RU2630457C1 - Method of obtaining biologically active substance with prebiotic effect based on medusomyces gysevii - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method of obtaining a biologically active substance with a prebiotic effect from dense biomass (zooglosses) of Medusomyces gysevii involves its extraction a, continuous washing under running water for 10-15 minutes, a grinding into individual fragments of size 2…3 cm² and a subsequent lyophilization. In this case, fragments of dense biomass (zoolei) of Medusomyces gysevii are placed in a low-temperature refrigerating chamber at a temperature of -38…-40°C for 48-50 hours, after which it is dried to a moisture content 8…10% at operating pressure in sublimate 70…80 Pa, condenser temperature -45…-50°C and the total duration of the drying cycle is 30-35 hours. Then, the dry substance is grinded to a particle size of 50…150 mcm, is placed in a glass container, after which it is tightly clogged and sterilized in an autoclave at a temperature of 110°C for 15 minutes.

EFFECT: invention makes it possible to obtain an effective biologically active substance from a byproduct of beverages, starter cultures, concentrates from Medusomyces gysevii having a prebiotic effect which can be used as a component of feeds and premixes for warm-blooded animals in order to normalize the composition and functions of the own microflora of the gastrointestinal tract.

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Description

FIELD OF THE INVENTION

The invention relates to biotechnology and agriculture, in particular to a method for producing a biologically active substance based on dense biomass (zooglya) Medusomyces gysevii (Kombucha), which has a prebiotic effect, and can be used as a component of feed and premixes for warm-blooded animals in order to normalize the composition and functions of the microflora of the gastrointestinal tract.

State of the art

A known method of obtaining a biologically active beverage based on the culture fluid Medusomyces gysevii and its concentrate, which includes incubating zoogley kombucha under aerobic conditions on a nutrient solution of a sugar-containing product in tea infusion at a temperature of 12 ... 40 ° C for 2-110 days. After removing the zooglya, the resulting culture fluid is incubated under aerobic conditions, stirring it at a speed of 0.1 ... 2 m / s at a temperature of 12-40 ° C for 40-80 hours. Next, the finished biologically active drink is evaporated in the temperature range 45 ... 55 ° C until a mass fraction of solids of more than 60 masses is obtained. % (see patent for invention RU 2500299, class A23L 2/395).

The disadvantage of this method is the presence of a large amount of waste in the production and the absence of a prebiotic effect on the product.

A known method of preparing a drink with biological activity, and a drink obtained by this method. The method consists in fermenting initially sugar-containing liquid using a yeast culture (baker's yeast, or wine yeast, or other yeast cultures) for a period of time from 1 hour to 14 days at a temperature of from 20 to 40 ° C, and then re-fermentation in the presence of tea culture fungus for a period of time from 10 hours to 30 days (see patent for invention RU 2337592, class A23L 2/38, A23L 2/70).

The disadvantage of this method is the complexity of the process and the absence of a prebiotic effect on the product.

A known method for producing a composition having biological activity, and a method for producing zoogley kombucha. In the first embodiment, the preparation of a biologically active composition occurs by mixing a sugar-containing substance, tea fungus culture fluid and chaga infusion, and fermenting the resulting mixture. In this case, it is advisable to additionally maintain the fermented mixture under anaerobic conditions and / or at a temperature of no higher than 10 ° C. According to the second embodiment, the preparation of a biologically active composition is carried out by mixing a sugar-containing composition, a component containing kombucha culture and chaga infusion and fermenting the resulting mixture, according to which a pre-fermented composition is used as the sugar-containing composition. It is preferable to use kvass as a pre-fermented composition. It is also advisable to further ferment the mixture under anaerobic conditions and / or at a temperature not exceeding 10 ° C. According to the third option, the preparation of a biologically active composition occurs by preliminary preparation of the zoo glue of kombucha, mixing the sugar-containing substance with liquid and chaga infusion, and fermenting the resulting mixture in the presence of zoogley of kombucha. Preferably, the preparation of zoogley is carried out by growing in a liquid medium other than the liquid used in mixing. It consists in the fact that 100 grams of sugar was added to a solution of blackcurrant juice (100 grams of pureed berries with 1000 ml of water) and a tea mushroom culture was added to the resulting composition. After 7 days of fermentation at a temperature of 25 ° С, a zoogley of Kombucha 5 mm thick was formed on the surface, which was a red-tinted film. The film was separated from the culture fluid, squeezed in a press and dried for 3 hours at a temperature of 40 ° C. In this case, before drying, the zoogley was ground on a mini-cutter (which is not mandatory). It is also advisable to further ferment the mixture under anaerobic conditions and / or at a temperature not exceeding 10 ° C (see patent for invention RU 2280394, class A23L 2/52, A23L 2/38).

The disadvantage of this method is the complexity of the process and the absence of a prebiotic effect on the product.

The closest in technical essence and accepted by the authors for the prototype is a method for producing a biologically active biomaterial, which consists in the fact that the zoo glue of Kombucha is selected as a natural compound, obtained by incubating it under aerobic conditions on a nutrient medium at room temperature for 14 ... 160 days. In this case, the nutrient medium is a solution of a sugar-containing product in the infusion of tea. Next, the obtained zooglium is extracted from the nutrient medium and dried at a temperature of not more than 55 ° C to a mass fraction of solids of more than 70 wt. % Zoogley incubation is carried out in traditional conditions. Zoogley growth time is determined depending on the amount of biomass required. A significant difference is the level of humidity required for the product, which is going to be manufactured further from this biomaterial, namely leather substitute, insoles, bactericidal patch, biologically active food additives (absorbent), used as a filler in the manufacture of medicines, as well as in food branches - as an additive in bakery and confectionery products (to slow down hardening, reduce calorie content), as a stabilizer of food emulsions, to improve the fluidity of the product, as an agent for the mind sheniya thickening properties of the starch (see. patent RU 2500198, Cl. A23L 2/00, A23L 2/30, A23F 3/16).

The disadvantage of this method is the lack of information on methods of sterilization, drying and standardization of the finished substance, the long periods of cultivation of zooglya, the absence of a prebiotic effect on the product and the incompletely disclosed potential of the raw material.

Disclosure of invention

The objective of the invention is to develop a method for producing a biologically active substance based on Medusomyces gysevii, which has a prebiotic effect on the organism of warm-blooded animals.

The technical result that can be achieved using the proposed method is to obtain a cheap and effective biologically active substance from a by-product of the production of drinks, starters, concentrates from Medusomyces gysevii - dense biomass (zoogley), the use of which by warm-blooded animals leads to selective stimulation of growth and the development of normal intestinal microflora, resulting in a beneficial effect on the body as a whole.

The technical result is achieved using the method of obtaining a biologically active substance with a prebiotic effect from dense biomass (zooglya) of Medusomyces gysevii, including its extraction, continuous washing under running water for 10-15 minutes, grinding into separate fragments of 2 ... 3 cm and subsequent lyophilization , for this, fragments of dense biomass (zooglya) of Medusomyces gysevii are placed in a low-temperature refrigerating chamber at a temperature of -38 ... -40 ° С for 48-50 hours, after which they are dried to a moisture content of 8 ... 10% at a working pressure ii sublimator at 70 ... 80 Pa, condenser temperature -45 ... -50 ° C and a total duration of 30-35 hours drying cycle. Then the dry substance is ground to a particle size of 50 ... 150 microns, placed in a glass container, then tightly cork and sterilized in an autoclave at a temperature of 110 ° C for 15 minutes.

Thus, the technical result is achieved due to the optimal parameters for the preparation of a biologically active substance, while the invention allows to utilize zooglya, which is inevitably formed in the production of drinks, starter cultures and concentrates from Kombucha.

The dense biomass (zoogley) of Medusomyces gysevii consists of microbial mass (acetic acid bacteria, yeast) and metabolites of microbial origin: mucous membranes, lignin, fiber, which includes bacterial cellulose, hemicellulose (Danielyan L.T., 2005: Jayabalan R. 2007 : Kozyrovska NO, 2012) and enzymatic fluid, up to 90% of the total volume. In practice, it is zooglea that remains an unused by-product after receiving the culture fluid, which is widely used in the food industry. Bacterial cellulose Medusomyces gysevii is synthesized by bacteria in the form of a chemically pure material (gel film). It has a three-dimensional structure formed by fibers that intertwine in a network in which bacteria are located. Cells of bacterial cellulose save cells from ultraviolet radiation, protect them from the attack of other microorganisms, as well as from the toxic effects of metals, such as copper. Bacterial cellulose is non-toxic, non-allergenic, highly absorbent, stimulates digestion and acts as an adsorbent. The dietary properties of bacterial cellulose are much higher than pure plant cellulose (Shamolina I.I., 1997; Pinevich A.V. 2007).

In addition, the choice of the drying method, namely, lyophilization, allows one to obtain a substance without losing its initial biological activity and preserve the rich chemical composition inherent in the enzymatic liquid: organic acids (malic, lactic, acetic, glucuronic, gluconic, citric, oxalic, pyruvic, phosphoric ), ethyl alcohol, sugars (monosaccharides, disaccharides), vitamins (C, B _1, PP, D), enzymes (amylase, sucrose, protease, catalase), lipids (phosphatides, sterols, fatty acids), pigments, purine bases, tannin substances, tarry substances, aldehydes, proteins, caffeine, saponins (Danielyan L.T., 2005: Jayabalan R. 2007: Kozyrovska NO, 2012). All substances present in zooglya provide a stimulating effect of the substance, which promotes self-healing of microbiocenosis.

In connection with the foregoing, as a potential raw material object, due to a wide range of biochemical components of microbial origin, we considered the dense biomass (zoogley) of the symbiont Medusomyces gysevii (Kombucha), as it is able to meet all the requirements for a substance to determine it as prebiotic: not hydrolyzed by digestive enzymes and not absorbed in the upper gastrointestinal tract; be a selective substrate for one and / or several genera of beneficial bacteria; have the ability to change the balance of intestinal microflora in the direction of a more favorable composition for the host organism; induce beneficial effects not only at the level of the gastrointestinal tract, but also at the level of the organism as a whole, i.e. system effects.

The implementation of the invention

Examples of a specific implementation of the method for producing a biologically active substance based on dense biomass (zoogley) of Medusomyces gysevii, which has a prebiotic effect.

Example 1. Zoogley Medusomyces gysevii is removed from the culture fluid, washed under running water, and packaged.

The resulting substance has disadvantages that are a high percentage of moisture in the product, the impossibility of accurately calculating the required dose, the impossibility of long-term storage, a low concentration of biologically active substances, and a low prebiotic effect.

Example 2. Zoogley Medusomyces gysevii is removed from the culture fluid, washed under running water, forcibly freed from moisture under a press, then dried in an oven at 55 ° C until the moisture content is not more than 30-40%.

The obtained substance has the disadvantages of a long drying period, high rigidity and stickiness of the finished product, difficult grinding, low concentration of biologically active substances, low prebiotic effect.

Example 3. Zoogley Medusomyces gysevii is removed from the culture fluid, continuously washed under running water for 10-15 minutes, crushed into separate fragments 2 ... 3 cm in size, placed in a low-temperature refrigerating chamber at a temperature of -38 ... -40 ° C at 48- 50 hours, after which it is dried using a freeze dryer to a moisture content of 8 ... 10% at a working pressure in the sublimator of 70-80 Pa, a condenser temperature of minus 45-50 ° C and a total drying cycle of 30-35 hours, the dry substance is crushed.

The resulting substance has disadvantages in that it contains live bacteria and fungi that are part of the Medusomyces gysevii symbiont, in an amount exceeding the permissible norms established in accordance with the standards and requirements of the Russian Federation for feed and feed additives.

Example 4. Carried out analogously to example 3, but after drying, the dry substance is crushed to particle sizes of 50 ... 150 microns, placed in a glass container, then tightly cork and sterilized in an autoclave at a temperature of 110 ° C for 15 minutes.

The substance obtained by this method does not contain living microorganisms, is easily transported and stored for a long time, has high biological activity and a high prebiotic effect.

Thus, the most optimal is the method of obtaining a biologically active substance based on Medusomyces gysevii with a prebiotic effect according to example 4.

Tests of a biologically active substance based on Medusomyces gysevii were carried out in order to confirm its prebiotic effect on the organism of warm-blooded animals. To do this, we studied the composition of the intestinal microflora of white rats with experimental antibiotic-associated dysbiosis.

All animal experiments were carried out in accordance with EU Directive 86/609 / EEC and in accordance with the legislation of the Russian Federation governing animal experiments.

The study was conducted on 60 male Wistar white rats aged 9 months, with an average body weight of 250 g, which were randomized and divided into 2 groups, control and experimental, 30 animals each. Animals were kept in a vivarium under standard conditions, with free access to water and feed. The air temperature in the room was plus 20 ... 22 ° C, air humidity - 40 ... 45%, the lighting cycle 12:12.

The material for bacteriological research was feces of rats. The collection of feces was carried out in a sterile, hermetically sealed container with a sterile spatula. The time from the moment the material was taken to its processing in the laboratory did not exceed 5 hours. For bacteriological analysis of feces, the industry standard for intestinal dysbiosis was used.

At the initial stage of the experiment, in all animals included in the experimental and control groups, a background bacteriological study of the contents of the large intestine was carried out. After that, they initiated the development of microbiocinosis disorders by oral administration of gentamicin with a syringe through a needle with olive at the end in doses of 10 mg 2 times a day, calculated on body weight, based on the average daily doses of an antibiotic for people for 10 days. In this case, the animals of the control group received the standard diet recommended by the Research Institute of Nutrition of the RAMS, and the animals of the experimental group each day during the experiment were supplemented with 120 mg of biologically active substance from zoogley Medusomyces gysevii per rat, taking into account the conversion factor per unit body surface.

The next selection of feces for bacteriological studies in experimental animals was carried out on the 5th day and 10th day.

The amounts of Bifidobacterium spp, Lactobacillus spp, Escherichia coli, Candida spp were counted. The research results are presented in table 1.

The results presented in table 1 show that in animals of the control group that are in the state of antibiotic-associated dysbiosis on a standard diet, already on the 5th day there is a significant decrease in the number of Bifidobacterium spp, Lactobacillus spp, Escherichia coli, which lasts until the end experiment, while in animals that received a biologically active substance from the zoogley Medusomyces gysevii, such significant changes did not occur. The amount of Candida spp. in animals of the control group, against the background of antibiotic-associated dysbiosis, on the contrary, it increases significantly until the end of the experiment, while in animals that received the biologically active substance from the zoogley Medusomyces gysevii per rat per day, it remains unchanged throughout the experiment.

Pathogenic microorganisms and microorganisms of the genus Proteus, Klebsiella, Citrobacter, hemolizing flora in all experimental rats were not detected before and after the experiment.

In addition, the use of a biologically active substance from the zooglya of Medusomyces gysevii has a positive effect on the general condition of the animals: during the experiment, they willingly took food, drank water, and moved actively in the cell. While the animals of the control group, on the contrary, showed a decrease in activity and a passive state, loss of appetite, a frequent number of individuals (11 rats) in this group showed frequent bowel movements with the release of liquid fecal masses.

After stopping the oral administration of gentamicin, the animals of the control group were divided into 2 subgroups of 15 rats each. Over the next 7 days, the animals of the first subgroup (control) received a standard diet recommended by the Research Institute of Nutrition RAMS, and the animals of the second subgroup (experimental) every day during the experiment, in addition to the main diet, were fed 120 mg of biologically active substance from Medusomyces gysevii zooglya per rat, taking into account the conversion coefficient per unit surface of the body. Feces for bacteriological studies in animals of both subgroups were selected on the 3rd and 7th day (at the end of the experiment).

The amounts of Bifidobacterium spp, Lactobacillus spp, Escherichia coli, Candida spp were counted. The research results are presented in table 2.

The results presented in table 2 show that after the cessation of gentamicin administration in animals of the 1st subgroup (control) on standard nutrition, a decrease in the number of Bifidobacterium spp, Lactobacillus spp, Escherichia coli and an increase in the number of Candida spp. lasts up to 3 days. At the end of the experiment on the 7th day, a significant increase in the number of Bifidobacterium spp, Lactobacillus spp was recorded, which indicates the natural self-healing of microflora.

In animals of the second subgroup (experimental), which received a biologically active substance from the zoogley Medusomyces gysevii, already on the 3rd day of the experiment, a steady tendency was observed to increase the number of Bifidobacterium spp, Lactobacillus spp, Escherichia coli and decrease Candida spp, which continues until the end of the experiment and brings group indicators closer to the initial data characteristic of healthy animals.

In rats of the 2nd subgroup (experimental), an improvement in the general condition of the body was observed already on the 2nd day after the start of its use of the biologically active substance from the zoogley Medusomyces gysevii, the animals resumed their activity, improved feed intake, and stools returned to normal. While in animals of the 1st subgroup, improvements began to appear only on the 5th day of the experiment.

The results obtained indicate that the biologically active substance from the zoogley Medusomyces gysevii, obtained by the proposed method, favorably affects the intestinal microflora of warm-blooded animals and their general condition.

The invention in comparison with the prototype and other known technical solutions has the following advantages:

- biologically active substance obtained using the present invention has a prebiotic effect on the organism of warm-blooded, stimulates the growth and development of normal intestinal microflora, resulting in a beneficial effect on the body as a whole;

- due to optimal processing conditions, the substance retains its original rich biochemical composition;

- The main methods, preparations, devices used in the preparation of the drug are readily available;

- the product obtained using the proposed method has a low cost, because zoogley is used in its production, which is inevitably formed in the production of drinks, starter cultures and concentrates from tea mushroom and is a waste of production;

- environmental cleanliness, lack of harmful impurities.

Literature

Patent RU 2280394. A composition of ingredients having biological activity (options), a method for its preparation (options) and a method for producing a zooleichain fungus / D.A. Zaitsev. - Publ. 12/16/2004.

Patent RU 2337592. A method of preparing a drink having biological activity, and a drink obtained by this method / D.A. Zaitsev. - Publ. 04/14/2005.

Patent RU 2500198. A method of obtaining biologically active biomaterial and biomaterial obtained by this method / V.X. Khachatryan. - Publ. 12/10/2013.

Patent RU 2500299. A method for producing a biologically active beverage, a beverage obtained by this method, a method for producing a biologically active beverage concentrate and a concentrate obtained by this method / B.X. Khachatryan. - Publ. 08.21.2012.

Danielyan, L.T. Kombucha and its biological features / L.T. Danielyan. - M.: Medicine, 2005 .-- 83 p.

Pinevich A.V. Miracle films, or the Word about bacterial cellulose / A.V. Pinevich // Bulletin of St. Petersburg University. - 2007. - No. 3. - S. 33-39.

Tarasenko, N.A. Briefly about prebiotics: History, classification, preparation, application / N.A. Tarasenko, E.V. Filippova // Fundamental research. - 2014. - No. 6. - S. 45-48.

Shamolina I.I. Prospects for the use of microbial raw materials in the production of fibrous and film materials / II. Chamoline // Chemical fibers. - 1997. - No. 1. - S. 3-10.

Jayabalan, R. Changes in content of organic acids and tea polyphenols during kombucha tea fermentation / R. Jayabalan, S. Marimuthu, K. Swaminathan // Food Chemistry. - 2007.P. 392-398.

Kozyrovska, N.O. Kombuchamicrobiome as a probiotic: a view from the perspective of post-genomics and synthetic ecology / N.O., Kozyrovska, O.M., Reva, V.B., Goginyan, J.P. de Vera // Biopolymers and Cell. - 2012. - Vol. 28., No. 2. - P. 103-113.

Claims (1)

A method of obtaining a biologically active substance with a prebiotic effect from the zooglya of Medusomyces gysevii, including its extraction, continuous washing under running water for 10-15 minutes, grinding into separate fragments of 2 ... 3 cm ² and subsequent lyophilization, for this, fragments of the zooglya of Medusomyces gysevii are placed in a low-temperature refrigerating chamber at a temperature of -38 ... -40 ° C for 48-50 hours, after which it is dried to a moisture content of 8 ... 10% at a working pressure in the sublimator of 70 ... 80 Pa, a condenser temperature of -45 ... -50 ° C and the total cycle times drying 30-35 hours, then the dry substance is crushed to a particle size of 50 ... 150 microns, placed in a glass container, then tightly cork and sterilized in an autoclave at a temperature of 110 ° C for 15 minutes.