RU2688117C1 - Method for evaluating pro- and antimicrobial properties of samples - Google Patents

Abstract

FIELD: measurement technology.

SUBSTANCE: invention relates to bio-measuring technologies. Method for evaluating the pro- and antimicrobial properties of samples is proposed. Method comprises incubating the test microorganisms Lactobacillus sp. in quantities of 5×10⁵ to 5×10⁶ viable cells per ml in a liquid nutrient medium pH 6.6–7.4 for 4–8 hours at a temperature of from 20 to 45 °C in the presence of test samples and in their absence. Measure pH, redox potential (E) and electrical conductivity (X) of the samples. Total degree of activation or inhibition (+/-) of the test microorganisms activity by the tested samples is determined using formula: ε_S=(ε_pH+0.4ε_E+0.6ε_X)/2, where ε_pH, ε_E and ε_X determined by the formulas ε_Y=100×(ΔYt-ΔYc)/ΔYc where ΔY=Ye-Yb are differences in pH, E or X values recorded at the beginning (Yb) and at the end (Ye) of the incubation of test samples in the presence of test samples (ΔYt) and in their absence (ΔYc).

EFFECT: method provides an objective assessment of the pro- and antimicrobial properties of samples, including those containing actively light-absorbing or light-scattering substances and material.

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Description

The invention relates to the field of bio-measuring technologies, and in particular to methods of evaluation with the help of test microorganisms of the pro- and anti-microbial properties of various samples (including samples containing actively light-absorbing or light-scattering substances and materials that interfere with the optical analysis of the overall composition of such samples).

Known "Method for determining the sensitivity of microorganisms to antimicrobial substances" (RU patent No. 2505813, IPC G01N 33/48, priority date 06.11.2012, published 01/27/2014), in which the antimicrobial activity of the tested substances in relation to a particular biomaterial is proposed to determine based on the number of colonies of microorganisms, initially contained in the said biomaterial and grown on a dense accumulative nutrient medium in the presence of the test preparation, as well as the time of appearance of the visible growth of these colonies. The disadvantages of this method are its duration (up to 3 days), large labor intensity, high materials consumption for nutrient media components, and also the fact that the antimicrobial activity of the tested substances is determined visually, and therefore, quite subjective.

The “Method for evaluating the effectiveness of antimicrobial effects of antiseptics on bacteria existing in the form of a biofilm” (RU patent No. 2603100, IPC C12Q, priority date 10.29.2015, published on 11/20/2016) is known, in which the effectiveness of the action of antiseptics on bacteria isolated from the patient’s clinical material and existing in a liquid nutrient medium (RPS) in the form of a biofilm, it is proposed to determine how the ratio of the working concentration of an antiseptic to its minimum inhibitory concentration. At the same time, it is proposed to record the effectiveness of a given concentration of a test antiseptic on a biofilm using a photometer — for discoloration and clouding of RIP with test microorganisms, incubated at a given temperature for a specified time (from 5 days or more) in the presence of the test antiseptic taken in the said concentration. The main disadvantages of this method, as in the previous cases, are its long duration and laboriousness, as well as low objectivity, because this method allows to evaluate the effect of test substances only on the growth rate of test microorganisms, but not on the intensity of their metabolism.

Closest to the proposed technical solution is the "Method for determining the bactericidal properties of serum" (RU patent No. 2489489, IPC C12Q 1/18, priority date 12/26/2011, published on 10.08.2013), in which the bactericidal properties of serum samples are determined by reducing the intensity luminescence incubated in LB-broth in the presence of test samples of the test transgenic Bacillus subtilis strain VKPM B-10548, effectively expressing the luxAB genes of the gram-negative marine microorganism Photobacterium leiognathi (as a result of which erment luciferase providing active illumination, the intensity of which depends on the total number of viable test microorganisms and their metabolic activity). When this analysis is as follows:

At the first (preparatory) stage, the Bacillus subtilis VKPM B-10548 strain is grown on LB agar in the presence of kanamycin at a final concentration of 40 μg / ml (which allows to exclude the growth of non-luminescent strains) at 37 ° C for 24 hours. The resulting biomass is washed off with sterile LB-broth, after which the resulting suspension is standardized to an optical density of 1.2 rel. units at 540 nm (measured in cuvettes with an optical path length of 1 cm);

at the second (main) stage, a suspension of bacterial cells and a test serum sample in a 1: 1 ratio are mixed and the mixture is incubated at 37 ° C for 30 minutes. At the same time, before and after incubation, 250 μl aliquots are taken from the test mixture into the cuvettes for the bioluminometer. And additionally add 2 µl of decanal in them at a final concentration of 7 × 10 ^-6 M (oxidized in the presence of molecular oxygen by the enzyme luciferase - which ensures the active luminescence of the test strain).

At the final stage, the emission level is measured in samples in the visible blue-green region of the spectrum (420–600). At the same time, the biochemiluminometer “Biotok-10M” can be used as a measuring instrument, as well as other domestic and foreign luminometers operating in the designated spectral region and providing appropriate measurement sensitivity.

The main disadvantages of this technical solution include:

- narrow field of application (analysis of bactericidal properties of only human and animal serum);

- the need to use a specially bred, transgenic, highly specialized strain of test microorganisms - which further limits the scope of this technical solution, since different testing organisms may be required for testing the pro and antibiotic properties of different chemical and physical factors (or even a combination of several different types and strains of test organisms);

- in addition, the strain Bacillus subtilis VKPM B-10548, used in the implementation of this technical solution, is not widely available, and therefore it is difficult to first obtain this strain, and then for a long time to maintain its purity, viability and specific properties (expressed, in particular , in the active synthesis of the enzyme luciferase);

- as well as the fact that the method under consideration does not have a very high objectivity, since the effect of test objects (which in this case are serum samples of blood of animals and humans) on test organisms is determined only by one indirect parameter (intensity of chemiluminescence of these objects) and using a test culture with a metabolism that is not typical in a number of parameters for most living organisms.

Due to the steady growth in the production and consumption by human society of various products, the problem of developing fairly rapid, objective, simple, cheap and widely available methods for evaluating the pro- and antibiotic properties of various substances, materials, preparations, food and other products, waste of various industries, etc.

In connection with the foregoing, the purpose of the claimed invention was to create a method that allows, in comparison with the prototype, a wider circle of users with more objectivity to evaluate the pro- and antimicrobial properties of a much wider range of tested samples (including samples containing actively absorbing or scattering interfering with the optical analysis of the overall composition of such samples).

This goal is achieved due to the fact that in the proposed method (which includes such operations as: preparing test samples, incubating these samples both in the presence and absence of the test samples, accompanied by measuring the properties of these samples at the beginning and end of their incubation, and the subsequent determination of pro- and anti-microbial properties of the test samples on the basis of said measurement) test samples before incubating Lactobacillus sp.v represent an amount of 5 × ¹⁰ May to ¹⁰ June 5 × viable cells per ml, sous pendirovannuyu in aqueous solution initially contains 35-45 g / L of dry milk and having a pH of 6.6-7.4; incubation of these samples in the presence of test samples is carried out for 4-8 hours at a temperature that is optimal for the development of the Lactobacillus strain chosen as the test (in the range from 20 to 45 ° C); the properties of the samples are determined by measuring the pH, redox potential (E) and electrical conductivity (X); after which the total degree of activation or inhibition (+/-) of the vital activity of test microorganisms by the tested samples is determined by the formula:

ε _S = (ε _pH + 0.4 ε _E + 0.6 ε _X ) / 2,

where ε _pH , ε _E and ε _X is determined by the formulas ε _Y = 100 × (ΔYt-ΔYc) / ΔYc,

where ΔY = Ye-Yb are the differences in pH, Е or X values recorded at the beginning (Yb) and at the end (Ye) of the incubation of test samples in the presence of test samples (ΔYt) and in their absence (ΔYc).

At the same time, the choice of Lactobacillus as a test microorganism is associated with the fact that lactic acid bacteria are widely distributed in nature, possessing a developed enzyme system, are actively involved in the natural decomposition of many natural and anthropogenic materials, are easily cultivated and, owing to all of the above, are widely used in various methods of biotesting, scientific research, as well as biotechnological processes.

The choice of the number of viable test microorganisms that must be present in the test samples before they are incubated in the presence and absence of test samples (from 5 × 10 ⁵ to 5 × 10 ⁶ cells / ml) is determined by the fact that with a smaller number of such microorganisms, the duration increases. incubation required to achieve maximum sensitivity of the proposed method of analysis; and with a larger number of test microorganisms, the intensity of their growth and metabolism decreases, which leads to a decrease in the sensitivity of the analysis.

The choice of the initial composition of the liquid nutrient medium, which is part of the test samples (aqueous solution with a pH of 6.6-7.4, containing 35-45 g / l of milk powder), is associated with the need to ensure optimal conditions for the development of test microorganisms in this medium - that, as in the previous case, it is necessary to increase the sensitivity and reduce the duration of the analysis.

The choice of incubation of test samples (4-8 hours at 20-45 ° C) is due to the fact that with longer incubation, the growth rate and metabolism of test microorganisms decrease (which leads to a corresponding decrease in the sensitivity of the assay); at other incubation temperatures, the growth rate and metabolism of test microorganisms also decrease (which leads to a decrease in the sensitivity of the analysis and the need to increase its duration); and with shorter incubation times, the test microorganisms do not have enough time to react to changes in their environment caused by the presence of test samples there (which leads to a decrease in both sensitivity and objectivity of the analysis).

The increase (compared with the prototype) of objectivity and applicability of the proposed method of analysis is ensured by the fact that during it an accurate, instrumental, quantitative assessment of changes in 3 different parameters (pH, E and X) characterizing the metabolic activity of conventional test microorganisms (Lactobacillus sp.) due to exposure to a wide range of test samples (including samples containing actively light absorbing or light scattering substances and materials interfering with optical analysis of the total leaving such samples). While in the prototype, the effect of the tested samples on test microorganisms was determined by only one parameter of the test sample (namely, the intensity of its chemiluminescence), which indirectly characterizes the metabolic activity of test microorganisms. Moreover, this method proposed to evaluate the bactericidal properties of a very narrow range of tested samples (namely, human and animal serum), and also, using a transgenic, highly specialized strain of test microorganisms (Bacillus subtilis VKPM B-10548) with a rather atypical metabolism.

And the availability of the proposed method of analysis to a wider circle of users is ensured, firstly, by the possibility of using such simple to use, affordable and cheap, portable devices, such as the Expert-001 ion meter and the Expert-002 conductometer, for recording the parameters of test samples. Secondly, the use of a minimum number of additional reagents and manual operations to sow and assess the growth and metabolic activity of test microorganisms in the presence of test samples when analyzing (while in the prototype it was proposed to use such specific chemical reagents as kanamycin, decanal, etc. ., as well as to carry out many additional manual operations for growing test microorganisms on LB agar and the subsequent preparation of a suspension of bacterial cells in LB broth). And thirdly, the ability to use for analysis such well-available and easily cultured test microorganisms as Lactobacillus sp. (While in the prototype it was proposed to use the Bacillus subtilis VKPM B-10548 strain as a test microorganism, which is poorly available and difficult to cultivate the need to maintain the required degree of purity and viability of this microbiological culture, necessary for the implementation of Bacillus subtilis VKPM B-10548 such specific properties as, in particular, the active synthesis of the luciferase enzyme).

The implementation of the proposed method of evaluation of Pro - and antimicrobial properties of the samples is as follows:

Step 1. Preparing a nutrient medium (PS), which is a sterile aqueous solution with a pH of 6.6-7.4, containing 35-45 g / l of dry milk. Then all containers with PS are stored in a tightly closed form, in the absence of light, at 2-4 ° C.

Stage 2. Selected test samples (TP); after which they are delivered to the laboratory and stored there until the beginning of the analysis in a dark place, in hermetically sealed form, at 2-4 ° C.

Step 3. Prepare the initial test sample (TO). For which, 1-10 vol. Sterile is inserted into one large container with PS. % starter containing PS suspended in viable cells of Lactobacillus sp. After that, the mentioned container is hermetically sealed and incubated for 12 hours at a temperature that is optimal for the development of the Lactobacillus strain chosen as a test (from the range of 20 to 45 ° C).

Then in the above capacity pH is measured. And according to the previously constructed calibration graph (representing the pH of the maintenance ratio as a function of the concentration of Lactobacillus sp. Cells contained in it), it is confirmed that the obtained pH value corresponds to the concentration of Lactobacillus sp. in TO from 5 × 10 ⁵ to 5 × 10 ⁶ cells per ml (cells / ml).

After that, if the measured pH value corresponds to the concentration of Lactobacillus sp. In THAT is less than 5 × 10 ⁵ cells / ml; the container with TO is incubated under the same conditions as before for another 6 hours; after which the pH is re-measured. And if the measured pH value corresponds to the concentration of Lactobacillus sp.in THEN is greater than 5 × 10 ⁶ cells / ml; then THEN in the same container is diluted with the required amount of PS.

Step 4. If we are interested in the concentration dependence of the pro- or antimicrobial action of the tested samples, or these samples have too high initial toxicity (so it is expected that, undiluted, they will inhibit the development of test microorganisms too much), then the required dilution of the original samples is made. . After that, each sample or its dilution is divided into 3-5 equal parts, each of which is placed in a separate container, which also adds a specified amount of TO, prepared at the 3rd stage of analysis.

Stage 5. All containers prepared at the 4th stage of the analysis, plus a certain number of control containers containing THAT without the addition of the tested samples (and, if necessary, THAT with a specified amount of a substance or mixture of substances whose pro- or antimicrobial activity is known in advance) are closed, mixed and placed in a thermostat, where they are incubated for 4-8 hours at a temperature that is optimal for the development of the Lactobacillus strain selected as a test (from the range of 20 to 45 ° C). At the same time, immediately before the start and after the end of this incubation, the values of such TO parameters (changing as a result of the growth and reproduction of test microorganisms there, as well as their conversion during the metabolic activity of some substances that make up the TO) into others, are recorded in all the mentioned containers as pH , redox potential (E) and electrical conductivity (X).

Step 6. After that, the total degree of activation or inhibition (+/-) of the vital activity of the test microorganisms by the tested samples is calculated by the formula ε _S = (ε _pH + 0.4ε _E + 0.6 ε _X ) / 2,

where ε _pH , ε _E and ε _X is determined by the formulas ε _Y = 100 × (ΔYt-ΔYc) / ΔYc,

where ΔY = Ye-Yb are the differences in pH, Е or X values recorded at the beginning (Yb) and at the end (Ye) of the incubation of TO in the presence of test samples (ΔYt) and in their absence (ΔYc).

The mentioned method can be implemented using such basic measuring devices as the Expert-001 ion meter (or another, which allows measuring pH and redox potential of liquid, water samples with a sufficient degree of sensitivity) and the Expert-002 conductometer (or another, allowing with a sufficient degree of sensitivity to measure the change in the electrical conductivity of liquid, water samples).

In more detail, the present invention is described in the following specific example. The probiotic properties of fine water-insoluble powders containing lignin, buckwheat and β-glucan, isolated from oyster mushroom (Pleurotis ostreatus), were studied. Each of these powders in the amount of 0.2 g was added (3 tubes in parallel) to 6 ml of test medium (TC), which was used as suspensions containing about 10 ⁶ viable cells of Lactobacillus acidophilus ATCC 4356, Lactobacillus helveticus ATCC 8018 or Lactobacillus kefiri ATCC 35411 in 1 ml of an aqueous solution, initially sterile, having a pH of 7.0 ± 0.2 and containing 40 g / l of skim milk powder. Then all these tubes (including 9 control tubes containing the same TS, but without adding any of the tested materials) were mixed, closed with cotton-gauze plugs and incubated at 30 ° C for 7 hours. At the same time, immediately before the start of incubation and immediately after its completion, the pH, redox potential (E) and electrical conductivity (X) were recorded successively at the vehicle in each of the tubes. Moreover, the values of pH and E were recorded with the help of ion-meter “Expert-001” with combined electrodes “ЭСК-10601/7” and “ЭРП-105”. And the X values were recorded using an Expert-002 conductometer with a UEP-P-S sensor operating at 1.6 kHz.

Further, the changes in each of the measured TS parameters were calculated using the formula: ΔY = Ye-Yb (where Yb and Ye are pH, E or X values recorded in each of the control and test tubes at the beginning and end of their incubation). Then, all obtained ΔY _i values were averaged (by test tubes containing the same materials in the same TS), and for each of the averaged values, a 95% confidence interval was calculated. Then, the total degree of activation or inhibition (+/-) of the life of the test microorganisms by the tested materials was calculated by the formula: ε _S = (ε _pH + 0.4ε _E + 0.6 ε _X ) / 2,

where ε _Y = 100 × (ΔYt-ΔYc) / ΔYc, a ΔYt and ΔYc are changes in pH, E or X values that occurred during the incubation of the TS in the presence of test materials (ΔYt) and in their absence (ΔYc). The results of these calculations are presented in table 1.

From the presented it is clear that in different species and strains of Lactobacillus, the reaction to the presence of the tested samples was, on the whole, largely similar. At the same time, lignin least activated the vital activity of test microorganisms (obviously, only by providing the possibility of sorption of test microorganisms on its surface). While for β-glucan and buckwheat, this effect manifested itself to a greater extent (since, in addition to sorption of test microorganisms on their surface, the latter obviously had the opportunity to use part of the substances that make up the tested materials in their metabolic processes).

Thus, the inventive method can expand the scope of evaluation of the pro- and antimicrobial properties of various samples (including samples containing actively light-absorbing or light-scattering substances and materials that interfere with the optical analysis of the overall composition of such samples), as well as increasing the objectivity of such an assessment and its availability for use by a wider range of users.

Claims (4)

A method for evaluating the pro- and antimicrobial properties of various samples, including the preparation of test samples containing a sufficient number of vital test microorganisms suspended in initially sterile liquid nutrient medium, incubating these samples both in the presence and absence of the test samples, followed by measuring the properties of test samples at the beginning and at the end of their incubation, and the subsequent evaluation of the pro- and antimicrobial properties of the tested samples, characterized in that the test microorganisms are dstavlyayut a Lactobacillus sp., the number of which in the test samples before incubation latter should be from 5 × ¹⁰ May to 5 × ¹⁰ June viable cells per ml of the liquid medium used to prepare the test samples, it is a sterile aqueous solution of pH 6 , 6-7.4, containing 35-45 g / l of dry milk, incubation of test samples is carried out for 4-8 hours at a temperature that is optimal for the development of the strain of Lactobacillus sp., Selected as a test (in the range from 20 to 45 ° C), the properties of the samples are determined by measuring Ia pH, redox potential (E) and electrical conductivity (X), after which the total degree of activation or inhibition of (+/-) test microorganisms test sample is determined by the formula: ε _S = (ε _pH + 0.4 ε _E + 0.6 ε _X ) / 2, where ε _pH , ε _E and ε _X is determined by the formulas ε _Y = 100 × (ΔYt-ΔYc) / ΔYc, where ΔY = Ye-Yb are the differences in pH, E or X values recorded at the beginning (Yb) and at the end (Ye) of the incubation of test samples in the presence of test samples (ΔYt) and in their absence (ΔYc).