Method to protect yeast saccharomyces cerevisiae against oxidising stress caused by exposure to hydrogen peroxide

FIELD: biotechnologies.

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.

7 ex

 

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.



 

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