RU2802074C1 - Method for cryopreservation of autologous vaginal lactobacilli - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method is proposed for preserving autologous lactobacilli in samples of vaginal discharge and pure cultures of certain types of autologous lactobacilli isolated from the microbiota of the vagina of women on dense universal and selective nutrient media, which consists in the fact that 1-3 ml of vaginal discharge is suspended with a concentration of lactobacilli of at least 10 ⁷ CFU /ml (GE/ml), in a ratio of 1:2-5 in a cryoprotective medium of a given composition, as well as pure cultures isolated from the vaginal microbiota, washed off from the surface of a dense nutrient medium from the specified cryoprotective medium in a final concentration of lactobacilli 4 units according to McFarland - 1.2× 10 ⁹ CFU / ml, mixed, poured 1 ml into 2 ml cryotubes, closed hermetically and frozen at appropriate temperatures in low-temperature storage facilities, using as needed, previously restored by thawing by heating in a water bath at a temperature of plus 37°C for 60-120 s.

EFFECT: invention ensures preservation of the titre and viability of lactobacilli during storage at low temperatures.

3 cl, 5 tbl, 2 ex

Description

The invention relates to medicine, namely to biotechnology, microbiology, obstetrics and gynecology, and is intended for cryopreservation of autologous vaginal lactobacilli.

The field to which the invention relates

The invention relates to the field of microbiology and can be used in the study of human microflora, microbiological diagnosis of dysbiotic disorders (bacterial vaginosis) and infectious diseases of the vagina, as well as in order to preserve bacteria in the working collection for microbiological research.

The invention can also be attributed to the field of biotechnology, in particular, to methods for preserving bacteria in human vaginal fluid and bacteria at low temperatures, including cryopreservation.

The invention can also be attributed to the field of obstetrics and gynecology, in particular, to methods for preserving the vaginal microbiota in order to subsequently create autoprobiotics for the treatment of infectious diseases and dysbiotic disorders of the female reproductive tract; cryopreserved normal vaginal microbiota can also be used for transplantation to treat infectious diseases and dysbiotic disorders of the female reproductive tract.

The microbiocenosis of the female vagina is a complex dynamically developing hormonally dependent ecosystem that undergoes changes throughout a woman's life [1]. A distinctive feature of the vaginal microbiota of the reproductive period is the multicomponent composition determined by its resident and transient components. The obligate resident microorganisms of the vagina of a healthy woman include lactobacilli, which absolutely dominate, making up 95-98% of the microbial pool. The main biological properties of representatives of the genus Lactobacillus, providing colonization resistance of the vaginal biotope, are adhesive and antagonistic activity, as well as resistance to high acidity (pH) of the environment. The antagonistic properties of lactobacilli are associated with the production of organic acids (lactic), hydrogen peroxide and antimicrobial substances of a protein nature, similar to antibiotics (bacteriocins) [2,3]. The transient component of the microbiota of healthy women, represented by opportunistic pathogens (OPM), colonizes the vagina in low titers due to competitive restraint, and its presence is considered as a variant of the norm. Under the influence of endogenous and exogenous influences on the body of a woman, a violation of the state of harmonious balance between lactobacilli and UPM can occur, which leads to an imbalance in the microflora and the development of an infectious process. The most significant vaginal infections caused by UPM are bacterial vaginosis, aerobic vaginitis, vulvovaginal candidiasis, and a combination of both. Opportunistic infections of the vagina are characterized by polyetiology and frequent recurrence. Currently, there is a significant variety of treatments for opportunistic infections of the vagina and vulva. However, the question of safe and effective prevention remains open. The use of probiotic preparations for the treatment and prevention of opportunistic vaginal infections has become widespread, however, most of the existing probiotics are based on lactobacilli of intestinal origin, which do not take root in the vaginal biotope, but only temporarily create conditions to support their own lactoflora. Currently, the possibility of using one's own protective microflora to restore the normobiota of the vagina is becoming more and more relevant [4, 5]. However, the technology for obtaining, long-term storage and assessment of the safety of the functional properties of autologous lactobacilli has not been developed enough.

The proposed technical solution makes it possible to preserve bacteria in a woman's vaginal fluid under natural conditions and bacterial cultures (protective lactobacilli) isolated from the vaginal microbiota at low temperatures, including cryopreservation. The technique meets the criteria for high bacterial survival, as well as maintaining the initial ratio of various groups of microorganisms after defrosting.

The invention relates to the field of medicine, namely industrial and clinical microbiology, obstetrics and gynecology, and can be used for the preparation of useful microorganisms for long-term storage, suitable for transplantation, as well as the creation of autoprobiotics.

The problem of preserving the human vaginal microbiota covers both medical and microecological aspects of preserving the biodiversity of microorganisms for subsequent use in industrial and biomedical technologies.

Currently, the most reliable way in many respects to preserve the human vaginal microbiota is freezing and preserving symbiotic microbial communities at low temperatures, including in liquid nitrogen.

However, despite significant advances in cryomicrobiology, the problem of freezing and preserving complex microbiocenoses at low (minus 80°–95°C) and extremely low (in liquid nitrogen minus 196°C) temperatures has not yet been solved. The lack of a generally accepted universal cryopreservation method for a wide range of microorganisms inhabiting the female reproductive tract makes the problem of cryopreservation extremely relevant in terms of preserving human microecological resources. It can already be unequivocally stated that low-temperature preservation of complex symbiotic communities of the vagina is possible, despite individual variability in the number and ratio of microorganisms in the microbiome, depending on the age, localization, lifestyle, and health status of a woman.

State of the art

A known method of freezing bacteria, involving the separation of bacteria by centrifugation, mixing a suspension of bacterial cells with a protective medium in a ratio of 1:1 containing glycerol, sucrose, sodium citrate and freezing the resulting mixture in liquid nitrogen in the form of granules. The bacterial concentrate obtained in this way retains its activity for 6 months at a temperature not higher than -18°C (USSR author's certificate No. 457727, 01/25/1975). The disadvantage of this invention is that when thawing the bacteria, it is necessary to use centrifugation to separate the bacterial cells from the cryoprotective medium. However, when using this method of cryofreezing during storage, the granules stick together, which makes it difficult to obtain the initial titer of bacterial cultures.

Known Method for long-term storage of natural symbiotic associations of human and animal microorganisms by freezing bacteria in granules using a sterile molten 10% gelatin solution in 0.9% saline (NaCl). The suspension of the contents of the large intestine is mixed with a 10% gelatin solution in a ratio of 1:1 to form, when frozen, gelatin balls with a sufficient number of bacterial cells. To create anaerobic conditions, nitrogen is blown through the prepared solution with cells. A suspension of bacterial cells using a dispenser of 50 and/or 100 μl is applied to a fluoroplastic plate cooled in liquid nitrogen vapor. The resulting frozen granules are "dumped" into liquid nitrogen, collected in nitrogen marked plastic tubes with screw caps and transferred to storage in Dewar vessels with liquid nitrogen (cryostorage) (RU 2123044, 10.12.1998).

The disadvantage of this method is the need to create anaerobic conditions by blowing cells through liquid nitrogen vapor; storage should be carried out only in liquid nitrogen, and therefore requires the availability of cryostorages; when thawed, bacterial cells should be freed from gelatin.

A known method of freezing lactic acid bacteria, involving mixing in a ratio of 1:1 lactic acid bacteria with a protective medium containing glycerol and sodium citrate and freezing the resulting mixture in liquid nitrogen in the form of granules, the difference of which from the previous invention is that the protective medium is prepared on the basis of phosphate buffer pH 7.2. At the same time, lactose and gelatin are additionally introduced into the protective environment, which ensure that the granules of lactic acid bacteria do not stick together after thawing (RU 2427624, 28.08.2011). The disadvantage of this method is that during thawing it is necessary to use centrifugation to separate bacterial cells from the cryoprotective medium, which makes it difficult to obtain the initial titer of bacterial cultures.

A known method of preserving lactic acid bacteria Lactobacillus delbrueckii (RU 2475527, 20.02.2013).

The invention relates to microbiology, in particular to the conservation of lactic acid bacteria Lactobacillus delbrueckii . The method involves obtaining a suspension of bacteria in a liquid nutrient medium, separating bacteria from the suspension by centrifugation to obtain biomass, mixing the resulting biomass with a protective medium containing sucrose and glycerin, and freezing the resulting mixture at a temperature (-18--20°C). Liquid nutrient medium contains components in the following ratio, wt.%: (10-12) - sucrose, (3-5) - malt sprouts and (4-6) - chalk. A mixture of biomass with a protective medium is cooled at a temperature of 4-6°C before freezing, kept at this temperature for 1 hour.

There is a known method of preserving bacteria in the fecal microbiota and bacterial cultures isolated from the fecal microbiota grown on dense agar nutrient or differential media. The method includes mixing 0.1 g of a sample in a 2 ml cryotube in a ratio of 1:2 with a cryoprotective medium consisting of 500 ml distilled water, 2 g bacteriological dry European agar, 2.5 g NaCl, 1 g yeast extract, L-cysteine 0.2 mg, casein pancreatic hydrolyzate 8.5 mg, dextrose 5 g with the addition of 10% glycerol, vortexing for 2-5 minutes. The capped cryovials are placed in the appropriate storage. The thawing of cryovials is carried out immediately before the research. The method ensures the preservation of the titer and viability of bacteria during storage at low temperatures (RU 2737321, 27.11.2020).

The disadvantage of this method is that it is used for cryopreservation of the species Lactobacillus delbrueckii - a species of lactobacilli that lives in the intestinal biotope and is rarely found in the composition of the vaginal microbiota (an associated species, always isolated in low titers), is not suitable for long-term cryopreservation of vaginal lactobacilli and their consortia due to relatively high storage temperatures (-18 - -20°C). In addition, the cryoprotective medium used does not provide for the storage of cultures of the specific vaginal species Lactobacillus iners.

Cryoprotectants are substances designed to prevent damage to bacterial cells during cooling, freezing, subsequent storage and thawing. The mechanism of the protective action of cryoprotectants is based on their creation of a stronger bond with water molecules, which prevents the formation of a regular ice crystal lattice and delays the onset of crystal growth. Cryoprotectors can have a toxic effect on cells, the magnitude of which depends on the temperature and duration of exposure of cells with a cryoprotectant. Therefore, as a rule, immediately after thawing, the cryoprotectant is removed from bacterial cells by successive washings and centrifugation. There is evidence of freezing the human intestinal microbiota to cryogenic temperatures of -70°C, -196°C in cryoprotective media with glycerol, dimethyl sulfoxide (DMSO), natural and synthetic polymeric substances. Glycerol and DMSO easily pass through the cell membrane and provide both intracellular and extracellular freeze protection. The choice of a cryoprotectant or their combination, as well as the choice of the correct mode of freezing and thawing, are the basis for the technological development of the preservation of microorganisms of the human symbiotic microbiota under cryopreservation at low temperatures, designed for long-term storage of microecological resources in a cryobank.

Currently, empirical approaches are mainly used to select the modes of freezing and thawing of specific objects, since the survival and resistance of bacteria when frozen in liquid nitrogen depends on a number of factors, in particular, on the type of bacterial cells and their concentration in suspension, and the sensitivity of bacteria to freezing. and thawing, depending on their taxonomic affiliation. Of great importance are the physicochemical properties, the quantitative content of bacteria and a number of other properties of the bacterial biomass to be frozen; the composition of the protective medium for cryofreezing (to preserve bacteria during freezing, it is essential to choose a protective medium, the nature and chemical structure of the cryoprotectant present in it, and the physicochemical features of its interaction with the components of the liquid and solid phases of the frozen bacterial cells); cooling mode (during freezing of bacterial cells, ice microcrystals are formed both inside the bacterial cells and outside, while the nature of their changes depends on the properties of the cryoprotectant, and mainly on the cooling rate); storage mode (at minus 70°С or in liquid nitrogen at minus 196°С). There are different points of view on the process of freezing living cells. Slow cooling is believed to result in the formation of extracellular ice, leading to dehydration of the bacterial cell before the freezing point of the cytoplasm is reached. With rapid cooling, bacterial cells freeze faster from the inside, dehydrate more slowly, which leads to the formation of ice crystals inside the cell. The best effect is usually achieved when freezing in liquid nitrogen at a temperature of minus 196°C. There is an opinion that the best results on the survival and recovery of bacterial cultures can be obtained in the case of slow cooling of the cells, for example at a rate of 1°C/min.

Slow cooling technology, as follows:

- ampoules or cryovials with culture are placed in an aluminum container, which is inserted into a cardboard or plastic box; the box is placed in the freezer of an automatic low-temperature refrigerator and a certain cooling rate is set; cells are frozen at a controlled cooling rate of 1°C/min to minus 30°C, and then at a much higher rate (15–30°C/min) to minus 150°C; after reaching this temperature, the ampoules are transferred into a vessel with liquid nitrogen and stored in it at a temperature of minus 196°C or above it in nitrogen vapor at a temperature of minus 150°C. With the help of more modern equipment, such as, for example, the BioArchive automated freezing and storage system, samples are preserved in the following way: cell material prepared for storage is placed in special plastic containers; plastic containers are placed in a program freezer, where the cooling process begins according to a special program that can be changed as needed. If there is no programmable refrigerator or an automated cooling system, slow cooling of samples is achieved in the following way: glass ampoules or plastic cryotubes with culture are placed for 1 hour in a freezer at a temperature of minus 60°C; then immerse them for 5 minutes in a bath of liquid nitrogen. The cooling rate in this case is approximately 1.5°C/min; after that, the samples are stored in a cryostorage with liquid nitrogen vapor (temperature minus 196°C).

An analysis of known technical solutions from the prior art showed that at the moment the closest analogue of the proposed technical solution has not been identified.

Disclosure of invention

The aim of the present invention is to create a technology to ensure the preservation of the initial titer of microorganisms after freezing and thawing; a method for long-term preservation of bacteria for advanced microbiome research, the creation of autoprobiotics, and the transplantation of beneficial vaginal microbiota.

The objective of the invention is to develop a technology for sampling vaginal fluid, isolation of pure cultures of protective lactobacilli, cryopreservation of human vaginal bacteria and their long-term storage with high preservation of the full biological properties of microorganisms for their subsequent use after thawing for the production of autoprobiotics, transplantation or research.

The technical result achieved by using the proposed invention is the creation of a model for sampling vaginal fluid and optimal conditions for preserving bacteria from the human vagina or bacterial cultures isolated from the human vagina grown on dense or liquid universal or selective nutrient media and their subsequent storage at low temperatures ( -80°C and -196°C), to ensure the safety of microorganisms after freezing and thawing (defrosting).

Below is a detailed description of the claimed method of cryopreservation of the vaginal microbiota, indicating in some cases specific parameter values that do not limit the claimed invention, but are presented only for a better understanding of the essence of the invention. The task and the technical result are solved by the fact that the method of cryopreservation of the vaginal microbiota includes the following steps:

1. Sampling of vaginal fluid.

Initial (native) samples of vaginal fluid are taken in the amount of 1-3 ml using a cotton (dacron) microbiological swab (pipette, applicator, Volkmann spoon) with an estimated concentration of the total bacterial mass of at least 10 ⁷ CFU / ml (GE / ml) according to according to a preliminary culture study or real-time PCR, containing 50-90% of biologically active species of Lactobacillus spp in an amount of 10 ^7-8 CFU / ml (GE / ml). The resulting vaginal fluid in a ratio of 1:2-5 is suspended in a special combined cryoprotective medium consisting of trypticase-soy broth (BBL, USA) (per 1 liter of distilled water: pancreatic hydrolyzate of casein - 17.0 g; papain hydrolyzate of soy flour - 3 .0 g; sodium chloride - 5.0 g; dipotassium phosphate - 2.5 g; dextrose - 2.5 g) or MPC broth (MRS Broth) (HIMEDIA, India) (per 1 liter of distilled water: proteose peptone - 10 g , meat extract - 10 g, yeast extract - 5 g, glucose - 20 g, tween 80 - 1 g, ammonium citrate - 2 g, sodium acetate - 5 g, magnesium sulfate - 0.1 g, manganese sulfate - 0.05 g, sodium hydrogen phosphate -2 g) with the addition of 10% glycerol as a cryoprotectant.

2. Fractionation of native material. You can make a dependent item

In case of insufficient amount of vaginal discharge, a flush is prepared by introducing 2-4 ml of sterile 0.9% saline (0.9% NaCl) into the vagina. The tube containing the flushing vaginal fluid is centrifuged at a speed of 5000 rpm. within 10 minutes, the supernatant is removed, the precipitate is resuspended in 1-2 ml of a cryoprotective medium of the same composition.

3. Preparation of cryoprotective medium.

Sterile glycerol (cryoprotectant) is added to the sterile growth medium in a volume of 10%. To prepare 100 ml of cryoprotective medium, 90 ml of sterile growth medium (trypticase-soy broth or MPC liquid medium) is used; 10 ml of sterile glycerol is added. Glycerin is sterilized by autoclaving for 15 min at a pressure of 10 ⁴ Pa.

1. Preparation of the bacterial mass for freezing and its storage.

The obtained vaginal samples in a cryoprotective medium are thoroughly mixed using a laboratory vortex mixer "Vortex" (2-3 minutes), poured into 1 ml cryovials and frozen at minus 80°C in a low-temperature refrigerator without using special freezing conditions (speed) or at - 196°C in liquid nitrogen using slow cooling technique.

When storing samples at -196° C. in liquid nitrogen, to obtain good results in terms of survival and recovery of bacterial cultures, slow cell cooling is used, for example at a rate of 1° C./min. The slow cooling technology is as follows: cryovials with vaginal biosamples in a cryoprotective environment are placed in a plastic container and transferred to the freezer of an automatic low-temperature refrigerator and a certain cooling rate is set; cells are frozen at a controlled cooling rate of 1°C/min to minus 30°C, and then at a much higher rate (15–30°C/min) to minus 150°C; after reaching this temperature, the cryovials are transferred to a storage facility with liquid nitrogen and stored there at a temperature of minus 196°C. If more modern equipment is available, for example, the BioArchive automated freezing and storage system, samples are preserved as follows: the cellular material prepared for storage is placed in special plastic containers, placed in a program freezer, where the cooling process begins according to a special program that can be changed according to need. If there is no programmable refrigerator, slow cooling of samples is achieved in the following way: cryovials with culture in a stainless steel tray are placed for 1 hour in a freezer at a temperature of minus 60°C; then immerse them for 5 minutes in a bath of liquid nitrogen. The cooling rate in this case is approximately 1.5°C/min; after that, the samples for storage are immersed directly in a cryostorage with liquid nitrogen (temperature minus 196°C).

2. Freezing and storage of pure cultures of protective lactobacilli.

Part of the native material - vaginal fluid is inoculated on dense (lactobacagar, FBSI "SSC PMB", Obolensk, Russia) selective for most lactobacilli nutrient media or, for example, Columbia agar (Oxoid, UK) (to isolate lactobacilli of the speciesLactobacillus iners) and cultivated under conditions of reduced oxygen content in CO₂incubator or in an anaerobic box in an atmosphere of a three-component gas mixture (N₂ – 80%; SO₂ - 10%; H₂ - 10%) followed by the isolation of pure cultures of lactobacilli. Pure cultures of autostrains of lactobacilli isolated on dense nutrient media and identified to species by the method matrix-activated laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), washed with 0.9% saline or liquid growth medium, prepare an inoculum with a concentration of 4 McFarland units (1.2 × 10⁹cfu/ml), centrifuged at 5000 rpm, the supernatant is removed and the pellet is resuspended in a cryoprotective medium (trypticase soy broth or MPC liquid medium supplemented with 10% glycerol). The resulting suspension is poured into 1 ml cryovials, hermetically sealed and frozen at -80°C or -196°C, as described above.

3. Thawing vaginal fluid with microbiological consortium or pure cultures of lactobacilli.

For quick recovery of cultures, samples stored in plastic cryovials (vaginal fluid or pure cultures or their consortium) are thawed by rapid heating in a water bath at a temperature of plus 37 ° C for 60-120 seconds with gentle shaking immediately before testing. The method ensures the preservation of the titer and viability of lactobacilli during storage at low temperatures.

Quality control of thawed vaginal microbiota samples and pure cultures of lactobacilli

To assess the quality of the preparation and storage of the resulting product (autologous lactobacilli) using the technology that is the subject of the invention, methods were used to assess the survival of frozen cultures and the main biological properties. For this purpose, a comparative assessment of the main biological properties that determine the colonization resistance of the vaginal biotope (the level of acid formation, the production of hydrogen peroxide) was carried out in 10 strains of lactobacilli isolated from the primary material (starting characteristics), which is important in the dynamic assessment of the indicated biological properties during storage and for assessment of the quality of the final product.

Example 1. Testing the storage capacity (survival of cultures) of lactobacilli after cryostorage.

Stored cultures of autologous strains of lactobacilli or their consortium in cryovials are subjected to periodic viability tests. The number of viable lactobacilli was determined by the final dilution method [7]. For quick recovery of cultures, samples stored in plastic cryovials (vaginal fluid or pure cultures or their consortium) are thawed by rapid heating in a water bath at a temperature of plus 37 ° C for 60-120 seconds with gentle shaking immediately before testing. To determine the number of viable lactobacilli, 10-fold serial dilutions were prepared from the thawed (reconstituted) bacterial suspension. 4.5 ml of 0.9% sterile saline solution or MPC liquid nutrient medium were poured into sterile test tubes. 0.5 ml of the initial bacterial suspension was added to the first test tube, then, in accordance with the rule for changing pipettes, 0.5 ml was transferred from a smaller dilution to a larger one. A total of 5 tenfold dilutions were prepared. 0.1 ml of the inoculum of each dilution was inoculated into Petri dishes with a nutrient medium for cultivating lactobacilli (lactobacagar) and rubbed with a sterile spatula from a larger dilution to a smaller one. Cups with crops were incubated at a temperature of 37±1°C for 48 h in an atmosphere of CO ₂ incubator or anaerobic boxes. After 48 hours, the grown colonies were counted. The determination of the number of viable cells in the samples was carried out in two repetitions. The total number of viable cells was calculated using the formula:

X = a×10 ⁿ⁺¹ , where

X is the number of colony-forming units of bacteria in 1 ml of the sample;

a is the number of colonies in the considered dilution.

n - considered dilution.

The results of assessing the viability of autologous strains of lactobacilli after cryopreservation, depending on the cryoprotective medium used and the temperature storage conditions, are presented in Table 1. The results of assessing the viability of autologous strains of lactobacilli after the 1st month of cryopreservation using trypticase-soy broth as the basis of a cryoprotective medium; Table 2. The results of assessing the viability of autologous strains of lactobacilli after the 1st month of cryostorage using the MPC liquid medium as the basis of the cryoprotective medium.

The results of evaluating the survival of pure cultures of lactobacilli isolated from the vaginal discharge of women using two different cryoprotective media for cryopreservation and two different freezing and storage conditions showed that there was no significant difference in the survival of these microorganisms depending on these factors.

The proposed method of preserving autologous strains of lactobacilli: freezing, storage and thawing (recovery), allows you to keep them in high titer and provide the possibility of restoring the viability of bacteria after storage at low temperatures.

Comparative evaluation of the biological properties of autologous strains of lactobacilli before and after cryopreservation

Example 2 . Evaluation of acid formation activity.

The activity of acid formation was determined by the titration method [6]. As a starter, a standardized starting concentration of each studied lactobacillus strain was used: Lactobacillus jensenii (3 strains), Lactobacillus gasseri (2 strains), Lactobacillus crispatus (4 strains), Lactobacillus rhamnosus (1 strain). For the starter, an inoculum was used prepared by washing with sterile saline (0.9% NaCL) pure cultures of lactobacilli grown for 48 hours on an agar nutrient medium of lactobacgar, the concentration of which corresponded to 2 units of the McFarland standard ( ^6x108 CFU/ml). The starter in a volume of 1% was added to test tubes with sterile milk 1.5% fat and cultivated for 48 hours at 37°C. 20 ml of distilled water and 3 drops of phenolphthalein were added to 10 ml of the obtained sample. Acidity was measured by titration in Turner degrees (T°) (T° is the value determined by the amount of 0.1 N alkali solution used for titration of 100 ml of the test sample).

The indicator of acid formation activity, expressed in Turner degrees /°T/, was calculated by the formula:

° T \u003d A × K × 10, where

A - the number of milliliters of 0.1N sodium hydroxide solution used for titration;

K - correction to the titer of 0.1 M sodium hydroxide solution;

10 - correction factor for the mass of the analyzed sample.

The average value of acid formation activity is calculated for two samples

The results of a comparative assessment of the level of acid formation by autologous strains of lactobacilli before and after cryopreservation are shown in Table 3. The activity of acid formation of vaginal strains of lactobacilli before and after cryopreservation using trypticase-soy broth as the basis of a cryoprotective medium and temperature regime - -80°C; Table 4. Activity of acid formation of vaginal strains of lactobacilli before and after cryopreservation using trypticase-soy broth as the basis of a cryoprotective medium and temperature regime - -196°C

The studies have shown that the acid-forming function of the preserved strains at -80°C and -196°C is comparable to their acid activity before freezing, which indicates a good preservation of their acid-forming function when using the proposed method for preserving autologous strains of vaginal lactobacilli.

Example 3 Evaluation of Hydrogen Peroxide (H ₂ O ₂ ) Production.

Estimate The ability to produce hydrogen peroxide was determined by cultivating bacteria on a dense nutrient medium containing the enzyme peroxidase and an indicator for oxygen [7]. For lactobacilli, lactobacillus was used, into which 5 mg of tetramethylbenzidine (benzidine) and 0.5 mg of horseradish peroxidase (per 20 ml of agar) were previously added. Cultures of lactobacilli were grown under reduced oxygen conditions (CO₂incubator) or under anaerobic conditions at 37°C for 24-48 hours. A quantitative inoculation of a suspension of 10 strains of vaginal lactobacilli was carried out: an inoculum of lactobacilli grown on a dense lactobacillus was prepared (1 unit according to McFarland - 3x10⁸cfu/ml), then three successive tenfold dilutions were made in physiological saline, and 0.1 ml of a suspension of the appropriate dilution was applied to the surface of the agar medium with horseradish peroxidase and benzidine, followed by distribution over the surface with a spatula. When taking into account the results, the presence of colonies that form hydrogen peroxide in blue was evaluated; colonies that do not produce H₂ABOUT₂- colorless. The results are shown in Table 5. Production of hydrogen peroxide by vaginal strains of lactobacilli before and after cryopreservation using trypticase-soy broth as the basis of a cryoprotective medium and temperature conditions of -80°C and -196°C.

The study showed that the indicators of hydrogen peroxide production by lactobacilli before cryopreservation and after cryopreservation -80°C and -196°C did not change.

Comparative evaluation of the biological properties of autologous strains of lactobacilli before and after cryostorage showed that storage conditions did not affect their biological properties.

Thus, the proposed method of preserving autologous strains of lactobacilli (preparation of cultures, freezing, storage and thawing) allows you to save their viability (high titer) and functional activity (biological properties).

List of cited literature

1. Farage M., Miller K., Sober J. Dynamics of the vaginal ecosystem - Hormonal influences. Infect. Diseases: Research and Treatment 2010, 3: 1-15.

2. Martin R., Suarez J. Biosynthesis and Degradation of H2O2 by Vaginal Lactobacilli. 2010 Appl. Environ. Microbiol 76: 400-405.

3. Eschenbach D. A., Davick P. R., Williams B. L., Klebanoff S. J., Young-Smith, K., Critchlow, C. M., Holmes K. K. (1989). Prevalence of hydrogen peroxide-producing Lactobacillus species in normal women and women with bacterial vaginosis. J Clin Microbiol 27: 251–256.

4. Wang Likui. The use of probiotics and autostrains of lactobacilli in the complex treatment of bacterial vaginosis: Abstract of the thesis. dis. … cand. honey. Sciences. - M., 2006. - 22 p.

5. Melnikov V. A., Stulova S. V., Tyumina O. V., Denisova N. G., Shchukin V. Yu. Autotransplantation of lactobacilli in the restoration of individual vaginal biocenosis. Fundamental Research, 2012, No. 1, pp. 64–67.

6. GOST 3624–92 Milk and dairy products Titrimetric methods for determining acidity.

7. Guidelines for the sanitary and epidemiological assessment, safety and functional potential of probiotic microorganisms used for food production. Guidelines MU 2.3.2.2789-10, Moscow, 2011.

Table 1

Culture of lactobacilli Initial inoculum of frozen culture
(CFU/ml) The number of grown colonies (CFU/ml) after freezing at -80°C in duplicate The number of grown colonies (CFU / ml) after freezing at -196 ° C in duplicate 1 2 1 2 Lactobacillus jensenii 1 1.2×10 ⁹ cfu/ml 5.3*10 ⁸ 4.5*10 ⁸ 6.0*10 ⁸ 5.7*10 ⁸ Lactobacillus jensenii 2 1.2×10 ⁹ cfu/ml 6.3*10 ⁸ 5.5*10 ⁸ 6.2*10 ⁸ 6.1*10 ⁸ Lactobacillus jensenii 3 1.2×10 ⁹ cfu/ml 7.2*10 ⁸ 6.5*10 ⁸ 6.9*10 ⁸ 6.7*10 ⁸ Lactobacillus gasseri 1 1.2×10 ⁹ cfu/ml 4.5*10 ⁸ 4.8*10 ⁸ 4.9*10 ⁸ 5.0*10 ⁸ Lactobacillus gasseri 2 1.2×10 ⁹ cfu/ml 3.9*10 ⁸ 3.7*10 ⁸ 4.0*10 ⁸ 4.1*10 ⁸ Lactobacillus crisratus 1 1.2×10 ⁹ cfu/ml 3.7*10 ⁸ 3.9*10 ⁸ 4.5*10 ⁸ 4.2*10 ⁸ Lactobacillus crisratus 2 1.2×10 ⁹ cfu/ml 6.3*10 ⁸ 6.3*10 ⁸ 6.5*10 ⁸ 6.7*10 ⁸ Lactobacillus crisratus 3 1.2×10 ⁹ cfu/ml 5.4*10 ⁸ 5.6*10 ⁸ 5.6*10 ⁸ 5.7*10 ⁸ Lactobacillus crisratus 4 1.2×10 ⁹ cfu/ml 6.0*10 ⁸ 6.3*10 ⁸ 6.5*10 ⁸ 6.3*10 ⁸ Lactobacillus rhamnosus 1.2×10 ⁹ cfu/ml 6.3*10 ⁸ 6.1*10 ⁸ 6.4*10 ⁸ 6.2*10 ⁸

table 2

Culture of lactobacilli Initial inoculum of frozen culture
(CFU/ml) The number of grown colonies (CFU/ml) after freezing at -80°C in duplicate The number of grown colonies (CFU / ml) after freezing at -196 ° C in duplicate 1 2 1 2 Lactobacillus jensenii 1 1.2×10 ⁹ cfu/ml 5.7*10 ⁸ 5.5*10 ⁸ 6.2*10 ⁸ 5.8*10 ⁸ Lactobacillus jensenii 2 1.2×10 ⁹ cfu/ml 6.5*10 ⁸ 6.0*10 ⁸ 6.3*10 ⁸ 6.4*10 ⁸ Lactobacillus jensenii 3 1.2×10 ⁹ cfu/ml 7.2*10 ⁸ 7.5*10 ⁸ 6.9*10 ⁸ 7.1*10 ⁸ Lactobacillus gasseri 1 1.2×10 ⁹ cfu/ml 4.3*10 ⁸ 4.5*10 ⁸ 4.9*10 ⁸ 5.1*10 ⁸ Lactobacillus gasseri 2 1.2×10 ⁹ cfu/ml 4.3*10 ⁸ 4.1*10 ⁸ 4.2*10 ⁸ 4.1*10 ⁸ Lactobacillus crisratus 1 1.2×10 ⁹ cfu/ml 3.8*10 ⁸ 3.9*10 ⁸ 4.4*10 ⁸ 4.3*10 ⁸ Lactobacillus crisratus 2 1.2×10 ⁹ cfu/ml 6.5*10 ⁸ 6.4*10 ⁸ 6.5*10 ⁸ 6.4*10 ⁸ Lactobacillus crisratus 3 1.2×10 ⁹ cfu/ml 5.3*10 ⁸ 5.6*10 ⁸ 5.6*10 ⁸ 5.8*10 ⁸ Lactobacillus crisratus 4 1.2×10 ⁹ cfu/ml 6.1*10 ⁸ 6.3*10 ⁸ 6.4*10 ⁸ 6.3*10 ⁸ Lactobacillus rhamnosus 1.2×10 ⁹ cfu/ml 6.4*10 ⁸ 6.3*10 ⁸ 6.4*10 ⁸ 6.5*10 ⁸

Table 3

Culture (sourdough/milk ratio
Volume / Volume = 1:100) Bacteria titer in starter culture, CFU/ml Acidity (Turner degree, (T °) (48 h) when stored at -80 ° C pH of milk after cultivation (48 h)
when stored at -80°С Before freezing After freezing Before freezing After freezing Lactobacillus jensenii 1 6×10 ⁸ cfu/ml 44 43 4.7 4.8 Lactobacillus jensenii 2 6×10 ⁸ cfu/ml 52 50 4.5 4.5 Lactobacillus jensenii 3 6×10 ⁸ cfu/ml 40 39 5.0 5.0 Lactobacillus gasseri 1 6×10 ⁸ cfu/ml 51 50 4.5 4.5 Lactobacillus gasseri 2 6×10 ⁸ cfu/ml 48 47 4.5 4.5 Lactobacillus crisratus 1 6×10 ⁸ cfu/ml 48 50 4.5 4.5 Lactobacillus crisratus 2 6×10 ⁸ cfu/ml 38 39 5.0 5.0 Lactobacillus crisratus 3 6×10 ⁸ cfu/ml 37 37 5.0 5.0 Lactobacillus crisratus 4 6×10 ⁸ cfu/ml 39 37 5.0 5.0 Lactobacillus rhamnosus 6×10 ⁸ cfu/ml 67 68 4.0 4.0

Table 4

Culture (sourdough/milk ratio
Volume / Volume = 1:100) Bacteria titer in starter culture, CFU/ml Acidity (Turner degree, (T °) (48 hours) pH of milk after cultivation (48 h) Before freezing After freezing Before freezing After freezing Lactobacillus jensenii 1 6×10 ⁸ cfu/ml 42 43 4.7 4.8 Lactobacillus jensenii 2 6×10 ⁸ cfu/ml 52 51 4.5 4.5 Lactobacillus jensenii 3 6×10 ⁸ cfu/ml 39 40 5.0 5.0 Lactobacillus gasseri 1 6×10 ⁸ cfu/ml 51 50 4.5 4.5 Lactobacillus gasseri 2 6×10 ⁸ cfu/ml 47 47 4.5 4.5 Lactobacillus crisratus 1 6×10 ⁸ cfu/ml 47 49 4.5 4.5 Lactobacillus crisratus 2 6×10 ⁸ cfu/ml 38 39 5.0 5.0 Lactobacillus crisratus 3 6×10 ⁸ cfu/ml 38 37 5.0 5.0 Lactobacillus crisratus 4 6×10 ⁸ cfu/ml 39 38 5.0 5.0 Lactobacillus rhamnosus 6×10 ⁸ cfu/ml 66 68 4.0 4.0

Table 5

Culture of lactobacilli Initial inoculum (cfu/ml) Production of H ₂ O ₂ (dilution 1.5×10 ⁶ CFU/ml) when stored at -80°C Production of H ₂ O ₂ (diluted 1.5×10 ⁶ CFU/ml) when stored at -196°C Before freezing After freezing Before freezing After freezing Lactobacillus jensenii 1 1.5×10 ⁸ cfu/ml + + + + Lactobacillus jensenii 2 1.5×10 ⁸ cfu/ml + + + + Lactobacillus jensenii 3 1.5×10 ⁸ cfu/ml + + + + Lactobacillus gasseri 1 1.5×10 ⁸ cfu/ml _ _ _ _ Lactobacillus gasseri 2 1.5×10 ⁸ cfu/ml _ _ _ _ Lactobacillus crisratus 1 1.5×10 ⁸ cfu/ml + + + + Lactobacillus crisratus 2 1.5×10 ⁸ cfu/ml + + + + Lactobacillus crisratus 3 1.5×10 ⁸ cfu/ml - _ _ _ Lactobacillus crisratus 4 1.5×10 ⁸ cfu/ml + + + + Lactobacillus rhamnosus 1.5×10 ⁸ cfu/ml - _ - _

Claims (3)

1. A method for preserving autologous lactobacilli in samples of vaginal discharge and pure cultures of certain types of autologous lactobacilli isolated from the microbiota of the vagina of women on dense universal and selective nutrient media, which consists in the fact that vaginal discharge in an amount of 1-3 ml with an estimated concentration of lactobacilli of at least 10 ⁷ CFU / ml (GE / ml) according to the data of a cultural study or real-time PCR, in a ratio of 1: 2-5 are suspended in a special combined cryoprotective medium consisting of 1000 ml of distilled water, pancreatic casein hydrolyzate - 17.0 g ; papain hydrolyzate of soy flour - 3.0 g; sodium chloride - 5.0 g; dipotassium phosphate - 2.5 g; dextrose - 2.5 g with the addition of 10% glycerol - a cryoprotectant, as well as pure cultures isolated from the vaginal microbiota, washed off the surface of a dense nutrient medium using a cryoprotective medium of the same composition in a final concentration of lactobacilli 4 units according to McFarland - 1.2 × 10 ⁹ CFU / ml, mixed with a vortex mixer, pour 1 ml into 2 ml cryotubes, hermetically close and freeze at appropriate temperatures in low-temperature storage, using as needed, previously restored by thawing by heating in a water bath at temperature plus 37°C for 60-120 s. 2. The method of claim 1, wherein the freezer at -80°C is used as storage for low temperature storage. 3. The method according to claim 1, in which a storage system in liquid nitrogen vapor is used as a storage for freezing and storage - a temperature of minus 196 ° C.