RU2792775C1 - Method for obtaining a biologically active additive based on whey and plant extract - Google Patents

Abstract

FIELD: food industry.

SUBSTANCE: invention relates to the food industry. The method for obtaining biologically active additives based on whey and plant extract is carried out as follows. Vegetable raw material is prepared and extracted with ethyl alcohol, which is used as plant cell cultures, common thyme callus culture and/or real ginseng root culture. The extraction of the biomass of the callus culture of common thyme is carried out at a temperature of 70°C and an ethyl alcohol concentration of 70%, the extraction of the biomass of the root culture of this ginseng is carried out at a temperature of 50°C and an ethyl alcohol concentration of 30% with a ratio of plant material: extractant 1:86 and a 4 hours extraction. The extract is filtered and the extractant is evaporated. The extract is dried on a spray dryer. Dry extract is mixed with auxiliary components: microcrystalline cellulose, aerosil, magnesium stearate, dry whey - curd and/or cheese. The resulting mixture is encapsulated.

EFFECT: obtaining an encapsulated form of a biologically active additive containing dry milk whey and an antioxidant complex of biologically active substances (BAS) of plant origin.

4 cl, 7 tbl, 3 ex

Description

The invention relates to biotechnology and can be used in the food and pharmaceutical industries to obtain a dietary supplement with antioxidant properties based on milk whey (curd and cheese) and extracts of common thyme (Thymus vulgaris L.) callus culture and real ginseng root culture (Panax ginseng) S.A. Meu).

Currently, the fastest, economically acceptable and scientifically sound way to solve the problem of human nutrition rationalization is the widespread use of biologically active food supplements (BAA) in everyday practice.

Milk whey (curd and/or cheese whey) is a by-product of the production of cottage cheese or cheese. Milk whey is often not used, but is discharged into sewers or water bodies, which worsens the ecological situation, leads to a technogenic load on wastewater treatment plants and the environment. At the same time, whey contains easily digestible components - proteins, carbohydrates (lactose), mineral salts, etc. [1].

According to the chemical composition, cheese and curd whey contains on average (wt.%): dry substances 6.0-6.5, lactose 4.2-4.5, protein substances 0.6-0.8, mineral salts 0.5 -0.6, as well as vitamins A, E, group B and others [2].

Due to the high mass fraction of moisture and the possibility of spoilage, whey must be fed to livestock within 12 hours. Within 2 days without special treatment, the nutritional value of whey decreases: the content of lactose decreases by 40%, protein by 14%, dry matter by 10%. Effective methods of using whey in dry and condensed form [3].

The use of liquid whey is impractical, since the assimilation of the main nutrients from whey is achieved only by 20% [4].

There is experience in the use of condensed whey in the form of a protein-carbohydrate concentrate. For example, the specified concentrate is recommended for the preparation of a feed mixture for raising dairy calves. The mixture includes whey, pea and oatmeal flour, fodder yeast, fish oil, table salt and other components. The concentrate is quite complex in composition and preparation. Feeding the mixture to young cattle increases the daily gain in live weight by 25-30% [5].

Antioxidants are molecules that fight oxidative processes in the body caused by free radicals. The main factors that increase the production of free radicals include: internal inflammation, polluted air, excessive exposure to ultraviolet radiation, cigarette smoke. Oxidative stress due to free radicals has been implicated in a variety of diseases, including cardiovascular disease, diabetes, Alzheimer's disease, Parkinson's disease, and eye problems such as cataracts and age-related macular degeneration [6]. Antioxidants are able to slow down these processes, and in some cases reverse them, restoring damaged cells. The fight against oxidative processes is the main task of antioxidants. As a consequence, they can: strengthen human tissue function, rejuvenate, enhance human immunity, improve metabolic function, reduce the risk of cancer, strengthen the function of lymphatic tissues, and resist peroxidation [7].

Antioxidants produced by the body itself are called endogenous, and those that come from outside are called exogenous. Secondary plant metabolites (flavonoids, polyphenols, glycosides, tannins, vitamins) are considered to be the most active in the fight against free radicals [8]. Common thyme (Thymus vulgaris L.) and real ginseng (Panax ginseng C.A. Mey) are of interest as sources of secondary metabolites with antioxidant properties.

Common thyme (Thymus vulgaris L.) is widely used in the food, cosmetic, and pharmaceutical industries [9]. Common thyme herb (Thymus vulgaris L.) contains [10]:

- essential oil (approximately 0.6%), which includes thymol (up to 42%), carvacrol, n-cymol, α-terpineol, borneol, geraniol;

- tannins;

- bitterness;

- gum;

- triterpene compounds - ursolic and oleanolic acids;

- flavonoids - apigenin, naringenin, cynaroside, cosmossiin, scutellarin;

- mineral salts;

- pigments - zeaxanthin, lutein, luteolin;

- sugars - glucose, galactose, arabinose, rhamnose, xylose, glucuronic acid, galacturonic acid;

- amino acids - cystine, alanine, leucine;

- fatty acids - palmitic, linoleic, linolenic acids;

- organic acids - citric, malic, malonic, oxalic acids. Also, gallic, hydroxybenzoic, chlorogenic, syringic, coumaric, benzoic, cinnamic, and rosmaric acids were found in the methanolic and ethanolic extracts of common thyme (Thymus vulgaris L.) [11]. The plant has biologically active properties due to the content of phenolic substances, among which special attention is paid to thymol, carvacrol and tannins. Due to the presence of these substances, common thyme (Thymus vulgaris L.) has anti-inflammatory, antimicrobial, and analgesic activity [11].

True ginseng (Panax ginseng S.A. Mey), or common ginseng, is a slow growing perennial plant of the Panax genus, which is often used as a component for functional foods and as a phytotherapeutic agent for the prevention, treatment of diseases (cancer, allergies, inflammatory diseases, diabetes mellitus) [12]. According to scientists, real ginseng (Panax ginseng S.A. Mey) is used as an adaptogenic agent to increase physical performance, vitality, immunity, resistance to stress and aging [13].

The main biologically active components of this ginseng (Panax ginseng S.A. Meu) include:

1. polysaccharides (glycans - ginsenan RA and ginsenan RV, consisting of L-arabinose, D-galactose, L-rhamnose, D-galacturonic acid and D-glucuronic acid);

2. saponins, especially ginsenosides, which can be classified as oligoglycosides of the dammarane, ocotillol and oleanan type, and divided into protopanaxadiol and protopanaxatriol;

3. flavonoids (kaempferol, trifolin and panasenoside);

4. polyacetylenes (panaxinol and panaxidol).

Ginsenosides (Re, Rg1, Rd, S-Rg2, Rb2, F1, Rc, S-Rh1, Rb3, R-Rg2, LC1) are the main active ingredients of real ginseng (Panax ginseng S.A. Mey), they belong to the class natural products of steroid glycosides and triterpene saponins and accumulate in the roots, berries and leaves of the plant [14].

Studies have shown that ginsenosides exhibit pronounced pharmacological effects such as relieving fatigue, improving immunity, slowing down aging, inhibiting cancer cell metastasis, regulating blood glucose levels, and protecting liver and kidney function [15]. Ginsenosides have antiallergic activity in mouse models of bronchial asthma and atopic disease. In addition, they suppress the development of the inflammatory process by inhibiting the activation of NF-κB, and increase immunity.

Polyphenols are natural, safe antioxidants that can prevent a number of diseases (cardiovascular, cancer, hypertension, diabetes, atherosclerosis). Many natural polysaccharides are highly valuable biomaterials characterized by biocompatibility, low toxicity, and biodegradable properties [16]. Plant-derived polysaccharides have various pharmacological properties, including antioxidant, antitumor, and immunomodulatory properties. Ginseng extract (Panax ginseng S.A. Mey) induces an improvement in the blood lipid profile, mainly by reducing the level of total and LDL-cholesterol [17].

With the development of science and technology in the field of human potential and high competition in the dairy market, we can expect the appearance of dairy products with the addition of antioxidants to enhance the immune effect.

From the existing level of technology, a method for obtaining dietary supplements for food is known (RF patent No. 2370151, publ. 20.10.2009). BAA for food is obtained on the basis of vegetable raw materials. Prepare honey extracts of calendula, dandelion, peppermint, licorice, taken in the form of infusions (decoctions), germinated in the serum of pumpkin seeds, chickpeas and milk thistle. The main disadvantage of this method of obtaining dietary supplements for food is the use of honey as an extractant. Firstly, some users of dietary supplements have an individual intolerance to honey. Secondly, honey is a rather expensive extractant. Also, this dietary supplement is characterized by low antioxidant activity due to the absence of phenolic acids and steroid-like compounds.

A known method of obtaining dietary supplements for food "ERAMIN" (RF patent No. 2435455, publ. 10.12.2011). The method for obtaining dietary supplements includes preparing plant materials, weighing the components of the recipe, preparing a solution from a dry mixture of alfalfa hay, trace elements and distilled water, extracting at a temperature of 180-190 ° C for 5-6 hours at a pressure of 6⋅10 ⁵ Pa, drying the extract in a drying cabinet at a temperature of 55-60°C for 4 hours. The disadvantages of this method include the high temperature and duration of extraction of plant materials, as well as low antioxidant activity.

A method for obtaining a biologically active food additive is described (RF patent No. 2449600, publ. 10.05.2012). BAA is a complex of vegetable raw materials, inosine, succinic acid and an auxiliary component - magnesium stearate. As components of plant origin, dry extracts of Eleutherococcus senticosus, fruits of milk thistle, tribulus terrestris and green tea are used. The disadvantages of this method include biologically active substances that are not balanced in their composition, low antioxidant activity due to the absence of phenolic acids and steroid-like compounds.

There is a method for obtaining a dry mix for preparing a sports drink (RF patent No. 2579211, publ. 04/10/2016). The method involves obtaining a dry biologically active additive, which contains, wt. %: dry cheese whey 25.0-27.0, dry extract of goji berries 22.2-24.2, dry extract of pineapple 20.1-20.4, flower pollen 11.1-12.2, royal jelly 4 ,3-4.5, fructose - the rest. The disadvantage of this composition is intolerance in some patients of individual components, such as royal jelly and pollen.

Also, this dietary supplement has low antioxidant activity due to the absence of phenolic acids and steroid-like compounds.

RF patent No. 2672914, publ. 11/21/2018 describes the production of whey with antioxidant properties. The invention describes the preparation of an antioxidant complex that contains: a vegetable-derived antioxidant and a milk hydrolyzate enriched with lactates during hydrolysis from whey and/or buttermilk and/or skim milk. The disadvantage of this method is the unbalanced composition of the vitamin-mineral complex, which does not have antioxidant properties.

There is a known method for obtaining dietary supplements in encapsulated form (RF patent No. 2756855, publ. 06.10.2021). The method includes obtaining a dietary supplement in a capsule form containing a complex of biologically active substances in the form of a dry extract as an active component, including the roots of a high lure, horsetail grass, dogwood fruits, fruits of a tripartite sequence, roots and rhizomes of elecampane, common blueberry shoots, gelega grass, leaves mulberries and strawberry leaves. The method for obtaining a dietary supplement in an encapsulated form includes obtaining a liquid extract followed by evaporation, purification of the extract followed by obtaining a dry extract, mixing with excipients and encapsulation. Among the disadvantages of the method for obtaining such a dietary supplement, ultrasonic extraction of plant materials should be mentioned, since this extraction method uses expensive equipment, high aggressiveness of ultrasound in relation to antioxidant substances. Water is used as an extractant; such an extractant is not able to release a sufficiently high amount of the BAS antioxidant complex.

A method for obtaining dietary supplements based on a plant source for the complex effective nutrition of dogs is described (CN patent No. 104026419A, publ. 10.09.2014). The method for preparing a complex dietary supplement of plant origin for dogs intended for self-feeding includes the following steps: adding skimmed milk powder, whey powder, sucrose and galactooligosaccharide to a container, uniform mixing and adding microcrystalline cellulose, mixing the nutrient with a plant source (walnut extract, pecan extract and banana extract), mixing again, then the whole mixture is tabletted. The disadvantages are the length of the process of evaporation of walnut, pecan and banana extracts, as well as low antioxidant activity due to the absence of phenolic acids and steroid-like compounds.

The patent (patent RO No. 134736A2, publ. 02/26/2021) describes a method for obtaining microencapsulated dietary supplements of various functionality based on extracts of anthocyanins from eggplant peel and lactic acid bacteria, and its application. According to the invention, the method consists in freeze-drying an ethanol extract from eggplant peel, having a high content of anthocyanin compounds, and selected lactic acid bacteria in a combination of matrices based on whey proteins and hydrolysates, carboxymethylcellulose and pectin, to obtain natural ingredients with in vitro biocompatibility, with high antioxidant and metabolic activity. The disadvantage of this method is the lyophilization drying of the eggplant peel extract, since lyophilization requires careful preparation of the dried raw material for drying, creating a vacuum for complete drying, drying time and rather high energy costs. Also, for this dietary supplement, low antioxidant activity was noted due to the absence of phenolic acids and steroid-like compounds.

There is a known method for obtaining dietary supplements with antioxidant properties (patent MD No. 1480Y, publ. 31.01.2021). The invention relates to the food industry and sanocreatology, namely to a biologically active food supplement with antioxidant activity. The food supplement according to the invention contains, in wt. %: dry extract of amaranth seeds 25, dry extract of wormwood leaves 35, dry extract of dihydroquercetin 10, dry extract of walnut shell 10, dry extract of dandelion roots 10 and activated carbon 10. The disadvantages of this method include high temperature and duration of extraction of plant materials, which leads to the destruction of antioxidant BAS.

The closest to the claimed method (the closest analogue) is a method for the production of a biologically active food supplement in encapsulated form (RF patent No. 2759731, publ. 11/17/2021). This method involves obtaining a dietary supplement in an encapsulated form containing a complex of biologically active substances in the form of a dry extract as an active component, including yarrow grass, marigold flowers, birch leaves, horsetail grass, licorice roots, nettle leaves, fennel fruits in a ratio of 2: 2 :1.5:1.5:1:1:1, and a complex of excipients, including a filler, binder, disintegrant, anti-adherent, sliding. The method for obtaining a dietary supplement in an encapsulated form includes obtaining a liquid extract followed by evaporation, purification of the extract followed by obtaining a dry extract, mixing with excipients and encapsulation. Dietary supplements are used as a source of flavonoids and glycisyrrhic acid. The disadvantage of this dietary supplement is the multicomponent composition, which leads to low stability due to the chemical interaction of the ingredients. Also, this dietary supplement is characterized by low antioxidant activity due to the absence of phenolic acids and steroid-like compounds; it also does not contain an additional source of lactose, protein, vitamins, macro- and microelements.

The technical problem to be solved using the developed invention is to develop a new method for obtaining a biologically active food supplement with antioxidant properties based on whey and cell culture extracts of common thyme (Thymus vulgaris L.) and real ginseng (Panax ginseng S.A. Meu) .

The technical result obtained by implementing the claimed method is to obtain an encapsulated form of a biologically active additive containing dry milk whey and an antioxidant complex of biologically active substances (BAS) of plant origin.

To achieve this technical result, it is proposed to use a method for obtaining encapsulated dietary supplements based on dry whey and extracts of cell cultures of common thyme (Thymus vulgaris L.) and real ginseng (Panax ginseng C.A. Mey), including successive steps: growing cell cultures of plants, water-alcohol extraction of dried biomass of plant cell cultures, filtration of the extract, evaporation of ethyl alcohol, spray drying of milk whey and extracts of plant cell cultures in vitro, mixing and encapsulation of dietary supplement components: dry whey, dry plant extracts and excipients.

Currently, special attention is drawn to the method of obtaining isolated plant cells and tissues with subsequent isolation of biologically active substances from them. Cell cultures of plants in comparison with traditional plant raw materials have a number of advantages: the possibility of year-round obtaining of raw materials, regardless of the natural conditions of the region and the climatic parameters of the year; the possibility of optimizing and standardizing growing conditions; the ability of plant cell cultures to synthesize a high level of secondary metabolites, surpassing the level of traditional plants.

The developed method is implemented as follows.

The callus culture of common thyme (Thymus vulgaris L.) and the root culture of present ginseng (Panax ginseng C.A. Mey) are preliminarily grown.

To determine and develop the qualitative and quantitative composition of dietary supplement capsules, the results of the quantitative determination of phenolic acids in the biomass of the callus culture of common thyme (Thymus vulgaris L.) and ginsenosides in the biomass of the root culture of this ginseng (Panax ginseng S.A. Meu), which are given in the table, were used. 1.

At the second stage, the process of water-alcohol extraction of the dried biomass of the callus culture of common thyme (Thymus vulgaris L.) and the root culture of real ginseng (Panax ginseng C.A. Mey) is carried out. The temperature for the extraction of the biomass of the callus culture of common thyme (Thymus vulgaris) is 70°C, for the biomass of the root culture of this ginseng (Panax ginseng SA Mey) - 50°C; the concentration of ethyl alcohol for the extraction of the callus culture of common thyme (Thymus vulgaris) is 70%, and for the root culture of this ginseng (Panax ginseng S.A. Meu) - 30% at a ratio of plant material / extractant 1:86, the duration of extraction for both cells cultures is 4 hours.

At the third stage, the extracts obtained are subjected to purification using a filtration unit - a druk filter operating under pressure of carbon dioxide, nitrogen and phlegmatized air.

The fourth stage involves the evaporation of the obtained extracts of the callus culture of common thyme (Thymus vulgaris L.) and the root culture of this ginseng (Panax ginseng S.A. Mey) on a rotary vacuum evaporator. In the evaporator, the extract is concentrated to 20% dry matter of the total volume.

The fifth stage consists in drying the extracts of callus culture of common thyme (Thymus vulgaris L.) and root culture of real ginseng (Panax ginseng C.A. Meu) and whey. Drying is carried out by spraying.

The sixth stage in the framework of the described method is the mixing and encapsulation of dietary supplement components: whey powder, dry plant extracts and excipients.

The optimal composition of dietary supplements of the encapsulated form based on dry whey and dry extracts of callus culture of common thyme (Thymus vulgaris L.) and root culture of real ginseng (Panax ginseng C.A. Mey), which have antioxidant properties, has been experimentally established. For encapsulation of dietary supplement components, gelatin capsules containing 700 mg of supplements are used. In addition to dry plant extracts and dry whey, dietary supplements include excipients: microcrystalline cellulose (MCC), colloidal silicon dioxide (aerosil) and magnesium stearate.

The required amount of phenolic acids in the dried biomass of the callus culture of common thyme (Thymus vulgaris L.) and ginsenosides in the dried biomass of the root culture of this ginseng (Panax ginseng C.A. Mey) based on the recommended doses specified in the guidelines MP 2.3.1.1915-04 "Recommended level of consumption of food and biologically active substances" is presented in table 2 [18]. As part of the daily portion of dietary supplements, the content of pharmacologically active compounds of a medicinal plant should be no more than 50-60% of a single therapeutic dose (RTD) when using this plant as a medicine. At the same time, the lower limit of the content of these compounds in dietary supplements should not be less than 10% of a single therapeutic dose.

The invention is illustrated by the following examples:

Example 1 A callus culture of common thyme (Thymus vulgaris L.) is prepared. At the first stage of obtaining a callus culture of common thyme, sterile seeds are used. Sterilization of common thyme seeds is carried out as follows: the seeds are washed with detergent, then placed in 70% ethyl alcohol for 1 min, then in 5% sodium hypochlorite solution for 50 min. After sterilization, the material is washed three times for 20 min in distilled sterile water. After sterilization, to obtain sterile seedlings, the seeds are planted on a nutrient medium with the following composition: macrosalts according to Gamborg (B5) × 20 - 50 ml; microsalts B5×20 - 10 ml; Fe-EDTA - 5 ml; sucrose - 30 mg; nicotinic acid - 1 mg; pyridoxine - 1 mg; thiamine - 10 mg; inositol - 100 mg; agar - 20; distilled water - 1000 ml. The first seedlings of common thyme are observed after 4-5 weeks. For the induction of callus cell cultures, 2-8 week old sterile seedlings with 2-4 true leaves are used. To do this, the leaves and stems of seedlings are cut into pieces and planted on agar media in Petri dishes with a diameter of 60 and 90 mm, as well as in jars with ventilated lids. The formation of primary calli is observed on the 7-14th day of cultivation. The nutrient medium for further cultivation of callus culture of common thyme is the nutrient medium of the following mineral and hormonal composition: macrosalts B5x20 - 50.00 ml; microsalts B5 - 10.00 ml; Fe-EDTA - 5.00 ml; thiamine - 10.00 mg; pyridoxine - 1.00 mg; nicotinic acid - 1.00 mg; sucrose - 30.00 g; inositol - 100.00 mg; kinetin - 2.00 mg; 6-benzylaminopurine (6-BAP) - 0.10 mg; p-indoleacetic acid (PUA) - 2.00 mg; 2,4-dichlorophenoxyacetic acid (2,4-D) - 2.00 mg; agar - 20.00 g; distilled water - 1000 ml. From the formed seedlings, stems and leaves are used as explants (8 days after the appearance of the first leaf). All explants are placed on a nutrient medium of a similar mineral and hormonal composition and incubated for about 25 days. The primary callus is separated from the remnants of plant explants and transferred to a fresh nutrient medium. The further crop growing cycle is 4-5 weeks.

Callus cultures of common thyme (Thymus vulgaris L.) are dried at a temperature of 25°C in a place protected from light and crushed. The dried plant material is crushed in a mill brand LZM-1M (Russia, Olis) and sieved through a sieve with a hole size of 1 mm. The process of extraction of the callus culture of common thyme (Thymus vulgaris) is carried out at a temperature of 70°C, an ethyl alcohol concentration of 70% at a ratio of plant material/extractant of 1:86 for 4 hours.

The extracts obtained are subjected to purification using a filtration unit - a druk filter operating under pressure of carbon dioxide, nitrogen and phlegmatized air.

After purification, the extract is subjected to evaporation on a rotary vacuum evaporator. In the evaporator, the extract is concentrated to 20% dry matter of the total volume.

Further drying of the extract is carried out on a spray dryer at a temperature of 90°C, an air flow rate (aspiration) of 100 m/h and a solution supply rate to the spray unit of 7 ml/min.

The dried extract of callus culture of common thyme is one of the components of dietary supplement. The second bioactive component is whey powder (curd and/or cheese whey).

Drying of milk whey (curd and/or cheese whey) is carried out on a spraying plant with the given parameters: drying temperature is 100°C, air flow rate (aspiration) is 100 m/h, and the solution supply rate to the spraying plant is 10 ml/min.

The next stage is the mixing of dietary supplement components. The mixing process involves the connection in a V-shaped mixer for bulk media of the following components: dry extract of common thyme callus culture (Thymus vulgaris L.), dry milk whey (curd and / or cheese) and excipients in accordance with the dietary supplement formulation (table 3) . After mixing the dietary supplement components, encapsulation takes place in gelatin capsules. The resulting capsules are packed in whole blisters of 10 cells.

For the finished dietary supplement, quality indicators are monitored, including antioxidant activity (table 6, 7).

Example 2. Similar to example 1, but the method involves the use of the root culture of ginseng present.

A root culture of present ginseng (Panax ginseng SA Mey) is prepared. At the first stage of obtaining the root culture of this ginseng, sterile rhizomes are used. The rhizomes are washed with detergent and placed in a 0.1% solution of sublimate (HgCl ₂ ) for 1 min. After sterilization, the material is washed three times for 20 min in distilled sterile water. Parts of the rhizome are cut with a scalpel into 5×5 mm segments and used as explants. For transformation, the explants are pierced with a sterile needle and placed on a B5 nutrient medium containing a suspension (OD ₆₀₀ =0.4) of Agrobacterium rhizogenes. Before transformation, the suspension culture of agrobacteria is grown on YEB medium for 24 hours in the dark at +23°C on a shaker with circular rotation (amplitude 5–10 cm, rotation speed 90 rpm). As a control, needle-pierced explants placed on B5 solid medium without application of a suspension of the microorganism are used.

After 12-48 hours of exposure to a bacterial suspension, the explants are washed with sterile water, moisture is removed from them (dry) with sterile filter paper, and placed on a B5 solid medium containing 500 mg/l of cefotaxime (Klaforan, UK). An antibiotic is added to remove the remnants of agrobacteria. After 30 days of cultivation, the frequency of transformation is estimated (the number of transformed explants to their total number).

Transformed explants are selected based on phenotypic characteristics (lateral branching, lack of geotropism, ability to grow in an environment without hormones). Subsequently, two passages of the obtained roots are carried out on a B5 solid medium containing no hormones, but containing a reduced dose of cefotaxime (250 mg/l). Cultivation takes place in the absence of light at a temperature of +23°C on a rocking chair with a frequency of 100 rpm, duration 5 weeks. The subsequent transplantation to a fresh medium is carried out as contamination with agrobacterium appears.

After 14 days, rhizogenesis is observed on individual explants. Ginseng seedlings are cut into 0.5 cm segments (0.5-1.0 g) and the Agrobacterium rhizogenes strain is immersed for 15 minutes. After infection, the explants are dried on sterilized filter paper and cultured at 23° C. in the dark in 1/2 Murashige-Skoog medium (MS) containing 500 mg/l cefotaxime. After 2 days of co-cultivation, the explants are transferred to 1/2 MS medium containing 500 mg/l cefotaxime to kill Agrobacterium. The cultivation cycle of ginseng root cultures is 5 weeks.

Root cultures of ginseng present (Panax ginseng SA Meu) are dried at 25°C in a place protected from light and crushed. The dried plant material is crushed in a mill brand LZM-1M (Russia, Olis) and sieved through a sieve with a hole size of 1 mm. The process of water-alcohol extraction of the root culture of real ginseng (Panax ginseng S.A. Mey) is carried out at a temperature of 50 ° C and an ethanol concentration of 30% at a ratio of vegetable raw materials / extractant 1:86 for a duration of 4 hours.

The stages of cleaning and drying the extract, obtaining dry whey, mixing the dietary supplement components and encapsulation are similar to example 1. The dietary supplement formulation based on the extract of the ginseng root culture is shown in table 4.

The results of determining the chemical composition and antioxidant activity of dietary supplements are presented in tables 6.7.

Example 3. Similar to example 1 and 2, but differs in dietary supplement formulation. This dietary supplement includes a mixture of dry plant extracts of common thyme callus culture (Thymus vulgaris L.) and real ginseng root culture (Panax ginseng S.A. Meu), whey powder and excipients, in accordance with the recipe (table 5).

The finished dietary supplement is subjected to quality control, including the analysis of antioxidant activity (table 6, 7).

The HPLC method is the basis for the quantitative determination of the antioxidant BAS complex. The results of the analysis are presented in table 6.

The antioxidant capacity of 2,2'-azino-bis-(3-ethyl-benothiazoline-6-sulfonic acid) diammonium salt (ABTS ⁺ ) (Angene, Great Britain) to remove radicals was determined according to the procedure [19]. The results of determining the antioxidant activity of dietary supplements are presented in table 7.

The defining essential distinguishing features of the proposed method for obtaining dietary supplements of encapsulated form based on whey and extracts of Thymus vulgaris L. and Panax ginseng C.A. Meu compared with the closest analogue (RF patent No. 2759731) are:

- Use of in vitro callus and root cultures of Thymus vulgaris L. and Panax ginseng C.A. Meu allows to obtain a high yield of antioxidant biologically active substances in comparison with traditional plant materials;

- Spray drying of extracts of callus and root cultures in vitro Thymus vulgaris L. and Panax ginseng C.A. Meu, allows you to reduce the duration of drying, which in turn contributes to the conservation of biologically active substances and reduce energy costs.

- The method involves the use of whey as an additional source of easily digestible protein, lactose, vitamins, micro and macro elements.

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Claims (7)

1. Method for obtaining a dietary supplement based on whey and plant extract, including successive stages of preparation and extraction of plant materials, filtering the extract, evaporating the extractant, drying the extract, mixing the dry extract with auxiliary components: microcrystalline cellulose, aerosil, magnesium stearate and encapsulating the resulting mixture, characterized in that plant cell cultures are used as plant materials: callus culture of common thyme and/or root culture of real ginseng, extraction is carried out with ethyl alcohol, while extraction of the biomass of the callus culture of common thyme is carried out at a temperature of 70°C and an ethanol concentration of 70%, extraction of the biomass of the root culture of present ginseng is carried out at a temperature of 50°C and an ethanol concentration of 30% at a ratio of vegetable raw materials: extractant 1:86 and an extraction duration of 4 hours, the extract is dried on a spray dryer, the biologically active additive contains additional Dry milk whey - curd and / or cheese whey - along with auxiliary components is carefully added. 2. The method according to p. 1, characterized in that the dietary supplement contains the following starting components, wt. %: whey powder 36.0 dry extract of callus culture biomass of common thyme ( Thymus vulgaris L.) 25.0 MCC 35.0 aerosil 1.2 magnesium stearate 2.8 3. The method according to p. 1, characterized in that the dietary supplement contains the following initial components, wt. %: whey powder 36.0 dry extract of real ginseng ( Panax ginseng CA Mey ) root culture biomass 27.4 MCC 33.5 aerosil 1.2 magnesium stearate 1.9 4. The method according to p. 1, characterized in that the dietary supplement contains the following initial components, wt. %: whey powder 32.3 dry extract of callus culture biomass of common thyme ( Thymus vulgaris L.) 12.0 dry extract of real ginseng ( Panax ginseng CA Mey ) root culture biomass 18.0 MCC 35.0 aerosil 1.2 magnesium stearate 1.5