RU2326938C2 - Consortium of lactobacteria strains and method of receiving preparation on its basis, which is used as dietary supplement or ferment for production of sour milk products - Google Patents

Abstract

FIELD: technological processes; medicine.

SUBSTANCE: consortium of lactobacteria strains contains Lactobacillus acidophilus 57S ("ВКПМ В-5863"), Lactobacillus piantarum "П-75" ("ВКПМ В-3962"), Lactobacillus casei stilb ("ВКПМ В-5724"), which are taken in proportion of 2:0.5:0.5. The method includes preparation of milk nutrient medium, its sterilisation and cooling down to the temperature of fermentation, introduction of inoculation dose of lactobacteria strains consortium in the quantity of not more than 5.0 mass %. The product is fermented during time, which is sufficient for formation of clot and the ready product is cooled at the temperature of not more than +6°C. As milk nutrient medium non-fat milk is used, normalised in solid residual down to 13.5-15%, lactopeptone and lithium citrate with the following proportion of components, (mass %): lactopeptone - 3.5-4.5, lithium citrate - 0.15-0.25 and non-fat milk, normalised in solid residual down to 13.5-15% - the rest.

EFFECT: increases shelf life and initial titre of lactobacteria consortium in prepared substance.

4 cl, 4 tbl, 5 ex

Description

Technical field

The invention relates to biotechnology and medical microbiology, and is a consortium of strains of lactobacilli, which can be used to obtain on its basis a drug used as a biologically active additive (BAA) or starter culture for the production of dairy products for medical and dietary purposes.

The functions of normal microflora in the body are vital and very extensive: synthesizing, regulatory, desensitizing, detoxifying, enzymatic, protective and immunostimulating.

Preparations and products containing lactobacilli are widely used for the prevention and treatment of dysbiosis and normalization of the intestinal microflora of people who have undergone stressful effects, antibiotic and chemotherapy.

State of the art

Probiotics and medical nutrition products are widely used abroad based on consortia of lactobacillus strains of the species Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus delbrueckii, Lactobacillus fermentum and Lactobacillus lactis No. 61 No. PC12, N Patent No. 68N.C. 35/72, published December 7, 2004; UK patent No. 883424, IPC C12C 1/20, published on November 29, 1961; UK patent No. 930107, IPC AK61/35, published on 07/03/1963), as well as on the basis of consortia strains of bifidobacteria and lactobacilli (Application US No. 20020090416, IPC A23L 1/00, publ. 07/11/2002; US patent No. 5866385, IPC С12N 1/21, А23С 9/123, publ. 02.02.1999) .

However, consortia of strains of lactobacilli and consortia of strains of bifidobacteria and lactobacilli of foreign developers are focused on the creation of mainly therapeutic and prophylactic drugs and dietary supplements in dry form (tablets, capsules). These consortia of strains have significant drawbacks if they are used for the preparation of preparations and fermented milk products in liquid form: short shelf life of these preparations and products, low organoleptic qualities that are not essential for preparations in dry form.

Known consortia of strains of bifidobacteria and lactobacilli used for the production of sour-milk and non-fermented products in liquid form (RF Patent No. 2180348, IPC C12N 1/20, publ. 2002; RF patent No. 2180914, IPC C12N 1/20, publ. 2002 g .; RF patent No. 2180915, IPC С12N 1/20, publ. 2002).

The main disadvantage of fermented milk products made on the basis of consortia of strains of bifidobacteria and lactobacilli is the insufficient shelf life (no more than 1 month) due to the fact that lactobacilli during storage of the product contribute to its rapid acidification. The titer of bifidobacteria in the acidified product quickly drops, which reduces the therapeutic properties of this product.

The closest technical solution (prototype) is a consortium of strains of lactobacilli Lactobacillus acidophilus (strain NCIMB 40354), Lactobacillus plantarum (strain NCIMB 40356); Lactobacillus casei (strain NCIMB 40357) used to produce fermented milk therapeutic and prophylactic products (UK patent No. 2251864, IPC С12N 1/20, publ. 07.22.1992).

However, the shelf life of such a consortium of strains of lactobacilli in fermented milk products is 30 days, which is not enough when preparing biologically active additives in liquid form.

Another closest technical solution (prototype) is a method of obtaining a preparation from 15 symbiotic cultures of probiotic bacteria of both bifidobacteria and lactobacilli, including strains of Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei (RF patent No. 2163445, IPC A23C 9 / 12, published on February 27, 2001), which can be used as a therapeutic and prophylactic drug or as a biologically active additive in liquid form. The method includes the propagation of each monoculture of probiotic bacteria on the medium separately at optimal temperatures in the range of 20-25 ° C for 12-16 hours until the formation of a clot in thermoses. Next, each monoculture is drained into a sterile container in a volume ratio of 1: 1 and the mixture is mixed. Then, 3-5% of a mixture of monocultures is added to sterile skim milk or milk with a fat content of 0.5-3.2%, mixed and placed in a thermostat for 5-7 hours at a temperature of 32-37 ° C until a clot forms and up to 10 ^8-9 cells in 1 g. The finished drug is refrigerated at 0-6 ° C.

However, the preparation obtained in this way has a low initial titer of the finished product (up to 10 ^8-9 cells per 1 g) and short shelf life. Lactobacilli during storage of the product contribute to its acidification, as a result of which the titer of lactobacilli and bifidobacteria in the acidified product drops rapidly, which reduces its therapeutic and prophylactic properties.

The technical result of the claimed technical solutions is to increase the shelf life of the inventive consortium of lactobacilli in prepared preparations, including dietary supplements, as well as providing a higher initial titer of finished products after their preparation.

The specified technical result is achieved by a new consortium of strains of lactobacilli Lactobacillus acidophilus 57S (deposited at the All-Russian Research Institute of Genetics at VKPM under No. 5863), Lactobacillus plantarum P-75 (deposited at the All-Russian Research Institute of Genetics at VKPM under No. B 3962); Lactobacillus casei Sat (deposited at the All-Russian Research Institute of Genetics at VKPM under No. B 5724) used to produce biologically active additives and fermented milk therapeutic and preventive products in liquid form. The amount of Lactobacillus acidophilus 57S, Lactobacillus plantarum P-75; Lactobacillus casei Sat correlates as 2: 0.5: 0.5.

The specified technical result is also achieved by the fact that in the method for producing a biologically active supplement of probiotic bacteria in liquid form, which includes preparing a milk culture medium, sterilizing it and cooling it to a fermentation temperature, introducing into the milk culture medium a seed dose of a combination of probiotic bacteria strains in an amount not more than 5.0 wt.%, ripening the product for a time sufficient to form a clot, and cooling the finished product at a temperature not exceeding + 6 ° C, according to the invention, as a combination of probiotic bacteria strains used lactobacilli Lactobacillus acidophilus 57S strains consortium (deposited at the Research Institute of Genetics in VKPM B under № 5863), Lactobacillus plantarum P-75 (deposited at the Research Institute of Genetics in VKPM B under № 3962); Lactobacillus casei Sat (deposited at the All-Russian Research Institute of Genetics at VKPM under No. B 5724), contained in a ratio of 2: 0.5: 0.5, and milk is used as a milk nutrient medium, normalized to the dry residue of SOMO 13.5-15% , in which lactopeptone and sodium citrate are additionally introduced in the following ratio of components (wt.%):

Lactopeptone 3.5-4.5 Sodium Citrate 0.15-0.25 Normalized milk return by dry residue СОМО 13.5-15% the rest is up to 100%.

Moreover, the fermentation is carried out at a temperature of (39-40) ° C for 4-5 hours until the acidity of the finished product is not less than 90 ° T.

In a standard skim milk medium, the concentration of proteins and lactose is artificially increased by 1.3-1.5 times by adding milk powder so that the dry skim milk residue (SOMO) reaches 13.5-15%. The higher the content of SOMO, the higher the content of casein, which forms a buffer system, linking a large number of acidic metabolites to caseinates. Thus, it is possible to restrain the increase in acidity of the medium with the growth of biomass. In addition, with an increase in the content up to 15% of SOMO in the milk image, the lactose content increases by 25-30%, which increases the growth properties of the medium, which increase the titer of lactobacilli. To prevent casein coagulation, a solution of sodium citrate is introduced into the milk base, which has strong stabilizing and buffering properties.

Lactopeptone is introduced to increase the growth properties of the milk nutrient medium, which is a whey protein hydrolyzate (α-lactalbumin, β-lactoglobulin, immunoglobulin, lactoferrin), which, after cleavage, give a large amount of essential and sulfur-containing amino acids.

As a result, it was possible to significantly increase the safety of the lactobacillus of the consortium. Optimization of the technology for producing biomass of lactobacilli on skim milk medium with a high content of SOMO and lactopeptone allowed us to obtain stable biomass with a high initial titer and a long shelf life.

Information about the deposit of strains of the consortium:

1. Lactobacillus acidophilus 57S (deposited at the All-Russian Research Institute of Genetics at VKPM under No. B 5863);

2. Lactobacillus plantarum P-75 (deposited at the All-Russian Research Institute of Genetics at VKPM under No. B 3962).

3. Lactobacillus casei Sat (deposited at the All-Russian Research Institute of Genetics at VKPM under No. B - 5724). The properties of the consortium are determined by a strain of lactobacilli Lactobacillus acidophilus 57S, the content of which in the consortium is about 70%.

Cultural and morphological features of the consortium

Lactobacillus cells are straight rods of medium length or large (1.0-1.5 × 3-14 microns), arranged in chains or individually, gram-positive, do not form spores and capsules. On dense media they form shallow deep colonies, usually in the form of a ball of filaments.

Strains do not dilute gelatin, do not form gas, do not produce catalase. They synthesize lactic acid, assimilate glucose, arabinose, lactose, do not assimilate raffinose, sucrose, maltose, mannitol.

The optimal temperature for the growth of strains of lactobacilli is (36-42) ° C.

When strains are added to skim milk in the amount of 3%, milk is fermented in 4-5 hours. The acidity of the consortium is 240-280 ° T.

Acid-forming activity: by 24 hours - 140 ° Turner, by 72 hours - 160-200 ° Turner.

The maximum titer of the consortium lactobacilli in the dairy medium (sterile skim milk) after 5-6 hours of growth is 1-2 × 10 ⁹ , after 90 days of storage in this medium - 1-2 × 10 ⁸ . When lactopeptone is added after 4-5 hours of growth, it is (3-7) · 10 ¹⁰ , after 90 days of storage in this medium - 2.5-6 × 10 ⁹

Antagonistic properties:

It is active against Shigella Sonne, Flexner, enteropathogenic Escherichia coli, staphylococci.

The ability of a consortium of strains of three types of lactobacilli to increase microbial mass in nutrient media, to ferment milk in a short time, to exhibit specific activity makes it possible to use it in the preparation of dietary supplements, sour-milk, non-fermented foods and bacterial preparations.

Research results confirming the beneficial properties of the inventive consortium of lactobacillus strains

To identify the technological features and properties of the inventive consortium of strains of lactobacilli, studies were conducted in the process of cultivating the inventive consortium, as well as its storage. Cultivation of the consortia was carried out in a dairy medium manufactured according to MUK 4.2.577-96 "Methods of microbiological control of baby food, therapeutic nutrition and their components." To increase the growth properties of the milk base, lactopeptone (wt.%) Was introduced:

Lactopeptone 3.5-4.5 Sodium Citrate 0.15-0.2 5 Milk return (base), normalized by dry residue 13.5-15% the rest is up to 100%

In a standard skim milk medium, the concentration of proteins and lactose is artificially increased by 1.3-1.5 times by adding milk powder so that the dry skim milk residue (SOMO) reaches 13.5-15%. The higher the content of SOMO, the higher the content of casein, which forms a buffer system, linking a large number of acidic metabolites to caseinates. Thus, it is possible to restrain the increase in acidity of the medium with the growth of biomass. The higher the lactose content, the higher the growth properties of the medium - an increase in the lactose content by 25-30% increases the titer of lactobacilli. To prevent casein coagulation, a solution of sodium citrate is introduced into the milk base, which has strong stabilizing and buffering properties.

In addition, to increase the growth properties of the milk nutrient medium, lactopeptone is introduced, which is a whey protein hydrolyzate (α-lactalbumin, β-lactoglobulin, immunoglobulin, lactoferrin), which, after cleavage, give a large amount of essential and sulfur-containing amino acids.

As a result, it was possible to significantly increase the safety of the lactobacillus of the consortium. Optimization of the technology for producing biomass of lactobacilli in skim milk medium with a high content of SOMO and lactopeptone allowed us to obtain stable biomass with a high initial titer and a long shelf life.

Table 1 presents the technological characteristics of the inventive consortium of strains of lactobacilli in a dairy environment (milk return, normalized by solids 13.5-15%) during cultivation and storage.

Table 1
Technological characteristics of the inventive consortium of strains of lactobacilli in a dairy environment during cultivation and storage Production sourdough Consortium-based Lactobacillus Biomass Types of strains of lactobacilli in the consortium The ratio of strains,% Cultivation time, hours Acidity, T ° The titer (CFU / ml) of ZhKB during storage Clot 1 day 72 days 1 day 60 days 72 days B - 5724
B - 3962
B - 5863 0.5: 0.5: 2 4 h. 50 m. 140 160 1 × 10 ¹⁰ 6 × 10 ⁹ 2 × 10 ⁹ Normal

The study of the antagonistic activity of the inventive consortium of strains of lactobacilli

The antagonistic activity of the strains of the consortium of lactobacilli (table 2) was studied by the in vitro method when co-grown with pathogenic test strains in the zone of growth inhibition of test microbes by the barcode method

table 2
The study of the antagonistic activity of lactobacilli in solid nutrient media The name of the test microbes Lactobacillus Lactobacillus casei Lactobacillus Lactobac ulus plantarum Lactobacillus Lactobacillus acidophilus E.coli 13 mm 12 mm 13 mm Sh.flexneri 12 mm 12 mm 12 mm S.aureus 15 mm 14 mm 14 mm Proteus vulgaris 11 mm 12 mm 12 mm Proteus mirabilis 12 mm 13 mm 13 mm

Studying the influence of a consortium of lactobacilli on representatives of normal intestinal microflora in an in vitro model.

These studies were carried out by the Head Testing Center at the State Research Institute of Nutrition RAMS. Objects of study - eubiotics manufactured by AOOT "BIOMED" named after I.M.Sechenova:

- Lactobacterin dry,

- Bifidumbacterin dry

Table 3
The results of studies of the impact of the proposed consortium of lactobacilli on normal intestinal microflora in an in vitro model Indicators Microorganisms-eubiotics, CFU / g of product Eubiotic + dietary supplement Control eubiotics Lactobacilli, growth on MRS medium microscopic preparation 1 × 109 Gy (+) sticks without spores, typical for lactobacilli 109 CFU / G, Gy (+) sticks without spores, typical for lactobacilli; absence of extraneous microflora cells Bifidobacteria
Microscopic preparation 1 × 108 Gy (+) sticks without spores, curved and with bifurcation, typical of bifidobacteria 1 × 108 CFU / g Gy (+) rods without spores, curved and with bifurcation, typical for bifidobacteria; absence of extraneous microflora cells Colibacterin E. Coli M-17 Growth in Malthus Enterobacteriaceae medium. Microbiological 1 × 109 Gy (-) sticks without spores, typical for colibacilli 1.3 × 1010 Gy (-) sticks without spores, typical for colibacilli; absence of extraneous microflora cells

From table 3 it is seen that the inhibitory effect of the consortium of lactobacilli on representatives of normal intestinal microflora in the in vitro model was not detected.

The efficacy of a consortium of lactobacillus strains was established as a result of preclinical studies conducted by the State Scientific Center “Vector” of the Ministry of Health of the Russian Federation on adult mice and young genetically standard colony ICR in accordance with the requirements of RD 42-28-8-89 “Preclinical trials of new immunobiological preparations. Key Points. ” The studies were carried out according to the scheme: general toxicity (chronic), embryotoxicity and effect on reproductive function, general pharmacological effect (effect on the digestive system, formation of adaptive effect).

In the study of chronic toxicity, all animals were viable, weight loss, impaired motor activity, inhibition of state and death were not observed. Moreover, the consortium has a stimulating effect on the growth, development and survival of animals. Hematological blood counts within the physiological norm. Histological studies of target organs showed no toxic effects on these organs. There was also no immunotoxic effect of the drug on the organs of the lymphoid system during their histological examination.

The high efficiency of the consortium of lactobacilli has been proven in the study of the reproductive function of pregnant females - there is a noticeable improvement in the condition of females, a decrease in late embryonic mortality after implantation (3.1%) versus (5.3%) in the control group, the best survival of newborn mice for this period up to 92.5% compared to control up to 80%. Testing the effectiveness of the physiological effects of the consortium on the digestive system of mice showed a stabilizing effect on the gastrointestinal tract and the formation of microbiocenosis of newborn mice due to the colonization activity of the consortium lactobacillus strains.

The consortium also contributes to the survival of mice in difficult situations (conditions of elevated temperatures - 32 + 2 ° C), acting as an adaptogen, contributing to the preservation of significant functional reserves of vital body systems.

Examples of a preferred embodiment of the invention

Example 1. Obtaining on the basis of the inventive consortium of lactobacilli BAA "TRILACT" or starter culture for the production of dairy products

The introduction of cultures of lactobacilli. For the preparation of primary (uterine) starter culture, lyophilized certified strains of lactobacilli Lactobacillus acidophilus 57S, Lactobacillus casei Sb and Lactobacillus plantarum P-75 or the same activated museum strains stored in test tubes in sterile skim milk are used.

In a vial with a lyophilized certified strain of lactobacilli, (0.2-0.5) ml of sterile saline solution is pipetted. After the lyophilic mass is dissolved with the same pipette, the contents of the ampoule are transferred (transferred) into a test tube with 9 ml of sterile skim milk and mixed well by pipetting. Crops are incubated in a TC26 thermostat at a temperature of (39.0-40.0) ° C for (20-24) hours. Next, 1-2 passages are carried out in glass bottles with a capacity of (0.1-0.25) l with sterile skim milk to fully activate their properties. Subsequent crops are incubated in an incubator at a temperature of (39.0-40.0) ° C for (5-6) hours.

After incubation, the culture bottles are transferred for cooling and storage in the refrigerator, after maturation for 10-12 hours they are used to obtain laboratory starter culture.

The primary (uterine) sourdough of lactobacilli can be stored in the refrigerator at a temperature of (6-8) ° C for no more than 7 days.

Preparation of laboratory and industrial starter cultures of lactobacillus strains. Laboratory starter cultures of lactobacilli are prepared in sterile skim milk from primary (uterine) starter culture separately for each strain of lactobacilli based on weekly requirements and are used in further production the day after its preparation.

Alternately, from vials with cultures of lactobacilli with a characteristic growth in sterile skim milk, a culture pipette of each strain in an amount of 3% inoculum to the volume of sterile skim milk is selected with a pipette. It is kept in the thermostat for (4-5) hours at a temperature of (39 ± 1) ° С.

The content of probiotic microorganisms in the obtained biomass of lactobacilli is lg 8-9 in 1 ml.

Getting a consortium of lactobacilli. To obtain a consortium of strains of lactobacilli, mechanical mixing of laboratory starter cultures of Lactobacillus acidophilus 57S, Lactobacillus plantarum P-75, Lactobacillus casei Sat in a ratio of 2: 0.5: 0.5 is performed.

The content of probiotic microorganisms in the obtained biomass of the association consortium is lg 9 in 1 ml.

Getting starter culture or dietary supplement “Trilact. The preparation of starter culture or biomass dietary supplement "Trilact" is carried out thermostatically in sterile glass bottles with a capacity of 15 liters. At the beginning, a milk nutrient medium is prepared under aseptic conditions on the basis of a milk back, normalized by dry solids of SOMO to 13.5-15.0%. Lactopeptone and sodium citrate are additionally introduced into the indicated milk medium in the following quantitative ratio (wt.%):

Lactopeptone 3.5-4.5 Sodium Citrate 0.15-0.25 Normalized milk return by dry residue 13.5-15% the rest is up to 100%

Under aseptic conditions, a ready-made production sourdough of a consortium of lactobacilli in an amount of 0.5 l is introduced into a glass bottle containing 9.5 l of normalized milk nutrient medium. The microbial mixture of lactobacilli is carefully mixed in a circular rotation of the bottle for (1-2) minutes. They are placed in a thermostat type TC-80M for (4-5) hours at a temperature of (39-40) ° С. The fermented mixture is left without stirring until a clot forms and an acidity of at least 90 ° T is achieved. After the formation of a clot, the bottles with the product are chilled to a temperature of (4 ± 2) ° C in refrigerators. To stabilize and strengthen the clot, the product is cooled down for (8-10) hours, followed by packing and storage of the finished product.

Example 2. The use of the consortium as a starter culture for the preparation of a symbiotic fermented milk product of therapeutic and functional nutrition - Bifacil

The leaven of the consortium prepared according to example 1, in a volume of 0.5-1%, is introduced into pasteurized milk together with bifidobacteria and thermophilic streptococcus in the amounts of starter cultures provided for the preparation of bifacil, mixed and cultured for 3-3.5 hours until a clot forms, after which the product is cooled and packaged in consumer packaging. The result is a therapeutic and prophylactic fermented milk product with a useful lactoflora content of at least lg 7-8 in 1 ml of the symbiotic product.

The resulting product has a pronounced immunocorrective effect.

Example 3. The use of a consortium of strains of lactobacilli to obtain a bacterial preparation

To the grown biomass of the consortium of Example 1, protective drying media (sucrose 10% and gelatin 5%) are added. The resulting suspension is poured into 2 ml ampoules or 5 ml into vials, frozen at t = minus 40-45 ° C and lyophilized from a frozen state in vacuum dryers. The resulting preparation contains at least 10 ⁹ living cells of lactobacilli.

The production technology of the consortium as part of the products does not require expensive equipment (fermenters) and allows you to get large quantities of lactobacillus biomass in a simple thermostatic way without pH correction and the use of gas exchange systems. At the same time, the concentration of lactobacilli in the biomass of the consortium of lactobacilli reaches a maximum value of lg 10. The acidity of the biomass of lactococorticium even increases when stored for 3 months in a liquid state, within acceptable limits. As a result, the proposed consortium is more technologically advanced and economical in comparison with other forms of consortia, and is very convenient in use, storage, and transportation.

Example 4. The study of the duration of storage of a consortium of strains of lactobacilli in a liquid dietary supplement "Trilact"

The duration of storage of a consortium of lactobacillus strains in the liquid dietary supplement “Trilact” was studied (table 4). A significant shelf life (up to 108 days) of the Trilact dietary supplement with a titer of at least 10 ⁸ CFU / ml is associated with ensuring the initial acidity of the drug of the consortium of lactobacilli no more than (120-140) ° T, obtaining the initial titer of lactobacilli (not less than 10 ⁹ CFU / ml) and using the original milk culture medium with the addition of lactopeptone.

Table 4
Data on the safety of the proposed consortium of lactobacilli in dietary supplement "Trilact" No. 1 day 60 days 108 days Titer (CFU / ml) Acidity (° T) Titer (CFU / ml) Acidity (° T) Titer (CFU / ml) Acidity (° T) one (2.5-3) × 10 ⁹ 100-115 (2.5-3) × 10 ⁹ 190-205 (1.1-2.5) × 10 ⁹ 210-220 2 (6-7) × 10 ⁹ 100-114 (2.5-3) × 10 ⁹ 180-190 (1.1-2.5) × 10 ⁹ 190-210 3 (7-6) × 10 ⁹ 110-120 (2.5-3) × 10 ⁹ 195-210 (2.5-3) × 10 ⁹ 210-225 four (3-5) × 10 ⁹ 115-122 (2.5-3) × 10 ⁹ 190-215 (1.1-2.5) × 10 ⁹ 210-230 5 (6-7) × 10 ⁹ 95-105 (2.5-3) × 10 ⁹ 173-180 (1.1-2.5) × 10 ⁹ 190-205 6 (5-6) × 10 ⁹ 97-110 (1.3-3) × 10 ⁹ 160-180 (1.1-2.5) × 10 ⁹ 180-204 7 (5-6) × 10 ⁹ 110-125 (1.3-3) × 10 ⁹ 180-190 (1.1-2.5) × 10 ⁹ 210-223 8 (5-6) × 10 ⁹ 95-110 (1.3-3) × 10 ⁹ 150-178 (1.1-2.5) × 10 ⁹ 200-221 9 (6-7) × 10 ⁹ 105-121 (1.3-3) × 10 ⁹ 175-193 (1.1-2.5) × 10 ⁹ 215-230 10 (6-7) × 10 ⁹ 100-110 (2.5-3) × 10 ⁹ 170-187 (2.5-3) × 10 ⁹ 180-195 eleven (6-7) × 10 ⁹ 90-115 (2.5-3) × 10 ⁹ 195-205 (1.1-2.5) × 10 ⁹ 110-225 12 (6-7) × 10 ⁹ 110-120 (2.5-3) × 10 ⁹ 170-189 (1.1-2.5) × 10 ⁹ 190-205 13 (6-7) × 10 ⁹ 100-117 (2.5-3) × 10 ⁹ 140-159 (2.5-3) × 10 ⁹ 165-180 fourteen (6-7) × 10 ⁹ 120-140 (2.5-3) × 10 ⁹ 200-228 (2.5-3) × 10 ⁹ 210-235 fifteen (6-7) × 10 ⁹ 100-112 (2.5-3) × 10 ⁹ 170-185 (1.3-2.5) × 10 ⁹ 180-191

Example 5. The use of dietary supplements "Trilact" for the correction of disorders of the intestinal biocenosis in somatic diseases, accompanied by a violation of the digestive system.

Clinical studies were conducted on the basis of the Novosibirsk Regional Children's Allergodermatosis Center, located on the basis of the Municipal Clinical Children's Clinical Hospital №1, clinical diagnostic and bacteriological laboratories of the hospital; Novosibirsk Center for Family Rehabilitation.

The most important role in the life of the human body is played by the normal intestinal microflora, suppressing the growth of pathogenic, putrefactive and gas-forming microorganisms, participating in the metabolism and synthesis of vitamins, stimulating the immune system. Violation of the qualitative and quantitative composition of normoflora, called dysbiosis, leads to diseases of the digestive system, hematopoietic system, immune system, development of hypotrophy, anemia, fermentopathy, and contributes to allergization of the body. Therefore, the prevention and treatment of disorders of intestinal biocenosis is an object of special attention.

BAA "Trilact" is a microbial mass of live lactobacilli, made according to example 1 and restores the normal composition of the intestinal microflora.

Under observation were 30 patients: children aged 6 months and adults under the age of 50 years, with intestinal dysbiosis not higher than 3 degrees, not suffering from viral hepatitis and who gave informed consent for the studies. The comparison group was 30. The clinical symptoms of the disease before treatment were malaise, weakness, headache, sleep disturbance (73.3%); dyspeptic syndrome in the form of a decrease in appetite, flatulence, nausea (53.3%); abdominal pain (50.0%); stool disorder in the form of chronic constipation (53.3%) and diarrhea (13.3%), alternating constipation and diarrhea (3.3%). 53.3% of the subjects had a skin syndrome in the form of urticaria and foci of atopic dermatitis in the subacute and exacerbation stages. All 30 people included in the experiment (100.0%) had dysbiotic intestinal disorders: dysbiosis I degree in 24 (80.0%), dysbiosis II degree in 6 (20.0%). 19 people out of 30 (63.3%) had an increased titer of opportunistic bacteria in the intestinal microflora, 20 (66.7%) had atypical and enzymatically inferior strains of E. coli.

The course of treatment using dietary supplements "Trilact" was 14 days at an age dose prescribed by the instructions for use:

- for children from 6 months to 1 year - on the recommendation of a doctor - up to 5 ml (1 teaspoon) per day for 2 doses,

- for children from 1 year to 7 years - 5 ml per 1 dose 2 times a day,

- children from 7 to 12 years old - 5 ml per 1 dose 3 times a day,

- Adults and children over 12 years old apply 10 ml (1 dessert spoon) for 1 dose 3 times a day.

The control group received standard treatment.

The study used a drug close to the expiration date. According to the results of evaluating the clinical effectiveness of the studied drug, it turned out that improvement in health in the form of normalization of sleep, the cessation of headaches and malaise was noted in 18 out of 22 people (81.8%) who had these symptoms, in 4 people they significantly decreased. A decrease in dyspeptic syndrome (the disappearance of nausea, a decrease in flatulence and an improvement in appetite) was observed in 9 of 16 (56.3%); cessation of abdominal pain in all 15 people (100.0%) with this symptomatology. Normalization of the frequency and consistency of the stool was observed in 17 out of 21 (80.9%) subjects, a clear positive dynamics of the skin syndrome was observed in 10 out of 16 (62.5%) children with atopic dermatitis and recurrent urticaria, bowel function improved, discomfort disappeared in 22 patients bloating and rumbling in the stomach. In 5 patients, diarrhea stopped on the first day, in 18 patients constipation was stopped. In all patients observed, the composition of the colon microflora improved. By the end of treatment, in 24 patients, the number of anaerobic microorganisms of bifidobacteria and lactobacilli significantly increased, the number of opportunistic microorganisms decreased.

Thus, the studied drug passed clinical trials and can be considered harmless and highly active in restoring intestinal normobiocenosis and accelerating sanogenesis in somatic diseases accompanied by impaired digestive system function.

The above examples show that the proposed consortium of strains of three types of lactobacilli actively propagates in nutrient media with the accumulation of industrial biomass in a short period of cultivation. The product ferments milk with the formation of a clot with good taste and organoleptic properties; has specific activity. A consortium of strains can be recommended as part of starter cultures for the preparation of Trilact dietary supplements, sour-milk therapeutic and dietary products, unfermented food products and bacterial preparations.

Industrial applicability

The above description of the invention sets forth the entire technological process for obtaining a consortium and products based on it, which can be produced both in dairy kitchens and in large dairy plants. Used media and reagents are publicly available. Tools and techniques for obtaining and growing the biomass of the consortium, including in the form of laboratory and industrial starter cultures, as well as tools and techniques for freezing and lyophilization of a dry bacterial preparation, are traditionally used in the medical industry and biotechnology.

Claims (4)

1. A consortium of strains of lactobacilli Lactobacillus acidophilus 57S (deposited at the All-Russian Research Institute of Genetics at VKPM under No. 5863), Lactobacillus plantarum P-75 (deposited at the All-Russian Research Institute of Genetics at VKPM under No. B 3962); Lactobacillus casei Sat (deposited at the All-Russian Research Institute of Genetics at VKPM No. B 5724), used to produce biologically active additives and fermented milk therapeutic and prophylactic products in liquid form. 2. A consortium of strains of lactobacilli according to claim 1, characterized in that the amount of Lactobacillus acidophilus 57S, Lactobacillus plantarum P-75; Lactobacillus casei Sat correlates as 2: 0.5: 0.5. 3. A method of obtaining a drug used as a biologically active additive or sourdough for the production of fermented milk products, including preparing a milk culture medium, sterilizing it and cooling it to a fermentation temperature, introducing into the milk culture medium a sowing dose of a combination of probiotic bacteria strains in an amount of not more than 5 , 0 wt.%, Ripening of the product for a time sufficient to form a clot, and cooling the finished product at a temperature not exceeding 6 ° C, characterized in that in quality a combination of probiotic bacterial strains uses a consortium of lactobacillus strains Lactobacillus acidophilus 57S (deposited at the All-Russian Research Institute of Genetics at VKPM under No. 5863), Lactobacillus plantarum P-75 (deposited at the All-Russian Research Institute of Genetics at VKPM under No. B 3962), Lactobacillus is deposited at the All-Russian Scientific Research Institute of Genetics and Medicine; VKPM No. B 5724), contained in a ratio of 2: 0.5: 0.5, and skim milk, normalized to 13.5-15% solids, using lactopeptone and sodium lemon -acid in the following ratio of components, wt.%: Lactopeptone 3.5-4.5 Sodium citric acid 0.15-0.25 Skim milk, normalized by dry residue up to 13.5-15% the rest is up to 100% 4. The method according to claim 3, characterized in that the ripening is carried out at a temperature of 39-40 ° C for 4-5 hours until the acidity of the finished product is not less than 90 ° T.