Method for evaluation of antioxidant activity of microorganisms

FIELD: medicine.

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.

1 ex

 

The invention relates to Microbiology and clinical laboratory diagnostics and can be used as an option to determine the antioxidant potential of microorganisms in the evaluation of antioxidant status and microecological features of the human body.

Description of the invention

The invention relates to Microbiology and clinical laboratory diagnostics and can be used as an alternative determination of antioxidant potential of microorganisms in assessing the microbial status of the human body. Typical pathological processes, such as hypoxia and inflammation, characteristic and developing in the majority of somatic and infectious diseases, severe injuries and wounds, always accompanied by excessive formation of reactive oxygen species, which leads to the strengthening of processes of lipid peroxidation, are part of cell membranes [1]. A significant contribution in maintaining the redox balance of the body makes normal microflora [2].

The known method EN No. 2112241, C16G01N 33/48, G01N 33/50 (date of publication of the claims 1998.05.27) determine the concentration of malondialdehyde using thiobarbituric acid (TBA). In this method for analysis using the minimum amount of serum or homogenate (0.2-0.5 ml), Rast is orange TBQ and incubation of the sample is carried out in the presence of Triton X-100, the mixture is stirred with a constant oscillation frequency (120 oscillations / min), the reaction is stopped dihydroquercetin; before determining the optical density of the sample add Trilon B and a mixture of ethanol and chloroform in the ratio (7:3). The method is based on the reaction between malonic dialdehyde (MDA) and thiobarbituric acid, which is at high temperature and acidic pH value proceeds with the formation of colored trimethylboron complex containing one molecule of MDA and two molecules thiobarbiturate acid. The maximum absorption of the complex have at 532 nm. The working solution thiobarbituric acid (TBA) prepared by dissolving weighed TBQ 864 mg in 100 ml of a mixture containing 1% solution of Triton X-100 and 8.2 M solution of ethanol.

However, this method has several drawbacks: it does not take into account the peculiarities of the metabolism of microorganisms to improve the solubility of the lipid fractions and 2-thiobarbituric acid is proposed to use a special reagent (polyoxyethylene-p-(tert-octyl) phenol (Triton X-100)and the reagent that require special conditions of storage and work (trichloromethane). Closest to the claimed method is detection of microorganisms protective actions in the effect of Fenton EN 2279079, C2 G01N 33/483, C12Q 1/02.

In the present method of detection of microorganisms protective actions in the effect of Fe the tone of the studied culture of microorganisms are grown in a liquid nutrient medium, separate the supernatant from the microbial cells by centrifugation, simultaneously prepare the control, representing a liquid nutrient medium, then each of the samples treated with chloroform and separate the supernatant from the chloroform by centrifugation, and then in the experimental sample and the control add test-culture of Staphylococcus aureus risk No. 201108, incubated separating the cells of the test culture by centrifugation and preparing microbial suspension in each of the samples add a solution of hydrogen peroxide and incubated separating the cells of the test culture by centrifugation, add to them a nutrient medium, incubated, measure the optical density of the grown crops in experimental and control samples, determine the degree of growth biomass of the test culture in the experience. The disadvantage of this method is the use as an indicator of a protective effect of microorganisms only the degree of increase in cell mass optical density. Therefore, the method does not take into account the kinetic parameters that occur when you add in the studied system of prooxidants. The method characterizes the duration and relative multistage. The results of the experiment require several stages of incubation, which should be chosen with consideration of the physiology of microorganisms. The considered method is not possible to determine quantitative characteristics, the OTP is concerned with antioxidant potential of microorganisms, members of the microbiocenosis of the person. Determination of protective action of the studied culture in the effect of Fenton, according to this method, it is possible only at presence in the environment of low concentrations of hydrogen peroxide.

The task of the invention is to improve the known method for determining the antioxidant activity, as well as the possibility of using the proposed method for the determination of antioxidant status in microorganisms that do not produce hydrogen peroxide and/or inactivating its enzymes.

The essence of this method is the determination of the antioxidant status of microorganisms for their ability to block the reaction of lipid peroxidation and/or to inhibit phospholipase.

Method for determination of antioxidant activity of microorganisms is that evaluated the antioxidant activity of broth cultures of microorganisms or the suspension of microorganisms in saline grown on solid nutrient medium. In the proposed method, as the oxidizable medium is a solution of lecithin. It is known that under the action of phospholipases from lecithin cleaved one of the fatty acids, turning it into lysolecithin. This product has a high toxicity in the cells, causing them to necrosis [1]. The proposed method Perek the red oxidation of lecithin is started by adding the cell suspension of microorganisms - producers of phospholipases, isolated from the subject, and in their absence, uses the well-known Museum strain phospholipase activity. Then, on Wednesday sequentially added to an aqueous solution of ascorbic acid, iron ions (II) (in the form of a solution of iron sulfate). After that analyzed the samples incubated for 30 min at a temperature of 42C, With constant shaking. After that, the sample make a concentrated phosphoric acid to pH 1, and a 1% solution of 2-thiobarbituric acid in dimethyl sulfoxide and ethanol (1:1). The reaction mixture is heated in a water bath at t=70C for 20 min, cooled in a dark place to a temperature of +4C and add 3 ml of n-butanol are added, thoroughly mixed, centrifuged at 3 thousand rpm, separate botanology phase and spectrophotometric, given the minimum transmittance at 550 nm. When spectrophotometrically the comparison cuvette cuvette is with n-butanol. In the inventive method uses several controls: control the maximum possible peroxidation reactions (ongoing studies)used for this sample of lecithin-initiated oxidation without antioxidant and control of inhibition of lipid peroxidation, where the specimen using equimolar quantities of substances with known antioxidant activity is updated. To compare the antioxidant status of substances in the hydrophilic phase used an aqueous solution of 6-methyl-2-ethyl-pyridine-3-ol, and substances with lipophilic properties as the reference sample is used tocopherol acetate - oil solution.

To quantify the antioxidant activity is expressed as arbitrary units of antioxidant activity (Ethe AoA)calculated by the formula

where

Ethe AoA- units of antioxidant activity,

IA- the intensity of the luminous flux (in units of bandwidth), passed through the layer of the absorbing solution of the sample at 550 nm,

IB- the intensity of the luminous flux (in units of bandwidth), passed through the absorbing layer of nutrient solution at 550 nm,

IC- the intensity of the luminous flux (in units of bandwidth), passed through the layer of the absorbing solution control with induced oxidation at 550 nm,

10 is the conversion factor in units of volume,

IE- the molar ratio trimethylboron complex in units of bandwidth 0,413 mmolcm-1,

ID- the intensity of the luminous flux (in units of bandwidth), passed through the layer of the absorbing solution control with known antioxidant at 550 nm.

Example 1.

Culture of Staphylococcus aureus isolated from Ki is echnolo microbiocenosis of the subject, grew up on a meat-peptone agar at 37C for 48 hours and Then suspended in sterile 0.9% sodium chloride solution and standardized by the turbidity standard to 109microbial cells in 1 ml

Isolated from feces of the patient's strain of Bifidobacterium breve, were grown in liquid nutrient medium, Blaurock. On the day of the studies prepared solutions: lecithin 0.1%of ascorbic acid 0,M, iron sulfate 0.07 m in bidistilled, svejeprokipachena and cooled to room temperature water.

In the experimental tube was added 0.5 ml of a solution of lecithin, 0.3 ml of the bacterial substrate, investigated the antioxidant activity - .breve, and 0.8 ml of ascorbic acid solution. The reaction of free radical formation was initiated by addition of 0.8 ml of a solution of iron sulfate. The tubes were placed in a thermostat at a constant shaking for 30 min In the quality control reference antioxidant used a 0.3 ml solution emoxipin. As a control of the maximum possible peroxidation used the test tube with the sample of lecithin, where a process has been initiated in the absence of an antioxidant. The investigated tubes were placed in a thermostat at 30 min, at a temperature of 42C, With constant shaking. After that, in the analyzed samples had consistently contributed 0.2 ml of concentrated phosphoric acid and 0.8 is l 1% solution of 2-thiobarbituric acid. The resulting reaction mixture was heated in a water bath at t=70C for 20 min, cooled in a dark place to a temperature of +4C and with thorough stirring, was added 3 ml of n-butanol. The resulting emulsion was centrifuged at 3 thousand rpm and gently separated botanology phase, which was spectrophotometrically on the spectrophotometer SF-2000, taking into account the unit of transmission at 550 nm. As the cell comparisons used a cell with n-butanol. Substituting these values into the formula, calculated that for the analyzed strain Bifidobacterium breve antioxidant activity was 3,03 Ethe AoA.

Sources of information

1. Shanin VY Clinical pathophysiology. Saint-Petersburg.: "Special literature", 1998, 569 S.

2. Shenderov B.A. Medical microbial ecology and functional nutrition 3 so / Bassendean. - M.: the Verge, 2001, Vol.3, 288 S.

The method of quantitative determination of antioxidant activity of microorganisms is that evaluated the antioxidant activity broth cultures of microorganisms or suspension of microorganism cells grown on solid nutrient medium, characterized in that as the oxidizable medium is a solution of lecithin, and as initiators of lipid peroxidation: sequentially added to a suspension of Staphylococcus aureus cells in physiological solution, the solution ASKO is benovoy acid, iron ions (II) (in the form of an aqueous solution of iron sulfate), incubated for 30 min at a temperature of t=42C, with constant shaking, then zachisliaut to pH 1 by adding concentrated phosphoric acid and 1%solution of 2-thiobarbituric acid in dimethyl sulfoxide and ethanol (1:1), heated in a water bath at t=70C for 20 minutes, cool, add 3 ml of n-butanol are added, thoroughly mixed, centrifuged at 3 thousand rpm, separate botanology phase and spectrophotometric, given the unit of transmission at 550 nm, where as the cell comparison using a cuvette containing n-butanol, and controls use of the test tube in which the substrate oxidation - lecithin in one added substance with known antioxidant activity, and the other initiated by peroxide oxidation is carried out without addition of antioxidant.



 

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