RU2506308C1 - CONSORTIUM OF PROBIOTIC STRAINS OF Lactobacillus rhamnosus AND Lactobacillus plantarum FOR PRODUCING BACTERIAL PREPARATION AND DIRECT ADMINISTRATION FERMENT FOR PRODUCING FERMENTED MILK AND FERMENTED BEET JUICE - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to microbiology and biotechnology. Disclosed is a consortium of Lactobacillus rhamnosus VKM B-2726D and Lactobacillus plantarum VKM B-2725D strains.

EFFECT: consortium is used to produce a bacterial preparation and a direct administration ferment for producing fermented milk and fermented beet juice as functional nutrition products.

15 tbl, 13 ex

Description

The invention relates to the field of microbiology and biotechnology, and in particular to strains of lactobacilli used in food and medical biotechnology.

An analysis of the health status of the population convincingly indicates a steady increase in the number of people suffering or prone to various diseases, called the "disease of civilization." These include cardiovascular, oncological, infectious, allergic diseases, diabetes, etc. According to epidemiological data and clinical observations, one of the main reasons for the development of these diseases is the imbalance in the modern human diet, which leads to a chronic deficiency in the intake of hundreds and thousands of macro- and micronutrients. This problem can be solved by introducing new functional foods into the human diet, i.e. products that along with nutritional properties also have the ability to restore, maintain and improve human health.

In recent decades, the concept of the active participation of the symbiotic microflora of a person in maintaining his health is gaining more and more popularity. Strains of probiotic microorganisms are used to create pharmacopoeial bacterial preparations and for the production of various functional nutrition products [1]. In connection with the foregoing, the development and improvement of the technology for the production of functional nutrition products using probiotic microorganisms is relevant.

It has been established that a huge role in the functional nutrition system along with dairy products belongs to vegetables, which are easily absorbed in the form of juices. To enrich vegetable juices with biologically active substances and improve their taste, they are subjected to fermentation using lactic acid fermentation. Lactobacillus fermenting juices can additionally provide probiotic effects that improve human health. Therefore, the selection of lactobacilli with pronounced probiotic properties for the fermentation of vegetable juices is of fundamental importance. Of particular note is the juice from beetroot.

A known strain of Lactobacillus cellobiosus 9K [2] used in the technology of preparing a drink from beets for diet food. The use of Lactobacillus cellobiosus 9K strain for fermentation of beetroot juice with pulp before the pressing stage allows increasing the yield of the drink and the content of biologically valuable components in it, such as betanidine, amino acids, and vitamins. However, the strain Lactobacillus cellobiosus 9K does not apply to the types of probiotic lactobacilli commonly used in medicine and the food industry in the Russian Federation.

Known consortia of strains of bifidobacteria and lactobacilli used for the production of fermented milk and non-fermented products in liquid form [3-6], however, there is no information about the possibility of using known consortia for the fermentation of vegetable juices, in particular beetroot.

The closest technical solution (prototype) is a consortium of strains of lactobacilli Lactobacillus acidophilus 57S (VKPM B-5863), Lactobacillus plantarum P-75 (VKPM B-3962), Lactobacillus casei Sat (VKPM B-5724), used to obtain the drug used in as a biologically active additive or yeast for the production of dairy products for medical and dietary purposes [7]. However, there is no information about the possibility of using this consortium for the fermentation of vegetable juices.

The objective of the invention is to expand the assortment of consortia of lactobacilli by obtaining a consortium that can be used for the manufacture of a bacterial preparation, as well as as a direct starter culture for the production of fermented milk and for the production of fermented beet juice.

The problem is solved by the fact that a consortium of probiotic strains Lactobacillus rhamnosus 2166 and Lactobacillus plantarum 2165 was obtained, used for the manufacture of a bacterial preparation and direct application starter for the production of fermented milk and fermented beet juice as functional food products.

The strains of Lactobacillus rhamnosus 2166 and Lactobacillus plantarum 2165 were isolated from home-made fermented milk sourdough used to obtain goat milk yogurt.

Microorganisms were identified by sequencing a gene encoding 16S rRNA.

Partial oligonucleotide sequence of a gene encoding 16S rRNA of Lactobacillus rhamnosus 2166:

Partial oligonucleotide sequence of a gene encoding 16S rRNA of Lactobacillus plantarum 2165:

The consortium strains were deposited in the All-Russian collection of microorganisms of the Institute of Biochemistry and Physiology of Microorganisms, RAS (Moscow Region, Pushchino).

The strain Lactobacillus plantarum 2165 has a registration number VKM B-2725D.

The strain Lactobacillus rhamnosus 2166 has a registration number VKM B-2726D.

The strains and consortium based on them are characterized by the following features.

Cultural and morphological features

When growing on the surface of an agarized MFC medium (Himedia, India) by the method of exhausting bar in a thermostat at 37 ° C for 18-20 hours:

- strain Lactobacillus plantarum 2165 - medium, white, round, convex colonies with a smooth edge;

- strain Lactobacillus rhamnosus 2166 - small, pale white, round, convex colonies with a smooth edge.

When grown on liquid MFC (Himedia, India) or milk at 37 ° C for 14-16 hours, the cells of the strain Lactobacillus plantarum 2165 are small short sticks ~ 3 μm in size, form short chains; cells of the strain Lactobacillus rhamnosus 2166 are very small short bacilli ~ 2 μm in size, form short chains.

When using a consortium of strains of Lactobacillus rhamnosus 2166 and Lactobacillus plantarum 2165, L.S. medium is used to differentiate these strains to determine their titers when plating from a single sample of the product. pH 6.1 (L.S. Differential Medium, Himedia) + 0.1 g triphenyltetrazolium chloride (TTC Himedia) per 1 liter of medium. In this medium, the colonies of the strain Lactobacillus rhamnosus 2166 acquire a red color with a bright red dot in the center, the colonies of Lactobacillus plantarum 2165 do not change their color, retaining a white color.

Physiological and biological characteristics

Both strains are facultative anaerobes, temperature optimum 37 ± 2 ° C, grow at 42 ° C, weak growth at 15 ° C. The optimal pH of the medium is 5.5-6.0.

Biochemical properties

The ability of the consortium strains to ferment carbohydrates was evaluated in an API test in accordance with the manufacturer's instructions (BioMerieux, France).

Both strains assimilate D-Ribose, D-Galactose, D-Glucose, D-Fructose, D-Mannose, L-Sorbose, D-Mannitol, N-Acetylglucosamine, Amygdalin, Arbutin, Esculin iron citrate, Salicin, D-Cellobiose, D Maltose, D-Lactose, D-Melicitose, D-Turanose.

Additionally, the strain Lactobacillus plantarum 2165 assimilates L-Arabinose, Methyl αD-mannopyranoside, D-Melibiosis, D-Sucrose, Gentiobiosis and has a taxonomic characteristic ID = 99.9%; T = 0.93.

The strain Lactobacillus rhamnosus 2166 additionally assimilates L-Ramnose, D-Sorbitol, Methyl-αD-glucopyranoside, D-Trehalose, D-Tagatose and has a taxonomic characteristic ID = 99.9%; T = 0.82 (taking into account the assimilation of ramnose).

Antibiotic sensitivity

Both strains are resistant to vancomycin. Lactobacillus plantarum 2165 is also resistant to ofloxacin and ciprofloxacin (Table 1).

Table 1 Resistance to antibiotic strains Antibiotic Minimum inhibitory concentration of the MIC antibiotic (mcg) Lactobacillus plantarum 2165 Lactobacillus rhamnosus 2166 Cephalaxin (Cf) 7.5 7.5 Ampicillin (Ar) 0.128 0.256 Gentamicin (G) 4,096 2,048 Streptomycin (S) 15.0 3.0 Chloramphenicol (C) 0.1 0.1 Ofloxacin (Of) 64.0 2.0 Ciprofloxacin (Cf) 120.0 0.25 Vancomycin (Va) 256,0 256,0 Tetracycline (T) 30,0 0.1

Method, conditions and composition of media for the propagation of strains of the consortium

The strains of Lactobacillus rhamnosus 2166 and Lactobacillus plantarum 2165 are grown at 37 ± 2 ° C in liquid medium MPC or in milk with 1.5% fat.

Commercially purchased milk with 1.5% fat is sterilized for 20 minutes by autoclaving at a pressure of 0.5 atm and a temperature of 110-120 ° C.

For the cultivation of individual colonies of strains using agar medium MPC containing 1.4% agar (Difco, USA).

Method, conditions and composition of media for storage of strains of the consortium

The strains of Lactobacillus rhamnosus 2166 and Lactobacillus plantarum 2165 can be stored:

- in sterile skim milk at 4 ° C with periodic reseeding once every 15-20 days;

- in a lyophilized state in sealed ampoules (protective medium during drying - sucrose 10%, pH 7.0) for 2 years at a temperature of 4 ° C;

- frozen at -70 ° C. Duration of storage is 6 months.

A consortium of strains can be stored:

- in a lyophilized state in sealed ampoules (protective medium during drying - sucrose 10%, pH 7.0) for 2 years at a temperature of 4 ° C;

- frozen at -70 ° C. Duration of storage is 6 months.

The invention is illustrated by the following examples.

Example 1. Cultivation of a strain of Lactobacillus plantarum 2165 in the laboratory

To obtain seed in a vial containing 10 mg of a lyophilized culture of Lactobacillus plantarum 2165, 0.5 ml of a sterile solution of 0.9% sodium chloride is added and the culture is grown on the surface of MPA agar medium by the method of exhaustion in a thermostat at 37 ° C for 18 hours . Then the isolated colony is placed in a bacteriological tube containing 7 ml of liquid medium MPC, and grown in an incubator at 37 ° C for 15 hours.

The resulting seed in an amount of 1% is introduced into vials containing 500 ml of liquid MPC medium and grown at 37 ° C for 18 hours. The titer of the obtained culture 4 × 10 ⁹ CFU / ml By adjusting the cell concentration to 1 × 10 ⁹ CFU / ml, a standardized starting biomass of Lactobacillus plantarum 2165 strain is obtained.

For long-term storage, cells are freed from the components of the growth medium by centrifugation, transferred to a drying medium (sucrose 10%, pH 7.0), packaged in 1 ml in penicillin vials and lyophilized. In the freeze-dried state, the starting biomass can be stored for two years at a temperature of 4 ° C.

Example 2. Cultivation of a strain of Lactobacillus rhamnosus 2166 in laboratory conditions

The cultivation of the strain of Lactobacillus rhamnosus 2166 is carried out according to example 1. Receive a culture with a titer of 2 × 10 ⁹ CFU / ml

Obtaining a standardized starting biomass of the strain Lactobacillus rhamnosus 2166 and its lyophilization is carried out according to example 1.

Example 3. Obtaining a bacterial preparation of a consortium of strains of Lactobacillus rhamnosus 2166 and Lactobacillus plantarum 2165

To obtain a bacterial preparation based on a consortium of lactobacillus strains, mechanical mixing of standardized liquid cultures of the strains Lactobacillus plantarum 2165 and Lactobacillus rhamnosus 2166 obtained in examples 1 and 2, respectively, is carried out. The cultures are mixed in equal volumes and get the starting biomass of the bacterial preparation - a consortium of strains of Lactobacillus rhamnosus 2166 and Lactobacillus plantarum 2165.

Other ratios of the starting strains (from 1: 100 to 100: 1) can be used to obtain the bacterial preparation, but a ratio of 1: 1 is preferred.

The resulting preparation is lyophilized according to Example 1. In a freeze-dried state, the bacterial preparation can be stored for two years at a temperature of 4 ° C. The standard cell concentration of each strain in the resulting biomass is 0.5 × 10 ⁹ CFU / ml.

The resulting bacterial preparation can be used, in particular, as a direct starter culture to obtain fermented products.

Example 4. Evaluation of the activity of acid formation of strains and consortium

The activity of acid formation is determined by the titrimetric method [8].

As a starter culture, starting biomasses of strains of Lactobacillus plantarum 2165, Lactobacillus rhamnosus 2166 and a consortium preparation are used.

The measurements are carried out for 48 hours at a ratio of sourdough / milk = v / v = 1: 100. The result is presented in degrees Turner (° T is a value expressing the amount of 0.1 N alkali, which went to the titration of 100 ml of the test sample).

table 2 The activity of acid formation of strains of Lactobacillus plantarum 2165, Lactobacillus rhamnosus 2166 and the consortium when fermenting milk No.
p / p Culture (sourdough / milk ratio = v / v = 1: 100) Bacterium titer in leaven, CFU / ml Clot formation (milk fermentation, yogurt) The acidity of the product, (degree Turner, ° T) product pH The titer of bacteria in the product, CFU / ml 10 h 14 h 48 h one Lactobacillus plantarum 2165 1.0 × 10 ⁹ + 71.0 ± 1.1 (48 h) 4.25 (48 h) 4.4 × 10 ⁸ (48 h) 2 Lactobacillus rhamnosus 2166 1.0 × 10 ⁹ + + 94.5 ± 1.2 (14 h) 4.0 (14 h) 1.4 × 10 ⁹ (14 h) 3 Lactobacillus plantarum 2165 0.5 × 10 ⁹ 8.6 × 10 ⁸ (10 h) & + + + 115.0 ± 1.2 (10 h) 3.8 (10 h) Lactobacillus rhamnosus 2166 0.5 × 10 ⁹ 2.2 × 10 ⁹ (10 h) Note: 10 hours, 14 hours, 48 hours - ripening time, hours.

The results shown in table 2 show that the strain of Lactobacillus plantarum 2165 fermented milk in 48 hours, the acidity of the product is 71.0 ± 1.1 ° T, the strain of Lactobacillus rhamnosus 2166 effectively fermented milk in 14 hours and provides an acidity of 94.5 ± 1.2 ° T (which corresponds to the standards of fermented milk drinks and yoghurts 70-140 ° T), clot formation upon inoculation of the consortium (0.5% Lactobacillus plantarum 2165 and 0.5% Lactobacillus rhamnosus 2166) occurs after 10 hours of fermentation, while the acidity product reaches 115.0 ± 1.2 ° T.

Thus, a consortium consisting of strains of Lactobacillus plantarum 2165 and Lactobacillus rhamnosus 2166 showed the best result when fermenting milk. Moreover, the resulting product has good taste properties and has a light sour-milk smell.

Studies have shown that the properties of the inventive consortium when used as a direct starter culture meet the technological requirements for the production of fermented milk products.

Example 5. Evaluation of the antioxidant activity of the lactobacilli of the consortium and sera of fermented milk products obtained with their use

To evaluate the antioxidant activity, starting biomasses of strains of Lactobacillus plantarum 2165, Lactobacillus rhamnosus 2166, a consortium and whey of fermented milk product are used.

The antioxidant activity of strains, consortium and whey of fermented milk product is determined according to the method proposed in [9].

Measurement of antioxidant activity was performed using ABTS [2,2'-azinobis- (3-ethylbenzothiazoline-6sulfonicasid]. Trolox solution (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylicacid) was used as an antioxidant standard. Measurements were carried out on an Ultrospec 3100 pro spectrophotometer (Amercham) at 734 nm.

The percentage of inhibition is calculated by the formula:

% = (1-A _f / A ₀ ) × 100,

where A _f is the optical density of the sample, A ₀ is the optical density of the ABTS solution.

The TEAC value (Trolox equivalent antioxidant capacity) is determined as a function of the percentage of inhibition of antioxidants to the concentration of Trolox under the same experimental conditions.

The results shown in table 3 show that the strains of Lactobacillus plantarum 2165 and Lactobacillus rhamnosus 2166, taken separately, as well as together in the consortium have a slight antioxidant activity.

Table 3 Antioxidant activity of lactobacilli, a consortium and whey of fermented milk products obtained with their use Culture TEAC (± SD) Cell sonicate (10 ⁹ CFU) Dairy product whey, ml * Lactobacillus rhamnosus 2166 3.45 ± 0.24 28.24 ± 1.60 Lactobacillus plantarum 2165 2.29 ± 0.20 9.64 ± 0.87 Lactobacillus plantarum 2165 & Lactobacillus rhamnosus 2166 6.84 ± 0.48 45.31 ± 2.36 * Serum antioxidant activity minus control (fresh milk).

The most pronounced antioxidant activity is found in whey of a fermented milk product obtained in the process of fermenting cow's milk using a consortium of strains of Lactobacillus rhamnosus 2166 and Lactobacillus splantarum 2165, which may be associated with a more efficient breakdown of milk proteins and the release of peptides exhibiting high antioxidant activity.

Example 6. The study of the resistance of strains to gastric and intestinal stress (the ability to grow in environments with low pH or in an environment containing bile)

In the study, the following methods are used.

In vitro imitation of gastric stress

Artificial Gastric Juice Composition:

NaCl (Sigma S9625) 2.2 g / l L-lactic acid (Sigma L 1750) 9.9 g / l (0.11 M) Pepsin (Sigma P7125) 3.5 g / l pH 2.7 ± 0.02 (add 35% NaOH) pH after 1/11 dilution 3.10 ± 0.10 (control for each culture)

1 ml of artificial gastric juice is added to 100 μl of the culture in the stationary phase (1/11 dilution). The cultures are incubated for 10 minutes, 30 minutes and 60 minutes at 37 ° C, 10% CO ₂ . Instead of artificial gastric juice, 1 ml of MPC is used as a control. Measurements are performed in duplicate for each culture.

After incubation, the culture (each replicate and controls) is diluted from 10 ² to 10 ⁹ or 10 ¹⁰ in MPC, dilutions are plated on plates with MPC-agar and incubated for 24-48 hours at 37 ° C, 10% CO ₂ . The titer of the culture is determined under stress and without stress by counting the number of colonies for dilutions.

In vitro imitation of intestinal stress

Artificial intestinal juice composition:

Bile salts (pig bile Sigma B8631) 3.3 g / l (final concentration 0.3%) NaHCO ₃ Carbonate Buffer (Sigma S8875) 16.5 g / l (final concentration 1.5%) pH 6.3

1 ml of artificial intestinal juice is added to 100 μl of the culture in the stationary phase (1/11 dilution). The cultures are incubated for 5 hours, at 37 ° C, 10% CO ₂ . As control use 1 ml of MPC. Measurements are performed in duplicate for each culture.

Cultures and controls are diluted from 10 ² to 10 ⁹ or 10 ¹⁰ in MRS, dilutions are plated on plates with MPC agar and incubated for 24-48 hours at 37 ° C, 10% CO ₂ . The titer is determined under stress and without stress by counting the number of colonies for dilutions.

Counting microorganisms in a milliliter of culture (CFU) is carried out according to the formula:

∑C / (n ₁ + 0,1n ₂ ) d,

where ∑C is the sum of all characteristic colonies counted on all plates containing from 15 to 300 colonies;

n ₁ is the number of cups in the lowest dilution (2 cups per dilution);

n ₂ is the number of cups in the highest dilution (2 cups per dilution);

d is the value of the first dilution (low dilution) taken for counting.

To determine the resistance of lactobacilli to gastric and intestinal stresses, cultures are used in the stationary phase of growth grown on liquid MPC medium, pH 6.5 for 18 hours at 37 ° C, 10% CO ₂ .

The results shown in tables 4 and 5 show that the strains of Lactobacillus rhamnosus 2166 and Lactobacillus plantarum 2165 are resistant to gastric and intestinal stress.

Example 7. The study of the antagonistic activity of strains of Lactobacillus rhamnosus 2166 and Lactobacillus plantarum 2165 and a consortium of strains

The study of antagonistic activity is carried out by the method of two-layer agar [10] on indicator cultures of Escherichia coli 25922 and Staphylococcus aureus FDA 29P.

The antagonistic activity of the studied cultures is expressed as the minimum inhibitory concentration of the antagonist (MIKA) - this is the minimum amount of the antagonist that inhibits the reproduction of the indicator culture. The indices for individual strains of Lactobacillus rhamnosus 2166 and Lactobacillus plantarum 2165 and a consortium of strains of Lactobacillus rhamnosus 2166 and Lactobacillus plantarum 2165 are presented in Table 6.

The research results indicate that the inventive consortium of strains exhibits a more pronounced antagonistic activity than individual strains of Lactobacillus rhamnosus 2166 and Lactobacillus plantarum 2165.

The probiotic properties of the strains and the consortium are stable and do not change during storage, during cultivation and under the influence of extreme factors, which indicates their industrial applicability.

Example 8. Inhibition of the early stage of the process of metastasis of cancer cells in vitro

For the study, cultures of Lactobacillus rhamnosus 2166, Lactobacillus plantarum 2165 and a consortium of strains in the stationary growth phase, as well as culture fluids (QL) obtained by culturing the strains and consortium, are used.

In vials with lyophilized strains and the bacterial preparation of the consortium obtained in examples 1-3, respectively, pour 1 ml of physiological saline, cooled to room temperature, shake and each solution is added to 100 ml of medium MPC, pH 6.5. The cultures are grown in MPC, pH 6.5 in an atmosphere of 10% CO ₂ for 24 hours at 37 ° C.

From bacteria washed twice in physiological saline, a suspension is prepared in a concentration of 1 × 10 ⁸ bacteria / ml. Bacterial concentrations of each strain and consortium are regulated at OD λ = 580 nm. Bacterial QOL, after adjusting the pH to 7.0-7.2, is sterilized by filtration (0.22 μm). The effect of strains of Lactobacillus rhamnosus 2166 and Lactobacillus plantarum 2165, a consortium of strains and QOL obtained by culturing the strains and consortium on the interaction of MCF-7 cancer cells (human breast cancer) with platelets was evaluated by the method described in [11]. The adhesion of intact MCF-7 cells is taken as 100% (control 1). Fucoidan LS inhibitor (Institute of Organic Chemistry, Moscow) at 20 μg / ml (control 2) and 100 μg / ml (control 3) are used as a positive control for inhibiting the interaction of MCF-7 cells with platelets.

The results obtained (Table 7) show that in the presence of fucoidan LS at a concentration of 20 μg / ml, 72 ± 8% of tumor cells bound to immobilized platelets compared with the intact control. With an increase in fucoidan LS concentration to 100 μg / ml, only 26 ± 4% of MCF-7 cells bind to platelets. A consortium of strains of Lactobacillus rhamnosus 2166 and Lactobacillus plantarum 2165 effectively inhibits the binding of MCF-7 to platelets - 16 ± 2%, however, the maximum activity was observed in QOL obtained after cultivation of the inventive consortium - 3 ± 0.5%.

In this regard, in subsequent experiments, the efficiency of in vivo metastasis inhibition in experiments in mice using the QOL of a consortium of strains was studied.

Table 7 Inhibition of the interaction of cancer cells MCF-7 with immobilized human platelets in vitro No. A drug The adhesion of MCF-7 cells on immobilized platelets,% one Control 1 (intact MCF-7) one hundred 2 Control 2 (fucoidan LS 20 μg / ml) 72 ± 8 * 3 Control 3 (fucoidan LS 100 μg / ml) 26 ± 4 * four Lactobacillus rhamnosus 2166 (cells) 28 ± 5 ** 5 Lactobacillus rhamnosus 2166 (QoL) 19 ± 2 ** 6 Lactobacillus plantarum 2165 (cells) 25 ± 3 ** 7 Lactobacillus plantarum 2165 (QOL) 18 ± 1.5 ** 8 Consortium (cells) 16 ± 2 ** 9 Consortium (QL) 3 ± 0.5 ** * p <0.05; ** p <0.01 differences between the control and the experimental group.

Example 9. Inhibition of the process of metastasis of tumor cells in vivo using culture fluid (QL) of a consortium of strains

In vials with lyophilized strains of Lactobacillus rhamnosus 2166 and Lactobacillus plantarum 2165, obtained according to examples 1 and 2, respectively, pour 1 ml of physiological saline, cooled to room temperature, shake and both solutions are added together in 100 ml of medium MPC, pH-6 ,5. The cultures are grown in MPC, pH 6.5 in an atmosphere of 10% CO ₂ for 24 hours at 37 ° C.

The obtained QOL of a consortium of strains is used to study the inhibition of tumor cell metastasis in vivo in accordance with the methodology [12].

It is known that T-lymphocytes are active participants in antitumor immunity. The immunodeficiency state of Nude mice allows them to be used as an object for the growth of various tumors of various haplotypes, including xenogenic ones. Nude nude mice created on the basis of the Balb / c line (haplotype H-2 ^d ) were inoculated with melanoma B 16 (haplotype H-2 ^b ). After surgical removal of the tumor node of such melanoma, an intensive growth of metastases in the lungs and pleura is usually observed. This model is used to study the effect of QOL of a consortium of strains of Lactobacillus rhamnosus 2166 and Lactobacillus plantarum 2165 on the development and growth of metastases in the postoperative period.

For research use 5 groups of 5 mice each. As a positive control, recombinant human protimosin alpha (rHu ProTα) with antimetastatic activity is used in concentrations of 3 μg and 0.3 μg. Animals are injected intraperitoneally in two weeks every other day:

Group 1 (control 1) - saline solution of 0.2 ml;

Group 2 (control 2) - rHuProTα 3 μg;

Group 3 (control 3) - rHuProTα 0.3 μg;

Group 4 - rHuProTα 0.3 μg + QOL of the consortium 0.2 ml;

Group 5 - QOL of the consortium 0.2 ml.

On day 14, all mice were subcutaneously injected with melanoma B 16 cells (1.5 × 10 ³ / mouse) and the corresponding preparations continued to be administered 3 times a week. Two weeks after vaccination, surgery is performed to remove the resulting tumor. The animals of each group continue to administer the appropriate drugs 3 times a week, while the dose of rHu ProTα is reduced 10 times. At the beginning of the death in the control group (on day 58 from the start of the experiment), animals of all groups are euthanized by asphyxiation in CO ₂ and metastases are counted.

The results obtained (Table 8) show that from the control group of mice in the lungs and pleura, 10 ± 2 to 14 ± 3 metastases were found, the average size of which was 50 ± 14 mm ³ . Moreover, in the experimental group of mice that received QOL, only one out of five mice showed 3 metastases in the lungs and 3 metastases in the pleura, the average size of which is 8.3 times smaller compared to the control group treated with saline, and is 6 ± 2 mm ³ . Synergies of the antimetastatic action of the QOL of the lactobacilli consortium and rHu ProTα (group 4) were also found. In this group, four animals out of five metastases are absent and only one mouse formed 3 metastases in the lungs, the size of which does not exceed 4 ± 1 mm ³ .

Example 10. The use of a consortium of strains of lactobacilli as a direct starter culture for the fermentation of beet juice in order to obtain beet kvass (SC)

Beets are sorted, washed, cleaned and ground to a homogeneous mass with a particle size of 6 × 3 × 1 (length, width, height, mm). Before being pasteurized, 0.25% ascorbic acid is added to the freshly ground beet pulp to increase the depth of extraction of coloring substances and treated at 75 ° C for 15 minutes.

The pulp is cooled to 37 ° C, add the lyophilized bacterial preparation obtained in example 3, mix.

Fermentation of pulp is carried out under static conditions at 37 ° C for 10 hours. A decrease in the pH of the resulting SC to 4.0 is noted. The pulp is separated from the SC by centrifugation.

Organoleptic evaluation of a laboratory sample of SC fermented using the inventive consortium showed that SC has a rich burgundy color corresponding to the color of beetroot and a pronounced beetroot taste, refreshing due to the sour taste of the content of lactic acid fermentation products.

SC obtained using a consortium of strains of Lactobacillus rhamnosus 2166 and Lactobacillus plantarum 2165 as a probiotic sourdough of direct application in technological parameters (Table 9) and in the content of vitamins in the finished product (Table 10) surpasses the known method [2].

Table 9 Technological indicators of SC obtained using a consortium of lactobacillus strains as a direct application probiotic sourdough Type of lactic acid bacteria Juice ripening time, hours The output of juice, ml / 100 g Solids content,% Active acidity, pH L. cellobiosus 9k [2] twenty 60 12.0 4.0 Consortium of Lactobacillus rhamnosus 2166 and Lactobacillus plantarum 2165 10 64 13,4 4.0 Table 10 Vitamin content in the finished product of beet juice fermentation - SC using a consortium of lactobacillus strains Type of lactic acid bacteria Vitamin content B1, mg% B2, mg% B3, mg% B6, mg% B9, mg% B12, mcg / ml PP, mg% E, mg% L. cellobiosus 9k 0.10 0.05 - - - 2.0 0.18 - Consortium of Lactobacillus rhamnosus 2166 and Lactobacillus plantarum 2165 0.10 0.05 0.09 0.06 1,0 2.0 0.22 0.10

Under the action of enzymes possessed by strains of the consortium Lactobacillus rhamnosus 2166 and Lactobacillus plantarum 2165, the biotransformation of the components of the starting vegetable raw material occurs. At the same time, the content of organic acids increases, partial hydrolysis of structural polysaccharides is noted, as a result, the composition of the pulp and beetroot juice changes during the fermentation process (Table 11).

An important consumer property of SC is its preservation of a high titer of viable cells (in terms of CFU / ml), the number of which in the finished drink (10 hours) is 6.3 × 10 ⁸ CFU / ml for Lactobacillus rhamnosus 2166 and 7.8 × 10 ⁸ CFU / ml for Lactobacillus plantarum 2165 and for 20 days of storage at 0-4 ° C slightly decreases to 7.5 × 10 ⁷ CFU / ml for Lactobacillus rhamnosus 2166 and 8.9 × 10 ⁷ CFU / ml for Lactobacillus plantarum 2165 (table .12).

Table 11 Changes in the chemical composition of beet juice and pulp before and after fermentation using a consortium of strains Indicators Before fermentation After fermentation Juice Betanin g / 100 g 1.08 ± 0.03 1.20 ± 0.04 Organic acids, g / dm ³ : apple 0.236 ± 0.09 0.965 ± 0.12 amber 0.021 ± 0.01 0.345 ± 0.07 Pulp Total pectin,% 0.19 ± 0.04 0.31 ± 0.05 Cellulose,% 2.36 ± 0.09 1.24 ± 0.08 Hemicellulose,% 1.85 ± 0.08 1.38 ± 0.05

Table 12 Preservation of live bacteria in fermented beet juice Strain CFU / ml After fermentation After 20 days of storage at 0-4 ° C Lactobacillus rhamnosus 2166 6.3 × 10 ⁸ 7.5 × 10 ⁷ Lactobacillus plantarum 2165 7.8 × 10 ⁸ 8.9 × 10 ⁷

This fact indicates that beet juice fermented by a consortium of strains of Lactobacillus rhamnosus 2166 and Lactobacillus plantarum 2165, as a direct probiotic starter, does not lose its probiotic properties in the final product - beet kvass during storage for 20 days at a temperature of 0-4 ° C. This indicator also indicates the ability of the symbiotic consortium of strains of Lactobacillus rhamnosus 2166 and Lactobacillus plantarum 2165 to survive well in saprotrophic conditions.

Thus, the use of a consortium containing the probiotic strains of Lactobacillus rhamnosus 2166 and Lactobacillus plantarum 2165 as a starter culture of direct introduction into beet juice and beet pulp for the production of SC allows increasing the depth of processing of raw materials, product quality, its biological value and taste.

Example 11. The influence of SC on the process of restoring normal levels of taurine in the heart of rats treated with β-alanine

It is known that a deficiency of taurine, which is a natural product of the exchange of sulfur-containing amino acids, in cells of any organ adversely affects its condition. In particular, it is important for tissue regeneration, has a protective effect during physical exertion, and has an antistress effect. Loss of taurine by heart cells leads to cardiomyopathy.

The aim of the study was to study the effect of SC on the process of restoring normal taurine levels in the heart of rats treated with β-alanine, which is an inhibitor of taurine transport.

For the study, 4 groups of male Wistar rats weighing 250-300 g, 15 in each group, are used.

To reduce rat taurine content, drinking water containing 3% β-alanine is obtained within 24 days.

Animals are provided with:

group 1 (control): water without 3% β-alanine;

group 2 (control): from 4 to 28 days - water containing 3% β-alanine;

group 3 (control): from 4 to 28 days - water containing 3% β-alanine, from 29 to 35 days - water without 3% β-alanine;

group 4 (experiment): from 4 to 28 days - water containing 3% β-alanine, from 29 to 35 days - water without 3% β-alanine + daily 2.5 ml of SC intragastrally.

The results (Table 13) show that SC accelerates the process of restoring the initial level of taurine in the heart of rats treated with β-alanine, and indicates the advisability of including SC as a functional food product in the diet of people, in particular athletes experiencing high physical and mental stress .

Table 13 Acceleration of the process of restoring the initial level of taurine in the heart of rats treated with SC Group The content of taurine in the heart of rats, mol / g dry weight 1 day 29 day 36 day 1 (control) 113 ± 6 114 ± 5 2 (control) 63 ± 3 * 3 (control) 67 ± 4 * 4 (experience) 112 ± 7 * p <0.05 - significance of differences in the content of taurine in the heart of rats in the compared groups 2 and 1, 4 and groups 3 and 1, 4.

Example 12. The influence of SC on learning and improving the memory of rats in the test of the conditioned reflex of active avoidance (URAI)

The influence of SC on the training of Wistar rats in the URAI test was studied in a shuttle test chamber (Hugo Basile, Italy). The device has two cameras, alternately receiving voltage. When the rat enters the "quiet" compartment, the current is turned off. The installation is equipped with a device that records the duration of the electric current supplied to the electrode floor, that is, the latent period until the rat enters the safe compartment.

For the study using 2 groups of rats of 10 animals in each group.

An experimental group of animals was injected intragastrally daily with 2.5 ml of SC daily for 28 days, and a physiological saline in the same dose was administered to the control group.

The measurement of the latency period of avoidance is carried out on 15, 16, 17 and 28 days of taking SC. Skill improvement is calculated as a percentage compared to the first training session.

The results shown in Table 14 show that after the third training session, the animals of the experimental group increased their learning rate by 29.7% versus 7.4% in animals of the control group, which indicates the stimulating effect of SC on short-term memory.

Table 14 The influence of SC on training Wistar rats in the URAI test Days of admission SK Training Session No. Latent avoidance period X (Xmax - Xmin) SC The control Indicators % Skill improvement Indicators % Skill improvement fifteen one 1518 (1544-1497) 0 1624 (1649-1598) 0 16 2 1237 (1286-1175) 18.5 1567 (1712-1521) 3,5 17 3 1067 (1185-952) 29.7 1504 (1547-1465) 7.4 28 four 472 (511-436) 68.9 702 (763-649) 56.8 Notes: X is the average value of the sample; Xmax = X + σn ^-1/2 tXmin = X-σn ^-1/2 t n is the sample size, σ is the standard deviation, t is the coefficient of student criterion n-1. The significance of pU differences according to the Wilcoxon-Mann-Whitney U criterion for all values in the experiment is 0.001.

Thus, the results of studies obtained on rats in the URAI test allow us to conclude that SC is a stimulator of short-term memory.

Example 13. The possibility of correction of oxidative stress in patients with diabetes mellitus using SC

40 patients (2 groups of 20 people each) suffering from type 2 diabetes mellitus (T2DM) were examined. The duration of the disease is 2-4 years.

Patients of the first group receive daily for 2 months 100 ml of SC on the background of basic hypoglycemic therapy, patients of the second group (control) use only basic hypoglycemic therapy.

Before and after a course of taking SC, total antioxidant activity (OAA), antioxidant enzyme activity of catalase (CAT), carbohydrate metabolism (fasting glycemia), lipid metabolism (total cholesterol and molecular product of lipid peroxidation - malondialdehyde (MDA)) are evaluated, as described in [13].

The results shown in Table 15 show that the consumption of SC as a functional food product helps to increase the overall antioxidant activity, realized, in particular, by increasing the activity of the antioxidant enzyme catalase, improving the compensation of carbohydrate and lipid metabolism. A decrease in blood plasma levels of MDA is due to the restriction of lipid peroxidation.

The results obtained indicate the feasibility of using SC as a functional nutrition product in the complex treatment of diabetes mellitus.

Thus, the claimed consortium of lactobacillus strains, consisting of Lactobacillus rhamnosus 2166 and Lactobacillu splantarum 2165, has high technological characteristics: the strains of the consortium actively develop in nutrient media, milk and beetroot juice, exhibit persistent symbiotic relationships as direct fermentation yeast for production and direct production vegetable products. This allows you to reduce the duration of the fermentation of milk and beet juice, increase the depth of beet processing, improve the quality of sugar, its taste, biological value.

It is shown that products obtained using the declared consortium can be recommended as functional nutrition products for the prevention and treatment of a wide range of diseases.

Literature

1. Shenderov B.A. Medical microbial ecology and functional nutrition. T.3. Probiotics and functional nutrition. M., 2001. Grant Publishing House, 287 pp.

2. KZ No. 34 "The method of obtaining a drink from beets for dietetic nutrition."

3. RU No. 2180348.

4. RU No. 2180914.

5. RU No. 2180915.

6. RU No. 2296797.

7. RU No. 2326938 (prototype).

8. GOST 3624-92. Milk and dairy products. Titrimetric methods for determining acidity.

9. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M. and Rice-Evans, C. (1999) Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med 26, 1231-1237.

10. Ermolenko E.I., Isakov V.A., Zhdan-Pushkina S.Kh., Tets V.V. Quantification of the antagonistic activity of lactobacilli. // Journal. microbiol., epidemic., immunobiol. - 2004. - 5. - P.94-98.

11. Cumashi A., Ushakova N. A, Preobrazhenskaya ME, D'Incecco A., Piccoli A., Totani L., Tinari N, Morozevich GE, Berman AE, Bilan M. I., Usov AI, Ustyuzhanina NE, Grachev AA, Sanderson CJ, Kelly M., Rabinovich GA, Iacobelli S., Nifantiev NE A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic, and antiadhesive activities of nine different fucoidans from brown seaweeds.// Glycobiology 2007, 17 (5 ): 541-552.

12. Khodyakova A.V. Immunobiological properties of recombinant human protimosin alfa. Diss. for the degree of candidate of biol.science. 2001, 92 p.

13. Harutyunyan A.V., Dubinina E.V., Zybina N.N. Methods for assessing free radical oxidation and the antioxidant system of the body. SPb., 2000, 104 p.

Claims (1)

A consortium of probiotic strains of Lactobacillus rhamnosus VKM B-2726D and Lactobacillus plantarum VKM B-2725D for the manufacture of a bacterial preparation and direct starter culture for the production of fermented milk and fermented beet juice as functional food products.