Method to protect yeast saccharomyces cerevisiae against oxidising stress caused by exposure to hydrogen peroxide
SUBSTANCE: method to protect yeast Saccharomyces cerevisiae against oxidising stress as a result of exposure to hydrogen peroxide includes growing of yeast culture under standard conditions till the end of the logarithmic or the start of the stationary phase of growth, incubation with a protective agent, exposure to hydrogen peroxide with subsequent determination of the number of survived cells. The protective agent is represented by soy inhibitors of proteases in concentration of 0.1-2.0 g/l. The time of incubation with the protective agent makes 5-6 hr, concentration of hydrogen peroxide is 700-750 mM. The invention provides for high extent of yeast cells production with no depressant action.
EFFECT: share of survived yeast cells during method realisation increases in comparison with the reference one 1,2-5,4 times.
The invention relates to biotechnology, namely to develop ways to protect yeast cells from oxidative stress due to exposure to hydrogen peroxide, and can be used in medical, chemical and microbiological industry.
There is a method of protection of Saccharomyces cerevisiae yeast cells from oxidative stress by trehalose, mannitol and galactose. The method includes adding one carbohydrate at a concentration of 500 mm in the cultural medium, incubation of yeast for 1 h, the effect of a mixture of hydrogen peroxide at a concentration of 2 mm and iron trichloride concentration of 1 mm for 1 h with subsequent determination of the number of surviving cells [N. Benaroudj, Do It, Lee, Goldberg A.L. Trehalose accumulation during cellular stress protects cells and cellular proteins from damage by oxygen radicals. // The Journal Of Biological Chemistry. 2001, vol. 276, No. 26, pp.24261-24267]. When using this remedy yeast achieved a 20-fold increase in the fraction of surviving cells compared with control cultures without added carbohydrate). The disadvantage of this method is used as the test culture mutant in relation to the synthesis of trehalose strain of the yeast Saccharomyces cerevisiae, as a result, this method does not reflect the protective effect of carbohydrates on a non-mutated strains of the yeast Saccharomyces cerevisiae.
The closest in technical essence and the achieved is the playing technique is a way of protecting the yeast Saccharomyces cerevisiae from the stress of the influence of various factors (the effects of oxidants, γ-radiation, the photo-oxidation) using as protective agents alkyloxybenzoic [konanykhine I.A., Shanenko E.F., Loyko N., Nikolaev Y.A., El-Registan GI Regulates the action of microbial AOB different patterns on the stress response of yeast. // Applied biochemistry and Microbiology. 2008. So 44, No. 5, s-575]. The method includes growing the yeast culture in standard conditions until late logarithmic or early stationary phase of growth, adding alkyloxybenzoic in concentration from 0.13 to 1.9 g/l, incubation of the culture with alkyloxybenzoic for 2 h, the impact of various stress agents with subsequent determination of the number of surviving cells. In conditions of oxidative stress (H2O2, 100 mm, 30 min) shows that C7-AOB in concentrations of 0.25-0.64 g/l increases the resistance of yeast cells to hydrogen peroxide in 2-5 times. In the next series of experiments, the stress induced γ-irradiation dose of 50 crad: at concentrations of alkyloxybenzoic of 1.40-1.65 g/l, the number of viable cells was increased 1.2-1.3 times in comparison with the control without making alkyloxybenzoic).
This method, selected as a prototype, has a number of disadvantages, the main of which is the relatively low degree of protection of yeast cells (increase survival 1.2-1.3 times) when exposed to the culture of irradiation, is also the when using higher compared with optimal concentrations of alkyloxybenzoic (more than 0.65 g/l) protective effect is lost and may even appear a dampening effect on the growth of yeast. In addition, the prototype used a relatively low concentration of hydrogen peroxide (100 mm) and not described protective effect of alkyloxybenzoic at higher concentrations stressor agent, although in terms of processes, the use and cultivation of yeast strains may be higher concentrations of hydrogen peroxide and a more severe impact of stressors.
The present invention is to develop a remedy yeast Saccharomyces cerevisiae from factors of oxidative stress for example hydrogen peroxide and hard ultraviolet radiation, allowing to achieve a high level of protection of yeast cells in the absence of inhibitory action on yeast cells.
The problem is solved in that the proposed remedy yeast Saccharomyces cerevisiae from stress agents, including growing the yeast culture in standard conditions until late logarithmic or early stationary phase of growth, incubation with a protective agent for 5-6 hours, exposure to various stress agents (for example, hydrogen peroxide and hard ultraviolet irradiation is possible) with subsequent determination of the number of surviving cells, and as protective agents use soy protease inhibitors at a concentration of from 0.1 to 2.0 g/L.
Unlike the prototype, this method will use a much higher concentration of hydrogen peroxide (700-750 mm, 7.0-7.5 times higher compared to the prototype), but achieved a protective effect when it is on the same level. When used as a stress agent hard ultraviolet radiation effect from the introduction of protease inhibitors exceeds the effect of making alkyloxybenzoic.
The invention is illustrated by the following examples.
Example 1. The yeast Saccharomyces cerevisiae was grown at a temperature of 28°C in a conical flask with a total volume of 250 ml with incubation on a laboratory shaker with constant stirring with a speed of 120 rpm./minimum Quantity of the nutrient medium was 50 ml Sample of culture was collected in a logarithmic or early stationary phase of growth, it added alkyloxybenzoic at a concentration of 0.25 g/l and incubated for 2 hours Resistance to oxidative stress was tested after 30 minutes incubation of the sample with hydrogen peroxide at a concentration of 100 mm. A sample of the culture subjected to oxidative stress, using the method of serial dilution subcultured in Petri dishes with agar medium and was carried out by counting colonies. It was shown that in the data conditions making alkyloxybenzoic increases the resistance of yeast cells in 2 times.
Example 2. The yeast Saccharomyces cerevisiae was grown at a temperature of 28°C in a conical flask with a total volume of 250 ml with incubation on a laboratory shaker with constant stirring at 150 rpm./minimum Quantity of the nutrient medium was 50 ml Sample of culture was collected in a logarithmic or early stationary phase of growth, the drug inhibitor Konitza at a concentration of 0.10 g/l and incubated for 6 hours Resistance to oxidative stress was tested after 30 minutes incubation of the sample with hydrogen peroxide at a concentration of 740 mm. Determination of the fraction of surviving cells was performed using the microscope with the dye methylene blue. It is established that the introduction of inhibitor Konitza at a concentration of 0.10 g/l under conditions of oxidative stress (H2About2, 740 mm, 30 min), the fraction of surviving cells is increased 2.1 times.
Example 3. Yeast were grown in the same conditions described in Example 2. The sample was added inhibitor Konitza at a concentration of 1.00 g/l and incubated for 6 hours After incubation, samples were transferred to a Petri dish, which was placed under a germicidal lamp, and perform the irradiation of the yeast suspension hard ultraviolet radiation for 30 seconds. After exposure the sample was counted the number of live and dead cells as described in Example 2 method. When making the inhibitor of Konitza at a concentration of 1.00 g/l when exposed to the culture of hard ultraviolet irradiation for 30 seconds, the percentage of surviving cells is increased in 2.8 times.
Example 4. The culture was grown under the conditions described in Example 2. The sample was added inhibitor Bauman-Birk at a concentration of 1.00 g/l, incubated with hydrogen peroxide and count live and dead cells as in Example 2. When introducing inhibitor Bauman-Birk at a concentration of 1.00 g/l under conditions of oxidative stress (H2O2, 740 mm, 30 min), the fraction of surviving cells is increased in 2,5 times.
Example 5. Yeast were grown in the conditions described in Example 2. Then the sample was added inhibitor Bauman-Birk at a concentration of 1.00 g/l and was carried out with the culture of the operations described in Example 3. After irradiation and counting of live and dead cells was found that the introduction of inhibitor Bauman-Birk at a concentration of 1.00 g/l when exposed to the culture of hard ultraviolet irradiation for 30 seconds, the percentage of surviving cells is increased 4.7 times.
Example 6. The culture was grown under the conditions described in Example 2. The sample was added inhibitor Bauman-Birk at a concentration of 1.00 g/l, incubated with hydrogen peroxide and count live and dead cells as in Example 2. When introducing inhibitor Bauman-Birk at a concentration of 1.00 g/l under conditions of oxidative stress (H2O2, 700 mm, 30 min), the fraction of surviving cells is increased in 2.4 times.
Example 7. The culture was grown under the conditions described in the Example 2. The sample was added inhibitor Konitza at a concentration of 1.00 g/l and incubated with hydrogen peroxide and count live and dead cells as in Example 2. When introducing inhibitor Bauman-Birk at a concentration of 1.00 g/l under conditions of oxidative stress (H2O2, 750 mm, 30 min), the fraction of surviving cells is increased 2.9 times.
As seen from the above examples, the implementation of this protection method of Saccharomyces cerevisiae cells is higher compared to the prototype of the effectiveness of protection from exposure (1.2-5.4 times). In addition, in the entire investigated range of concentrations insertion inhibitors was not observed inhibition of culture growth. Also, despite the increase in the concentration of hydrogen peroxide to a concentration of 700 to 750 mm, 7.0-7.5 times higher in comparison with the prototype, the effect of the introduction of soybean protease inhibitors on the level of the analogue. In conditions of oxidative stress (H2O2, 700-750 mm, 30 min) shows that the inhibitor Konitza increases the proportion of surviving cells in 1.5-3 times in comparison with control, inhibitor Bauman-Birk - 1.5-2.5 times. In the next series of experiments, the stress induced by irradiation hard with ultraviolet light under similar conditions of treatment with inhibitors at the same concentrations. In this case, the inhibitor Konitza increases the proportion of surviving cells in 1,2-2,7 RA is compared with the control, inhibitor Bauman-Birk - 2.3-4.7 times. The survival of yeast cells under irradiation is higher in comparison with the prototype, and the proportion of live cells treated with hydrogen peroxide, is at the level of the prototype despite the increase in the concentration of peroxide to a concentration of 700 to 750 mm (7.0-7.5 times higher compared to the prototype), indicating that higher antioxid based activity of protease inhibitors. In addition, unlike the prototype is not observed oppression of culture in the whole investigated range of concentrations of protease inhibitors.
The remedy yeast Saccharomyces cerevisiae from oxidative stress due to exposure to hydrogen peroxide, which includes the cultivation of yeast culture in standard conditions until late logarithmic or early stationary phase of growth, incubation with a protective agent, the effect of hydrogen peroxide with subsequent determination of the number of surviving cells, characterized in that as protective agents use soy protease inhibitors, and the time of incubation with the protective agent is 5-6 h, the concentration of the protective agent is 0.1 to 2.0 g/l, the concentration of hydrogen peroxide is 700-750 mm.
SUBSTANCE: composition for production of organosilicon sol-gel matrix for immobilisation of microorganisms in biosensor analysers is provided. The composition consists of 20% polyethyleneglycol solution in phosphate buffer solution, Tetraethoxysilane, and 0.2 mol/dm3 catalyst solution NaF, an additionally introduced hydrophobic additive - methyltriethoxysilane. The components are taken at a volume ratio of PEG:TES:MTES: NaF 4:(18-3.4) (2-16.6):1.
EFFECT: reducing the toxic effect of the matrix on the biomaterial and increase in its mechanical strength.
1 tbl, 1 ex
SUBSTANCE: present invention relates to microbiology and a method of detecting and counting viable Legionella pneumophila microorganisms in a sample. The described method involves: (1) contacting said microorganisms of said sample with at least one reducing compound which contains glutamate and pyruvate, and a culture medium which is a buffered charcoal yeast (BCYE) or GVPC agar culture medium, (2) incubating the product of step (1), and (3) detecting and determining the number of viable microorganisms. The reducing compound used directly or indirectly affects metabolism, reducing oxidative stress of the microorganism by reducing formation and/or breaking down reactive forms of oxygen.
EFFECT: disclosed method enables to accurately count Legionella pneumophila microorganisms in a stressed condition.
7 cl, 5 dwg, 5 ex
SUBSTANCE: method of qualitative assessment of biocorrosion damages of thin-walled sealed enclosures of aluminium-magnesium alloys in operation of spacecrafts and the suspension of spore materials fungi of implementation of the said method is proposed. The test and control samples of aluminium and magnesium alloys are prepared. The prepared samples are dried, sterilised. Fungal cultures - strains of microorganisms Paecilomyces variotii Bainier of All-Russian collection of microorganisms F-4039D, Ulocladium botrytis Preuss of All-Russian collection of microorganisms F-4032D, Penicillium chrysogenum Thorn of All-Russian collection of microorganisms F-4034D, Aspergillus sydowii (Bainier et Sartory) Thorn et Church of All-Russian collection of microorganisms F-4037D, Cladosporium sphaerospermum Penz. of All-Russian collection of microorganisms F-4041D are inoculated for growing spores into tubes with a sloped agar medium of Czapek-Dox. The tubes are thermostated at a temperature of (29+2)°C for 14-28 days until appearance of mature spores. Spore materials suspensions of individual cultures of the said fungi are prepared, which are then mixed in equal proportions. And the concentration of spores of each fungi species in the suspension should be in the range of 1-2 mln/cm3. The resulting suspension is applied to the sterilised test samples. Then they are dried and placed as one sample in each Petri dish on the surface of agar medium of Czapek-Dox. The control samples not treated by the spore material are also placed on the surface of the agar medium of Czapek-Dox in the Petri dishes. The Petri dishes with the test and control samples are placed in different desiccators at the bottom of which water is poured to maintain the humidity more than 90%. The desiccators are closed and incubated in the thermostat at a temperature of (29±2)°C. The exposition of test and control samples is carried out for 40, 120 and 180 days. Then the samples are removed from the Petri dishes, and the corrosion products and mycelium are removed from the surface of the samples washing them in running water. The samples are soaked in 70% ethyl alcohol for 30 minutes, then they are washed with detergent and dried. Then, using a scanning electron microscope a qualitative assessment of biocorrosion damages is carried out. According to the change in the appearance of the surface of the samples the initial stages of biocorrosion and its type is evaluated, as well as the distribution of corrosion damage on the surface of the samples, and the dependence of the biocorrosion process on time is determined.
EFFECT: inventions enable to carry out the qualitative assessment of the initial stages of biocorrosion damages of thin-walled sealed enclosures of aluminium-magnesium alloys with the thickness of more than 2 mm.
2 cl, 9 tbl
SUBSTANCE: invention relates to means of controlling quality of organic and inorganic products, and can be used to evaluate safety of food and fodder products, natural water and waste water, ground, soil, determining the maximum allowable concentration of contaminants, as well as the impact of human activities on the environment, including oil extraction and refining products. The apparatus consists of a computer with a software system and a biodetector. The biodetector has a housing 1 inside of which there is a displacement controller for a board 2 with containers 3 for test objects, a light source - light-emitting diodes 4, an optical system with a television camera 5 and a lens 6 mounted on a support 7. The apparatus is provided with a light-proof casing 8 for closing the top of the board with containers for test objects, the inner surface of which is coated with white paint. The optical system with a television camera 5 and a lens 6 is mounted on the support 7 by a turning mechanism 9 in form of a ball head. Inside the housing 1 there is a stepper motor 10 for rotating the board 2 with containers 3.
EFFECT: invention increases measurement accuracy while reducing the measuring time, simplifying the design and reducing the size of the apparatus.
2 cl, 2 dwg
SUBSTANCE: device comprises a through cell equipped with holes, where at least one hole represents an inlet hole for intake of fluid medium from the specified technological flow, and at least one hole is an outlet hole for discharge of fluid medium from the specified through cell. To one of specified holes an RK probe is attached, possibly, an OVP probe, a cleaning accessory. The first pipeline is connected to the inlet hole. Possibly, the second pipeline is connected to the outlet hole. A valve is connected to the specified through cell. With the help of the specified devices and methods they measure volume microbiological activity and surface microbiological activity in a process flow of water by means of measurement of dissolved oxygen concentration.
EFFECT: method improvement.
45 cl, 10 dwg, 2 tbl, 4 ex
SUBSTANCE: method includes the following stages: contact of a sample with a source of nutrition for cells, containing antioxidant, representing pyroracemic acid or its salt, and an inhibitor of cell proliferation, which is selected from ciprofloxacin and cefalexin; contact of the specified sample with fluorescent-marked oligonucleotide probes, capable of specific hybridisation at least with one section of ribosomal nucleic acids, which belong to microorganisms of Legionella pneumophila kind and type; and detection and quantitative determination of a fluorescent signal.
EFFECT: provided method and set make it possible to more accurately and reliably detect and calculate viable microorganisms of Legionella pneumophila type, having excluded natural fluorescence of microorganisms from calculation.
6 cl, 2 dwg, 6 tbl, 2 ex
SUBSTANCE: present invention relates to chemical industry and agriculture and is a plant growth stimulating agent.
EFFECT: invention enables to produce an agent capable of regulating plant growth or differentiation.
16 cl, 22 ex, 11 dwg
SUBSTANCE: method for prediction of bacterial translocation into blood in generalised periodontitis provides recovery of symbiont strains from gingival pocket biocenosis and comparison of their hemolytic activity (HA), antilysozyme activity (ALA) and growth.
EFFECT: invention enables planning and implementing targeted preventive remedial measures in generalised periodontitis.
3 dwg, 5 tbl, 2 ex
SUBSTANCE: tear is taken by sterile disks made of a porous material to form a microbial lawn and to determine lysocyme activity by lysocyme diffusion from the disk into the microbial lawn. It is followed by incubation in a thermostat for at least 24 hours. A zone of microbial growth retardation is determined by measuring a diameter of the zone of microbial growth retardation. The diameter of the zone of microbial growth retardation in healthy infants is 28-30 mm, while the diameter of the zone of microbial growth retardation in infants suffering inflammatory eye diseases is 20-24 mm.
EFFECT: method enables determining lachrymal lysocyme activity in infants, including newborns.
SUBSTANCE: antioxidant activity of a broth culture of microorganisms or a cell suspension of microorganisms grown on a solid nutrient medium is evaluated. An oxidable medium is presented by a lecithin solution, while peroxide initiators are as follows: Staphylococcus aureus cells, ascorbic acid, ferric (II) ions (in the form of an aqueous solution of ferric sulphate). Antioxidant activity is evaluated by an ability to inhibit lipid peroxidation by adding concentrated orthophosphoric acid, and 1% 2-thiobarbituric acid in dimethyl sulphoxide or ethanol (1:1). The stained complex is extracted in n-butanol, and after a butanol phase separated, it is spectrophotometered with regard to transmission unit at 550 nm wherein a reference tray is a tray with n-butanol. It is combined with preparing two controls wherein test tubes containing an oxidation substrate - lecithin are added with known antioxidant activity, while in the other one initiated peroxidation is enabled with no antioxidant added.
EFFECT: derived values of the antioxidant status are calculated in units of antioxidant activity.
SUBSTANCE: what is declared is a method for inducing cell death ensured by inducing heat shock response in a cell in a combination with inhibition of an adaptive heat shock response. The invention may be used in medicine for preventing cancer, treating intracellular parasitic infections and inflammation-related conditions.
EFFECT: higher clinical effectiveness.
8 cl, 17 dwg, 9 ex
SUBSTANCE: clinical effectiveness is estimated on an simulation model and provides creating a model of bacterial biofilm grown on biolumenescent bacteria Vibrio fischeri. The biofilm is exposed to antibiotics and ultrasound to estimate an antimicrobial effect. The bacterial viability variation may be controlled by varying luminous intensity of the biolumenescent bacteria with using devices, e.g. lumen meters. The antimicrobial effect is estimated by a degree of luminous intensity inhibition as compared to the reference.
EFFECT: method may be applicable in pre-clinical experiments to specify the effective method of treating chronic bacterial infections complicated by bacterial biofilm formation, reduced costs and labour inputs of experiments.
25 dwg, 5 ex
SUBSTANCE: invention relates to microbiology. Biological or artificial fluid medium containing the selected microorganism is centrifuged. Supernatant fluid is filtered. A series of diluted samples corresponding to increase in filtrate dilution of up to 10-15 is obtained. The samples are exposed to an electric, magnetic and/or electromagnetic excitation field. Electric signals detected by a solenoid and the digital record of said electric signal after passing through an analogue-to-digital converter are analysed. Diluted samples for which characteristic electric signals, whose amplitude is 1.5 times higher than that of the background noise signals emitted by water, are obtained are selected. Test tubes with equal volume of diluted samples are placed in protective jackets to protect the diluted solutions from external electromagnetic fields. The solution contained in test tube T1 is used as the standard solution. Test tube T2 is placed in the immediate proximity of the sample or is brought into contact with sample X which is presumed to contain the selected microorganism (e.g., E.coli). The obtained electromagnetic signals are compared. Suppression of the signal indicates presence of the microorganism in sample X.
EFFECT: group of inventions enables to obtain reagents and a system for detecting microorganisms in a sample, and detect infection in humans or animals.
3 ex, 8 dwg
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to field of experimental medicine and oncology. Method of experimental vaccine producing for treatment of Erlich's adenocarcinoma by inoculation of experimental animals with tumour cells of Erlich's adenocarcinoma, includes regulation of vitality of obtained culture cells, and regulation of cells vitality being realised by irradiation of cell culture "in vitro" by electromagnetic field of optical range of wavelength 750-1000 nm, for 10-20 hours with density of falling power 0.4-0.6 mWt/cm2.
EFFECT: method makes it possible to increase cell resource during vaccine production and reduce financial expenditures on initial raw material.
SUBSTANCE: claimed is method of removing protein molecules of external S-layer from surface of purple membranes of halobacteria Halobacterium salinarum cells, under impact of ultrasound. Method consists in the following: suspension of halobacteria cells is subjected to ultrasound impact, with frequency 880 kHz and energy density in medium 0.1 Wt/cm3, for 10 min at temperature 20°C.
EFFECT: method makes it possible to purify surface of purple membranes from bound with it protein complexes without breaking membrane integrity and without application of any other extraneous substances.
2 dwg, 4 ex
SUBSTANCE: method includes filling work chamber with suspension of tumour cells and their aggregates. After that carried out is complex processing of suspension of tumour cells and their aggregates by high-amplitude low-frequency ultrasound and ozone/NO - containing gas mixture, introduced immediately into volume of insonifies and bubbled suspension, as well as ozone/NO-containing solution, which is formed at barbotage of suspension with ozone/NO-containing gas mixture.
EFFECT: invention makes it possible to increase efficiency of process of disintegration of suspension of tumour cells and their aggregates for maximally possible separation from them of biologically active substances.
6 dwg, 1 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to biotechnology and specifically to biophysical methods of acting on biological objects. The method is realised with a single pulse in a vector potential field, as well as with a single pulse and/or packet of separate pulses in a magnetic vector potential field with field strength of 0.2-500 mT. Duration of the single pulses is in the interval of 1·10-6-0.6·102 s, the pulse rise duration is in the interval of 0.0001-0.5 times the pulse duration and the pulse drop duration is in the interval of 0.001-15 times the pulse duration.
EFFECT: invention can be used in different fields of engineering to stimulate growth of biological objects.
7 cl, 4 tbl, 4 ex
SUBSTANCE: mixture of yeast suspension with sterile wort is treated through vibration at frequency of 5-11 Hz and amplitude of oscillations of 4-5 mm for 15-30 minutes with ratio of yeast suspension to wort equal to 1:40-50. The source of vibrations is placed inside the mixture at a distance of 1/3-2/3 from the surface of the mixture.
EFFECT: increased mass of cells of pure yeast culture and increased number of active (live) cells in the ready wine starter, faster wort fermentation which leads to a shorter process of preparing wine materials.
2 tbl, 4 ex
SUBSTANCE: method is based on exposure of an object to be processed to electromagnetic radiation. The EHF radiation of frequency 20 - 95 GHz at power flux density no more than 10 mcWt/cm2 is used. Exposure time is at least 20 minutes.
EFFECT: effective inhibition of the vital activity of various microorganisms exhibiting pathogenic properties with respect to a human body.
5 cl, 3 tbl
SUBSTANCE: invention refers to method of obtaining new tetracycline-sensitive strains of genus Bifidobacteriaum sp., containing mutant gene tet in chromosome, as well as new antibiotic-sensitive strains obtained from antibiotic-resistant probiotic strains.
EFFECT: extension of arsenal of tetracycline-sensitive strains.
8 cl, 9 tbl, 13 ex
SUBSTANCE: alcohol (8-C) strain Saccharomyces cerevisiae No 8 having a high generative activity was deposited in the Russian National Collection of Industrial Microorganisms (RNCIM) under the registration number RNCIM B-3855 and can be used in production of alcohol.
EFFECT: invention enables to increase the alcohol yield and to reduce the formation of byproducts.