RU2798521C1 - Synbiotic agent based on viable biomass of meyerozyma (pichia) guilliermondii vkpm y-4316 yeast strain - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: synbiotic agent based on a viable biomass of the yeast strain Meyerozyma (Pichia) guilliermondii VKPM Y-4316 is proposed. The synbiotic agent has the following composition per 100 g: protein — at least 18 g, fats — at least 0.5 g, carbohydrates — at least 55 g, humidity — not more than 15%, vitamin B1 (thiamine) — 0.1 mg, vitamin B2 (riboflavin) — 0.8 mg, energy value — at least 300 kcal. An agent contains a probiotic component — at least 109 CFU of the above yeast per g of substrate, a prebiotic component: soluble and insoluble dietary fiber — at least 60% by mass fraction. It has an adsorption activity of at least 65 mg/g, which is comparable to about 1/3 of the activity of activated carbon.

EFFECT: production of food, pharmaceutical and feed compositions, as well as medicinal products for humans and animals with synbiotic action, adsorption and antagonistic activity.

1 cl, 12 dwg, 4 ex

Description

Technical field

The invention relates to biotechnology, the field of nutrition and human and/or animal health. It can be used in the production of synbiotic preparations based on the yeast Meyerozyma (Pichia) guilliermondii VKPM Y-4316 and has a wide range of applications in the food, feed industry, pharmaceuticals and veterinary medicine.

State of the art

The impoverishment of the microbiological profile of the indigenous intestinal microbiota, the presence of dysbiotic symptoms in the population caused by changes in the qualitative and quantitative composition of the microbiota, and, as a result, the reduction of colonization resistance is now recognized as a serious problem in biomedicine. The species composition of the intestinal microbiota according to the results of scientific research [1-4] is unique for each individual by 2/3 and contains bifidobacteria, lactobacilli, yeast, enterococci, bacteroids, staphylococci. These microorganisms, including those related to opportunistic pathogens, form symbiotic systems with a common metabolism, which make it possible to assimilate the available nutrient substrates with the formation of substances useful for the macroorganism. Violation of the composition of the microbiota as a result of various factors leads to its possible transition from a normal physiological profile to a pathological one. In order to prevent and correct such conditions, functional preparations and products with pre, pro- and synbiotic effects are widely used.

Probiotics are live microorganisms that provide health benefits to the host when administered in adequate amounts. The main functions of probiotics include: support of colonization resistance, metabolism of food substrates and utilization of final metabolites, production of substrates necessary for the macroorganism, as well as regulation of local and adaptive immune response [5]. If prebiotic constituents, such as fructooligosaccharides and galactooligosaccharides [6, 11, 15], are added to the target biological product, dietary fiber, which leads to specific changes in the composition and/or activity of the gastrointestinal microbiota, thus benefiting the health of the host organism, one can talk about the synbiotic effect of drugs [7].

Numerous microorganisms have been described in the literature for beneficial use in humans in the digestive tract and for nutritional purposes. Known industrial strains of lactobacteria and bifidobacteria that have the ability to influence the physiological and immunobiological processes in the body, which are used for the preparation of medicines and food (drugs, "Linex", "Bifiform", "Bifidumbacterin", "Narine", "Acidophyllin" , EU patent EP2990045B1; US patent US10716817B2, US patent US20160129055A1, RF patent 2176668 dated 04/01/2001; RF patent 2524117 dated 08/07/2013 and so on).

Compositions for providing colonization resistance of human intestinal microbiocenosis and methods for their preparation are known [9, 10]. The compositions consist of a combination of metabolites of the probiotic strain Bacillus subtilis VKPM No. B-2335 or additionally include sterilized dried culture liquids containing metabolites of Enterococcus faecium L-3, Lactobacillus delbrueckii TS1-06 and Lactobacillus fermentum TS3-06. Oat flakes or a complex of polysaccharides (β-glucan and mannan) are used as a prebiotic component. Also included are zeolite, calcium stearate or aerosil. Their use makes it possible to normalize the intestinal microbiocenosis by selectively stimulating the growth and reproduction of lactobacilli and bifidobacteria of the indigenous component of the normal flora of the biotope and providing specific antagonistic activity against pathogenic and opportunistic microorganisms.

The disadvantages of the claimed compositions are limited biological activity, expressed in insufficient levels of specific antagonistic activity against a number of pathogenic and opportunistic microorganisms, which is associated with the use of only inactivated microorganisms in its composition, that is, the absence of a probiotic effect, the impossibility of obtaining a quick therapeutic effect, because with stimulation of indigenous microflora, its growth does not occur immediately and requires a long time of taking the compositions, the absence of detoxification activity. It should also be noted the need for complex production lines to obtain compositions and the complexity of the production process due to the large number of components.

It has also been observed that quite a number of microorganisms, in particular lactic acid bacteria, have a pro-inflammatory effect. Such a pro-inflammatory effect may be particularly harmful and undesirable, for example, in the case of autoimmune diseases or immunodeficiency.

Great potential in the creation of such functional products can be realized using yeast. Yeast cell walls contain mannano-oligosaccharides (MOS) - prebiotic substances that are indigestible in the upper intestine and fermented by indigenous microflora in the colon to form short-chain fatty acids, which promotes the proliferation and differentiation of intestinal epithelial cells. A series of biomedical studies [8] conducted in China on the mechanisms underlying the positive effect of MOS on the modeling of intestinal health in piglets showed that the introduction of MOS in the diet reduces the quantitative content of proteobacteria in the intestine, promotes the growth of beneficial microorganisms, such as Bifidobacterium and Lactobacillus species, adsorbs and removes mycotoxins from the body of animals. These model animals are very similar to humans in anatomy (monogastric animals), genetics, physiology and are used for a preliminary assessment of the effect of biological products on humans. In this case, it is advisable to use the entire yeast biomass, without the stage of separation of the cell walls containing MOS, due to the content of high-value protein and vitamins in it.

As a widely used yeast-based probiotic, the Enterol drug is known, which has proven its effectiveness, immunomodulatory effect and is included in the recommendations of international medical associations, the Saccharomyces cerevisiae var.boulardii strain is used. Clinical studies [12] have shown that the drug allows you to remove diarrhea pathogens from the body, helps to restore the digestive function of the intestine, and helps to neutralize toxins that damage the intestine.

Also known is the remedy obtained under the patent of the Russian Federation 2 490 324 dated 20.08. 2013 [16], which refers to the yeast strains Saccharomyces cerevisiae CNCM I-3856 and Saccharomyces cerevisiae var. boulardii CNCM I-3799 used as a probiotic useful in the manufacture of food or pharmaceutical compositions. Also proposed is a composition containing the yeast strain Saccharomyces cerevisiae CNCM I-3856 and/or Saccharomyces cerevisiae var. boulardii CNCM 1-3799 and/or at least one of the parental mannoproteins (MOC) EL 05 and EL 06 of the yeast strain Saccharomyces cerevisiae CNCM I-3856. EFFECT: invention helps to reduce pain in the intestines, induce anti-inflammatory action in the absence of pro-inflammatory action, hinder and reduce adhesion and colonization of the gastrointestinal tract by pathogenic and/or invasive bacteria.

The main disadvantage of these preparations and compositions is that the active strain is not eubiotic, passes through the digestive tract without colonization and is completely eliminated 5 days after administration. In addition, the preparations do not contain a prebiotic component, which reduces the effectiveness of the impact on the autoflora of a particular individual.

In a viable state, yeast is also included in a variety of products, including lactic acid products: tan, ayran, koumiss, kvass, kefir. However, in such products, the number of yeast colony-forming units is at a low level of 10 ⁴ -10 ⁵ CFU per cm ³ , which does not allow demonstrating the therapeutic and prophylactic properties of biological products and products.

In addition, in a rapidly changing epidemiological situation, the issue of detoxification of the body, support, and stimulation of immunity is also acute.

There are drugs [13,14] with immunomodulatory, enterosorption, antitoxic and anti-inflammatory activity, containing sterile bran and microbial cells of the yeast strain Saccharamyces cerevisae (vini) VCMP Y-511 grown on them in the amount of 10 ⁹ ÷10 ¹⁰ microbial bodies per 1 g bran, subsequently inactivated, and the method of its production. The method differs in that during fermentation, the solid-phase substrate is moistened with water without taste and odor, with a turbidity of less than 0.1, pH 8.7, not containing chlorine, and permanganate oxidizability of not more than 1.6 mg/dm ³ , hardness of 1.5 mg /DM ³ , total mineralization is not more than 384 mg/DM ³ , and heat treatment during cultivation is carried out by uniform heating of the medium.

The disadvantages of these drugs are their limited biological activity, since they do not contain live probiotic microorganisms capable of colonizing the intestinal microflora in dysbiosis, the lack of evidence of a lactogenic, bifidogenic effect and a detoxifying effect on widespread staphylococcal enterotoxins. Also obvious is the high energy consumption of operations for the preparation of the moisturizing component.

The closest prototype to the synbiotic agent we offer in terms of technical essence and the achieved result is RF patent 2,771,136 dated April 26, 2022 (clause 5 of the formula), in which the agent (basic substance "Minikor") was obtained with a content of dead yeast cultures of at least 10 ⁹ ÷10 ¹⁰ microbial bodies per unit of substrate, which has a lactogenic and bifidogenic effect, a detoxifying effect on staphylococcal enterotoxins, a prebiotic effect and a high biological value.

The disadvantage of this invention is limited biological activity, since the biological product does not contain live probiotic microorganisms capable of colonizing the intestinal microflora in dysbiosis and having an antagonistic effect on opportunistic bacteria, that is, it has only a prebiotic effect and provides nutritional support to the body.

The essence of the invention

The purpose of the invention is the creation of a new synbiotic agent of complex action, as well as the expansion of the arsenal of effective drugs/products for the normalization of the microflora of the human and animal gastrointestinal tract and/or the sorption of toxins.

The purpose and technical result is achieved by obtaining a synbiotic agent based on a viable biomass of the strain Meyerozyma (Pichia) guilliermondii VKPM Y-4316 of the following composition per 100 g: protein - not less than 18 g, fats - not less than 0.5 g, carbohydrates - not less than 55 g, humidity - no more than 15%, - characterized in that it contains at least 10 ⁹ CFU of the above yeast per g of substrate, the prebiotic component: soluble and insoluble dietary fiber - at least 60% by mass fraction, vitamin B1 (thiamine) - 0 .1 mg / 100 g, vitamin B2 (riboflavin) - 0.8 mg / 100 g, and with an adsorption activity of at least 65 mg / g. The technical result is confirmed by the conducted research.

The synbiotic agent is able to stimulate the growth of indigenous microorganisms of the genera Bifidobacterium and Lactobacillus, and also has detoxifying, antagonistic properties on representatives of the genera Escherichia, Enterobacter, Klebsiella and has a high biological value.

The possibility of using the invention is illustrated by examples that do not limit the scope and essence of the claims associated with them.

Implementation of the invention

Example 1. Obtaining a synbiotic agent for humans and animals of the claimed composition

A synbiotic agent is obtained by solid-phase cultivation of the strain Meyerozyma (Pichia) guilliermondii VKPM Y-4316 on wheat bran in a thin layer 1÷3 cm high with the substrate moistened with water up to 60% and subsequent natural drying of the resulting agent at a temperature of 35÷40°C.

To do this, wheat bran was sterilized in an autoclave for 30 min at 120°C, cooled to room temperature, and moistened to 60% with liquid inoculum (Y-4316) based on pre-sterilized water. Seeding dose - 10 ⁷ ÷10 ⁸ CFU/g of substrate. The seeded wheat bran was cultivated for 48 hours at a temperature of 30±2°C.

In solid-phase fermentation, it is necessary to take into account the degree of dispersion, porosity, bulk density, etc. At the same time, the indicators of the efficiency of growth and metabolism of microbial cultures can change markedly. In order to show a layer height parameter acceptable for fermentation, a biological preparation with the optimum moisture content (60%) was obtained and cultivated in the layer height range from 1 cm to 6 cm in glass cuvettes. Next, the number of viable yeast cells after fermentation was determined by the method of limiting dilutions and in accordance with GOST 10444.12-88. The research results are presented in Fig. 1.

The maximum productivity falls on the variant with a substrate layer height of 3 cm and reaches 4.6×10 ⁹ CFU/g. However, it should be noted that at a layer height of 1 and 2 cm, a high increase in biomass is also achieved. At the same time, with air access to the culture layer from both sides, the layer thickness can be increased up to 6 cm.

It should be noted that the level of oxygen access in aerobic microorganisms can significantly affect the metabolic pathways for obtaining valuable products, such as amino acids, B vitamins, as well as the use of fats and carbohydrates in the composition of the substrate. In this regard, the preparation of the synbiotic agent is advantageously carried out in a thin layer 3 cm high, and the research data on the metabolic products of the yeast used are only relevant for the indicated layer height.

When developing a technology for obtaining potential synbiotics, it is important to choose such a drying mode in order to least injure the living cells of microorganisms. We studied the temperature range of natural drying - from 30°C to 50°C in a thin layer of 1÷3 cm with constant air access.

Drying was carried out to a moisture content of not more than 15%. The number of viable cells of yeast cultures was determined by seeding on dense nutrient media (Koch method). The experimental data are shown in Fig. 2.

According to the research results, a drying mode was chosen at a temperature of 35÷40°C, in which the effect on viable cells of yeast cultures is not so traumatic, only 15÷25% of yeast cells died. However, drying at a temperature of 40°C was faster, so it is advisable to use this mode of drying. It should also be noted that the height of the layer affects the safety of the cells during the drying process, the optimal layer height is 3 cm.

The resulting synbiotic agent was examined at the Moscow Center for Hygiene and Epidemiology for such indicators as the content of solids, protein, carbohydrates, fats, ash, B vitamins (B2 and B1), and the total calorie content was measured. The research data are presented in Fig. 3.

Shown in FIG. 1, fig. 2 and FIG. 3 data indicate the active accumulation of useful metabolites by the new yeast strain during protein fermentation and the use of fats: protein - at least 18 g, fats - at least 0.5 g, carbohydrates - at least 55 g, vitamin B1 (thiamine) - 0, 1 mg / 100 g, vitamin B2 (riboflavin) - 0.8 mg / 100 g, - and colony-forming units at the level of 4.6 * 10 ⁹ CFU / g, maintaining a larger amount of CFU when dried to a moisture content of not more than 15% - 3, 0*10 ⁹ CFU/g, which corresponds to the claimed technical result.

Example 2. Use of the obtained agent as a synbiotic

Synbiotics include two components: prebiotic and probiotic.

Benefits of prebiotics include improving gut barrier function and human immunity, reducing subpopulations of opportunistic bacteria (eg, clostridium). Indigestible substances provide a beneficial physiological effect for the body by selectively stimulating the favorable growth or activity of a limited number of local bacteria. Dietary fiber - food components that are not digested by food enzymes of the human body, but are processed by beneficial intestinal microflora.

The resulting synbiotic agent was examined in an accredited testing laboratory of Liga Cert LLC for the presence of a prebiotic component. According to the research results, the content of soluble and insoluble dietary fibers in the synbiotic agent is 73.2±11% by mass fraction, which corresponds to the claimed technical result.

At the same time, in the Russian Federation, in accordance with TR CU 022/011 "Food products in terms of their labeling", the recommended level of daily intake of dietary fiber for an adult is 30 g, that is, taking 50 g of a synbiotic agent per day will fully satisfy this need.

Probiotic properties are determined by the yeast Meyerozyma (Pichia) guilliermondii VKPM Y-4316 included in the composition of the synbiotic agent in an amount of at least 10 ⁹ CFU of the above yeast per g of substrate, with the lactogenic and bifidogenic effect described in RF patent 2 771 136 (clause 1 of the formula), detoxification and antagonistic action against staphylococci.

In addition, the mechanism of action of probiotic strains is based on maintaining colonization resistance, that is, the ability of probiotic strains to prevent colonization of the gastrointestinal tract (GIT) by opportunistic and pathogenic microorganisms by inhibiting their activity and ability to reproduce due to competition for nutrients.

In addition to the detoxifying and antagonistic action against staphylococci described in RF patent 2 771 136, the culture of a viable strain of yeast Meyerozyma (Pichia) guilliermondii VKPM Y-4316 was tested for the presence of antagonistic activity against opportunistic cultures of microorganisms: E. coli, E .cloacae, K.oxytoca, E.aerogenes. The yeast strain formed stunted zones with all opportunistic cultures used (FIG. 4). However, the greatest value was achieved when acting on E. coli, perhaps this is due to the fact that the yeast consumes sugars from the nutrient medium, thereby partially inhibiting its growth. The described data confirm the claimed technical result.

Example 3 Adsorptive Activity of a Synbiotic Agent

Mannans, which are part of the structure of the cell walls of yeast of the genus Pichia, block the attachment of pathogens to the epithelium of the gastrointestinal tract, which prevents the development of infections. In the gastrointestinal tract, they work as "cleaners". For the development of most diseases of the gastrointestinal tract, it is necessary for pathogenic bacteria to gain a foothold on the mucous membrane of the digestive tract. Their attachment occurs by binding specific proteins on the surface of bacteria (pectins) to MOS on the surface of the epithelium. The addition of bioavailable mannans presumably prevents infection by blocking bacterial pectins. The cell wall of yeast is layered. From the outer and inner sides, it is just covered with mannanoproteins. Pathogenic bacteria attach to the surface of yeast cells, mistaking them for intestinal epithelial cells, and are excreted from the human body.

To confirm the adsorption activity, studies were carried out in accordance with the standard values and according to the methods (GOST 4453-74) used to determine the widely recognized adsorbent with proven efficiency - activated carbon.

Adsorption activity was determined by the indicator methylene blue.

To do this, 0.15 g of methylene blue indicator is weighed (the result of weighing in grams is recorded to the third decimal place), placed in a volumetric flask with a capacity of 1000 cm ³ and dissolved in 200 cm ³ of hot distilled water, then the solution is cooled, brought to the mark with distilled water ( get a working solution of mass concentration of 150 mg/DM ³ ) . The working solution should be stored in a hermetically sealed dark glass container for no more than two weeks. Comparative solutions are prepared to construct a calibration curve. To do this, in 10 volumetric flasks with a capacity of 100 cm ³ each enter 0.5; 1.0; 2.0; 3.0; 4.0; 5.0; 6.0; 7.0; 8.0; 9.0 cm ³ of a working solution of methylene blue with a mass concentration of 150 mg/dm3, after which the volumes are adjusted with water at a temperature of (20 ± 2) ° C to the mark. The resulting solutions contain 1 dm ³ , respectively, 0.75; 1.50; 3.00; 4.50; 6.00; 7.50; 9.00; 10.50; 12.00; 13.50 mg / dm ³ methylene blue. The optical density of the prepared reference solutions is measured on a photoelectrocolorimeter using a light filter with a wavelength (K) from 650 to 670 nm in cuvettes with a light-absorbing layer thickness of 10 mm. Distilled water is used as a control solution. Based on the data obtained, a calibration graph of the dependence of the optical density on the mass concentration of the reference solution is built (Fig. 5).

For analysis, a solution of an indicator of mass concentration of 1500 mg / dm3 is prepared, 1.5 g of the indicator is placed in a volumetric flask with a capacity of 1000 cm ³ and dissolved in 200 cm ³ of hot water (70-80 ° C). Then the solution is cooled, the volume of the solution is adjusted to the mark, and mixed. A portion of a synbiotic agent weighing 0.01 g, previously dried according to GOST 12597-67, is placed in a conical flask with a capacity of 100 cm ³ , 25 cm ³ of a solution of methyl orange mass concentration of 1500 mg / dm ³ is added, closed with a stopper and shaken on an apparatus for shaking the liquid in vessels for 60 minutes. After shaking, the suspension is transferred to centrifuge tubes and centrifuged for 15 minutes. Carefully pipette 1 cm ³ of the clarified solution and transfer to a 100 cm ³ volumetric flask. The solution in the flask was diluted with distilled water to the mark. Determine the optical density of the solution after dilution. According to the obtained value of optical density, using a calibration curve, find the residual mass concentration of methylene blue in a dilute solution.

The adsorption activity of the synbiotic agent according to the indicator (X) in milligrams per 1 g of the product is calculated by the formula:

where C ₁ - mass concentration of the initial solution of the indicator, mg/DM ³ ; C ₂ - mass concentration of the solution after contact with activated carbon, mg/DM ³ ; K is the dilution factor of the solution; 0.025 - the volume of the indicator solution taken for clarification, dm ³ ; m is the mass of the sample of activated carbon, g.

The result of the analysis is taken as the arithmetic mean of the results of two parallel determinations, the absolute discrepancy between which does not exceed the permissible discrepancy equal to 10 mg per 1 g of the synbiotic agent.

The results presented in Fig.6 show that the adsorption activity of the synbiotic agent is 65.5 mg/g, that is, about 20% of the dye (methylene blue) is adsorbed. The described data confirm the claimed technical result. Taking into account the norms of this indicator for an effective adsorbent - activated carbon, used as a drug for various diseases of the gastrointestinal tract - which are 225 mg / g, we can conclude that the synbiotic agent has a significant adsorption activity comparable to about 1/3 of the activity of activated carbon. coal.

Example 4 Preclinical Studies

The safety of probiotics must comply with strict microbiological standards, which are determined by the Uniform Sanitary and Epidemiological and Hygienic Requirements for Goods Subject to Sanitary and Epidemiological Supervision and are controlled by Rospotrebnadzor.

To determine the safety, toxicological studies were carried out on laboratory animals in vivo in accordance with GPM.1.7.2.0001.15 “Safety of probiotics in in vivo tests”, MUK 4.2.2602-10 “System for pre-registration preclinical study of drug safety. Selection, verification and storage of industrial strains used in the production of probiotics”:

• determination of strain virulence;

• determination of strain toxigenicity;

• determination of strain toxicity;

• determination of the harmlessness of the strain;

• determination of dermonecrotic properties of the strain;

• study of the acute toxicity of a synbiotic agent derived from the strain.

The study was carried out in the laboratory of preclinical studies of the Research Institute of Pharmacology of Living Systems, Belgorod State National Research University.

In the study of the safety of the yeast strain Meyerozyma (Pichia) guilliermondii VKPM Y-4316, test doses of the test substance (10 ⁹ , 10 ¹⁰ , 10 ¹¹ CFU/ml) were contained in a volume of 1.0 ml per animal. Before each administration, the suspension of the prepared test substance was stirred 6-8 times. The suspension was injected into the stomach of each mouse using a specially made atraumatic probe at a rate of 0.1 ml/s. The number of mice in each group - 5 goals. The general condition of the animals did not change during the entire observation period. No animal mortality was noted. No behavioral features in experimental mice other than healthy intact animals were noted. The intensity and nature of motor activity did not change during 5 days of observation and did not differ from the indicators of healthy intact animals. Seizures were absent in all animals under observation. The coordination of movements and the tone of skeletal muscles in the experimental groups were not disturbed during 5 days of the experiment and did not differ from the corresponding indicators of healthy intact animals. The response to tactile, pain, sound and light stimuli was adequate in all experimental animals; no differences were observed between the experimental and healthy intact animals. The frequency and depth of respiratory movements in experimental mice were visually indistinguishable from healthy intact animals. The condition of the hair and skin of the experimental animals did not change during the course of the experiment. Within 5 days after the administration of the test substances, the visible mucous membranes remained pale pink in color, their humidity did not change, there were no symptoms of bronchitis, conjunctivitis and rhinitis. The frequency of defecation and urination, the consistency of fecal matter and the color of the urine of experimental animals did not change as a result of the administration of the studied substances at all doses. The body weights of mice observed after a single intragastric administration of the test substances are shown in FIG. 7.

As follows from the presented results, by the fifth day of the experiment, the experimental animals showed a positive dynamics of body weight, there was no decrease in the group body weight of animals compared to the initial one, which, together with the absence of lethality, allows us to consider the studied strain passed the test in the safety test in accordance with the general Pharmacopoeia article OFS.1.7.2.0001.15 "Safety of probiotics in in vivo tests".

The study of virulence was carried out with a single intraperitoneal injection to male white mice of test doses of the test substance - 10 ⁸ , 10 ⁹ , 10 ¹⁰ CFU/0.5 ml in a volume of 0.5 ml for each animal. The tests were carried out on healthy animals weighing 12-14 g, quarantined and not previously used in experiments. In the experiment, 10 mice were used for each test dose. The test substances in the indicated volume were administered intraperitoneally to each mouse at a rate of 0.1 ml/s. During the next five days of observation of the animals, there were no deviations in the general condition. Thus, the general condition of the animals did not change during the entire period of observation of them (see paragraph on the definition of harmlessness). No animal mortality was noted. The body weights of mice observed after a single intraperitoneal injection of the test substances are shown in FIG. 8.

As follows from the presented results, by the fifth day of the experiment, the experimental animals showed a positive dynamics of body weight, there was no decrease in the group body weight of animals compared to the initial one, which, together with the absence of signs of intoxication and lethality, allows us to consider the studied strain as having passed the test in the virulence test in in accordance with the general pharmacopoeial article GPM.1.7.2.0001.15 "Safety of probiotics in in vivo tests".

The toxicity test was carried out with a single intraperitoneal injection to male white mice of test doses of the tested yeast strain, inactivated by heat - 0.5×10 ⁹ , 10 ⁹ , 2×10 ⁹ CFU/0.5 ml in a volume of 0.5 ml per animal. The tests were carried out on healthy white mice weighing 12-14 g, quarantined and not previously used in experiments. The conditions of keeping and feeding ensured the normal life of the animals. In the experiment, 10 mice were used per test dose. Before testing, the group weight of all mice was determined. Weighing was carried out immediately before the experiment. Test doses were prepared by diluting the starting materials with 0.9% sodium chloride solution so that the required test doses were contained in a volume of 0.5 ml. Test doses were administered to each mouse intraperitoneally at a rate of 0.1 ml/s.

During the next five days of observation of the animals, there were no deviations in the general condition. Thus, the general condition of the animals did not change during the entire period of observation of them (see paragraph on the definition of harmlessness). No animal mortality was noted. The body weights of mice observed after a single intraperitoneal injection of the test substances are shown in FIG. 9.

As follows from the presented results, by the fifth day of the experiment, the experimental animals showed a positive dynamics of body weight, there was no decrease in the group body weight of animals compared to the initial one, which, together with the absence of signs of intoxication and lethality, allows us to consider the studied strain as having passed the test in the toxicity test in in accordance with the general pharmacopoeial article GPM.1.7.2.0001.15 "Safety of probiotics in in vivo tests".

Determination of the toxigenicity of the test yeast strain was carried out with a single intraperitoneal injection of test doses of the filtrate into male white mice. The tests were carried out on healthy outbred mice of the same sex weighing 12-14 g, quarantined and not previously used in experiments. In the experiment, 10 mice were used per test dose. The conditions of keeping and feeding ensured the normal life of the animals. Before testing, the group body weight of the mice was determined. Weighing was carried out immediately before the experiment. The filtrate was administered to mice intraperitoneally in test doses of 0.1; 0.5; 1.0 ml. A group of mice treated with a sterile liquid medium in a maximum volume of 1.0 ml served as a control.

During the next five days of observation of the animals, there were no deviations in the general condition. Thus, the general condition of the animals did not change during the entire period of observation of them (see paragraph on the definition of harmlessness). No animal mortality was noted. The body weights of mice observed after a single intraperitoneal injection of the test substances are shown in FIG. 10.

As follows from the presented results, by the fifth day of the experiment, the experimental animals showed a positive dynamics of body weight, there was no decrease in the group body weight of animals compared to the initial one, which, together with the absence of signs of intoxication and lethality, allows us to consider the strain under study as having passed the test in the toxigenicity test in in accordance with the general pharmacopoeial article GPM.1.7.2.0001.15 "Safety of probiotics in in vivo tests".

Determination of dermonecrotic properties of the test strain was carried out on two white-skinned rabbits weighing 2-2.5 kg. Suspensions of the studied strain at concentrations of 10 ⁹ , 10 ¹⁰ , 10 ¹¹ CFU/ml were injected intradermally in a volume of 0.1 ml into the back area using disposable sterile syringes with a thin needle. The animals were monitored daily for 4 days. The appearance of swelling, redness, necrosis was noted. 0.9% sodium chloride solution was used as a sensitivity control.

During the next four days of observation of the animals, there were no deviations in the general condition. During the first day, no local changes were observed at the injection site. On the second day, there was slight swelling at the injection site at all concentrations tested. On the third day of observation, a slight reddening was noted, which also persisted on the fourth day of observation. Subcutaneous hematoma was observed in one of the rabbits at the injection site of the test strain at a concentration of 10 ¹¹ CFU/ml. At the same time, no necrotic changes were observed in any of the studied concentrations (Fig. 11). Thus, according to the results of the study in accordance with the guidelines of MUK 4.2.2602-10, the studied strain does not have dermonecrotic activity.

When studying acute toxicity, male white laboratory mice weighing 18-22 g were intragastrically injected with a synbiotic agent in the form of a suspension in 1% starch paste in the maximum allowable volume for this type of animal and their body weight - 1.0 ml, three times with an interval of 6 hours between introductions. To obtain a suspension, 0.1 g of the synbiotic agent was dissolved in 2 ml of 1% starch paste. At this concentration, the suspension retained the ability to pass through the lumen of the probe for intragastric administration. Thus, each test mouse received 3 ml of a suspension containing 150 mg of the synbiotic under investigation, which corresponds approximately to a dose of 7500 mg/kg. Since it is assumed that the synbiotic agent is not metabolized and its action is not species-specific, cross-species conversion factors do not apply (i.e. more than 550 g per adult). The control group of mice was injected with 1% starch paste in the same way. The animals were observed for 7 days.

During the next seven days of observation of the animals, there were no deviations in the general condition. Thus, the general condition of the animals did not change during the entire period of observation of them (see paragraph on the definition of harmlessness). Animal mortality was also not observed, therefore, due to the impossibility of administering to animals a dose of the studied synbiotic agent greater than 7500 mg/kg, determining LD ₅₀ and achieving higher values of the maximum tolerated dose is not possible. The body weights of the mice observed after administration of the test biological product are shown in FIG. 12.

As follows from the presented results, during the experiment, the experimental animals showed a positive dynamics of body weight, no decrease in body weight compared to the initial one was observed. In addition, there were no significant differences from the animals of the control group. That is, the use of a synbiotic agent at a dose of 550 g/day per adult is harmless.

5 mice from each group were euthanized 24 hours and 7 days after the administration of the study biological product. Conducted necropsy of the bodies of animals. During necropsy, visually distinguishable changes were not found in any of the experimental animals. The results of macroscopic and microscopic examination of the internal organs of mice treated with the studied synbiotic agent 24 hours and 7 days after administration showed that this agent did not cause dystrophic, inflammatory, locally irritating and other pathological changes in internal organs. These data confirm the possibility of using the resulting agent as a safe synbiotic.

List of sources

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Claims (1)

A synbiotic agent based on a viable biomass of the yeast strain Meyerozyma (Pichia) guilliermondii VKPM Y-4316, characterized in that it contains 109 CFU of the above yeast per g of substrate, and has the following composition per 100 g: protein -18.8 ± 0.2 g, fats - 0.6 ± 0.3 g, carbohydrates - 65.3 ± 1.1 g, vitamin B1 (thiamine) - 0.14 ± 0.04 mg, vitamin B2 (riboflavin) - 0.82 ± 0.2 mg, prebiotic component: soluble and insoluble dietary fiber - 73.2 ± 11% by mass fraction, and has an adsorption activity of 65.5 mg / g.

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