речовини, НАДФ+-залежна глутаматдегiдрогеназна активнiсть, катiони кальщю та магнiю.
ВЛИЯНИЕ КАТИОНОВ КАЛЬЦИЯ И МАГНИЯ НА АНТИМИКРОБНУЮ И АНТИАДГЕЗИВНУЮ АКТИВНОСТЬ ПОВЕРХНОСТНО-АКТИВНЫХ ВЕЩЕСТВ Acinetobacter calcoaceticus 1МВ В-7241
Т. П. Пирог, И. В. Сидор, Д. А. Луцай
Национальный университет пищевых технологий, Киев, Украина
E-mail: [email protected]
Целью работы было исследовать влияние Ca2+ и Mg2+ на НАДФ+-зависимую глутамат-дегидрогеназную активность (ключевой энзим биосинтеза поверхностно-активных аминоли-пидов Acinetobacter calcoaceticus ИМВ В-7241) с последующей модификацией состава среды и определением антимикробной и антиадгезивной активности поверхностно-активных веществ.
Штамм ИМВ В-7241 выращивали в среде с этанолом. НАДФ+- зависимую глутаматдеги-дрогеназную активность экстракта анализировали по образованию глутамата при окислении НАДФН. Поверхностно-активные вещества экстрагировали из супернатанта культураль-ной жидкости смесью Фолча. Антиадгезивную активность определяли спектрофотометриче-ским методом, антимикробную — по показателю минимальной ингибирующей концентрации.
Установлено, что при наличии в среде 10 мМ Ca2+ и Mg2+ НАДФ+-зависимая глутаматдеги-дрогеназная активность экстракта повышалась в 1,5 раза по сравнению с таковой без катионов. Увеличение концентрации сульфата магния до 0,2 г/л или добавление CaCl2 (0,1 г/л) в среду культивирования сопровождалось повышением этой активности в 2,4 и 3,0 раза соответственно, а также увеличением антимикробной и антиадгезивной активности синтезированных поверхностно-активных веществ. Минимальная ингибирующая концентрация поверхностно-активных веществ, синтезированных на модифицированных средах, по отношению к некоторым бактериям была в 1,3-3,5 раза, их адгезия на абиотических поверхностях, обработанных такими поверхностно-активными веществами — в среднем на 5-17% ниже, а степень разрушения биопленки на 7-13% выше по сравнению с показателями, установленными для поверхностно-активных веществ, образуемых на базовой среде.
Приведенные данные свидетельствуют о возможности регуляции антимикробной и антиадгезивной активности поверхностно-активных веществ в процессе культивирования продуцента.
Ключевые слова: Acinetobacter calcoaceticus ИМВ В-7405, поверхностно-активные вещества, НАДФ+-зависимая глутаматдегидрогеназ-ная активность, катионы кальция и магния.
