RU2614116C2 - Method of producing probiotic composition - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, food and medical industry and can be used in production of probiotics for direct consumption or biologically active additive to food products, in production of fermented milk products of preventive purpose, intended particularly for normalization of cholesterol level, and useful microflora of gastrointestinal tract and increasing general body resistance. Invention involves method of producing probiotic composition for lowering of blood cholesterol, containing dry biomass of probiotic bacteria strains B. bifidum GG-72 VKPM Ac-1884, L. fermentum LFM-2 VKPM V-10368, L. rhamnosus LC-52GV VKPM V-9475, L. plantarum GVI-1 VKPM V-8556, L. acidophilus AST-44. Above method involves separate cultivation of strains B. bifidum GG-72 VKPM Ac-1884 on Blaurock medium, strain L. fermentum LFM-2 VKPM V-10368, L. rhamnosus LC-52GV VKPM V-9475, L. plantarum GVI-1 VKPM V-8556 and L. acidophilus ACT-44 B. bifidum GG-72 VKPM Ac-1884, L. fermentum LFM-2 VKPM V-10368, L. rhamnosus LC-52GV VKPM V-9475, L. plantarum GVI-1 VKPM V-8556, L. acidophilus AST-44 VKPM-V-9647 on MRS broth at 37±1 °C up to stationary phase of development, concentration, mixing with protective medium, drying and mixing of dry biomass of probiotic strains in equal quantities.

EFFECT: method of producing probiotic composition is disclosed.

2 cl, 8 dwg, 6 tbl

Description

The invention relates to biotechnology, food and medical industry and can be used to obtain probiotics intended for direct consumption or as a biologically active additive to food products, in the production of fermented milk prophylactic products, intended, in particular, to normalize cholesterol and beneficial microflora human gastrointestinal tract and increase the overall resistance of the body.

Probiotic microorganisms are well known in the art.

Lactic acid bacteria, including Lactobacillus and Bifidobacterium, are commonly used as probiotics, but others, including the Lactococcus, Propionibacterium, Bacillus, Saccharomyces, Streptococcus, and Escherichia strains, can also be used.

The most accessible and common probiotics in food. However, powders, tablets and capsules containing probiotics are also known. Thus, probiotics can be used in various types of prophylactic products.

Probiotic microorganisms may be administered to a subject to provide a beneficial effect, such as, for example, improving intestinal microflora. Probiotics can suppress the number of viable unwanted microorganisms by producing antibacterial compounds by competing for nutrients or for adhesion sites. In addition, they can alter microbial metabolism by increasing or decreasing enzymatic activity, or they can stimulate the immune system by increasing antibody levels or increasing macrophage activity. So, it is known that probiotic microorganisms can participate in the fulfillment of vital functions in the human body (see Synbiotics in food technology: Rogov I.A. et al., M: MGUPB, 2006, Amerkhanova AM Role of probiotic microorganisms in modern preventive technologies and restorative medicine and the possibility of increasing the effectiveness of drugs based on them.New drugs, 2007, No. 4, p. 4-7).

It is known that the use of probiotics positively affects the maintenance and / or restoration of intestinal health and prevents the occurrence of dysbiosis of any etiology (see, for example, RU 2536939 C2, 12/27/2014). The possibility of using probiotics for the prevention of colorectal cancer, or for the prevention and treatment of inflammatory bowel disease (IBD), or for the prevention and treatment of vaginal infections and inflammatory conditions in the vagina, or for replenishing and restoring the balance of non-pathogenic intestinal bacterial flora is also known. . RU 2364623 C2, 08.20.2009).

The use of probiotics as part of fermented milk products is widespread (RU 97108832 A, 04.27.1999, KZ 24649 10.17.2011 A4, WO 2010/055499, 05.20.2010). The advantages of probiotics over fermented milk products with probiotic properties are that they can be consumed with various foods.

So, from the prior art there is a known method for producing the probiotic "Simbiter-2" and a method for the production of a fermented milk product with its use (RU 97108832 A 04/27/1999). A method for producing a probiotic involves co-culturing in a milk medium bifidobacteria of various species, acetic acid bacteria and propionic acid bacteria, which are mixed with a previously prepared consortium of lactic acid bacteria in a ratio of 4: 1. Using such a probiotic, a fermented milk product is obtained on the basis of milk, or buttermilk, or a mixture of milk with buttermilk, or a mixture of milk with whey, or a mixture of buttermilk with whey. The disadvantage of this method of obtaining probiotic is that when co-culturing different types of bifidobacteria, which differ in the dynamics of development, it is not always possible to obtain a probiotic of stable quality, where all types of bifidobacteria used will be presented in the right amount. The use of this probiotic is limited only by its use for the production of sour-milk products of a certain composition and having a short shelf life.

The prior art also known a method of obtaining a dry bacterial preparation "Bifacid" (RU 2083666 C1, 07/10/1997), which provides for the preparation of a nutrient medium, heat treatment, cooling to incubation temperature, separate cultivation of specific strains of acidophilic bacteria and bifidobacteria, mixing them in equal ratios, freeze-drying. The disadvantage of this method of obtaining a dry bacterial preparation is the use of culture media that are most favorable for the accumulation of cells of specific strains of acidophilic bacteria and bifidobacteria and do not contain ingredients that enhance the probiotic effect of the drug in the human body.

As they study probiotic bacteria, scientists report revealing functional properties in them, such as their ability to participate in the regulation of blood cholesterol (Getz GS Animal models of atherosclerosis, Arterioscler Tromb Vase Biol, Vol 32, No. 5, 2012, p. 1104- 1115, Khamagaeva IS et al. Study of the cholesterol metabolizing properties of probiotic microorganisms. - Dairy Industry, 2011, No. 10, p. 56, Kumar et. Al. Cholesterol-lowering probiotics as potential biotherapeutics for metabolic diseases, Experimental diabetes research, 2012 , T. 2012, p. 14). Cardiovascular diseases are one of the main causes of death among middle and older age groups of people in different countries of the world. Elevated blood lipids are recognized as the main cause of cardiovascular disease, in particular coronary heart disease and other health problems in developing countries. First of all, clinical trials show a relationship between cholesterol and the risk of coronary heart disease (Getz GS Animal models of atherosclerosis, Arterioscler Tromb Vasc Biol, Vol 32, No. 5, 2012, p. 1104-1115, BM Bondarenko Role of opportunistic bacteria in chronic inflammatory processes of various localization. –Tver: LLC Triad Publishing House, 2001, 88 pp.).

A known composition comprising an effective amount of at least one of the strains selected from the group consisting of Lactobacillus plantarum CECT 7527, Lactobacillus plantarum CECT 7528 and Lactobacillus plantarum CECT 7529 or their mutant strains, where mutant strains are obtained using deposited strains as starting material and where the mutant strains retain or further improve the cholesterol-lowering activity of the parent strains used to lower cholesterol (RU 2012118639, 11/20/2013).

The authors of the present invention in the article "Cholesterol-reducing probiotic bacteria in dairy products." The dairy industry, No. 5, 2014, two consortia of bacteria have been proposed that reduce the concentration of cholesterol in the nutrient medium.

However, at the moment there is a need to find more effective probiotic strains or compositions of probiotic strains to lower cholesterol, since known probiotic strains and compositions do not provide the desired level of cholesterol reduction.

An object of the present invention is to provide a highly effective probiotic composition that reduces cholesterol.

Thus, the technical result is to increase the ability of the probiotic strains included in the composition to lower cholesterol.

The authors of the present invention, it was unexpectedly discovered that the composition of the nutrient medium affects the ability of probiotic cells to lower cholesterol concentration. In addition, the intensity of lowering the concentration of cholesterol by probiotic strains at different stages of their development on selected media was studied. It was unexpectedly discovered that the developmental stage affects the ability to lower cholesterol concentration by probiotic strains.

The essence of the proposed technical solution consists in a method for producing a probiotic composition for lowering cholesterol containing dry biomass of probiotic strains of bacteria B. bifidum GG-72 VKPM Ac-1884, L. Fermentum LFM-2 VKPM B-10368, L. Rhamnosus LC-52GV VKPM B-9475, L. Plantarum GVI-1 VKPM B-8556, L. Acidophilus AST-44 VKPM B-9647, including separate cultivation of B. bifidum GG-72 VKPM Ac-1884 strains on Blaurock medium, L. Fermentum LFM-strains 2 VKPM B-10368, L. Rhamnosus LC-52GV VKPM B-9475, L. Plantarum GVI-1 VKPM B-8556 and L. Acidophilus AST-44 VKPM B-9647 on MRS broth at 37 ° C ± 1 ° C to the stationary phase of development, conc Centering, drying and mixing dry biomass of probiotic strains in equal amounts.

The task was solved as follows.

To begin with, experiments were conducted to study the ability of probiotic strains to lower cholesterol. The following strains were selected as probiotic strains: B. adolescentis BGV-11, B. bifidum GG-72, L. Acidophilus AST-44, L. Fermentum LFM-2, L. Plantarum GVI-1, L. Rhamnosus LC-52GV ; L. Acidophilus ACT-41, L. Acidophilus 887.

The nature of their growth in a culture medium containing cholesterol with an emulsifying agent was tested (Figure 1). It turned out that cholesterol in combination with Tween-80 in all strains increases the cell concentration from 1.2 times (B. adolescentis BGV-11) to 11.2 times (L. Rhamnosus LC-52GV). On average, the growth increase in all studied strains was 3.8 times. Strain B. bifidum GG-72 increased growth by 2.2 times in the presence of cholesterol and tween-80.

The research results showed the presence of different ability to lower cholesterol concentration in the studied strains (Figure 2). It was revealed that the strains were most capable of lowering cholesterol concentration under these conditions: B. adolescentis BGV-11 - by 33.6 ± 0.9%, B. bifidum GG-72 - by 37.6 ± 0.38%; L. Acidophilus AST-44 - by 16.0 ± 0.41%, L. Fermentum LFM-2 - by 29.6 ± 0.73%; L. Plantarum GVI-1 - 30.6 ± 1.4%, L. Rhamnosus LC-52GV - 27.4 ± 2.1%; the smallest L. Acidophilus AST-41 - by 10.6 ± 2%, L. Acidophilus 887 - by 13.4 ± 0.6%.

In addition, probiotic cultures were studied on CaCo-2 cells to determine the ability to adhere. Data were obtained on the number of cells of the studied probiotic strains capable of adhesion to CaCo-2 intestinal epithelial model cells. The adhesion level was estimated based on the number of bacterial cells attached to 1000 CaCo-2 cells (see Table 1).

Thus, it turned out that the three strains are the most promising for two indicators at once (adhesion to CaCo-2 cells and a decrease in cholesterol concentration in vitro) - these are strains of B. bifidum GG-72, B. adolescentis BGV-11, L. Rhamnosus LC -52GV. Probiotic strains were the second most promising: L. Plantarum GVI-1, L. Fermentum LFM-2. And the least promising were three strains: L. Acidophilus AST-41, L. Acidophilus AST-44; L. Acidophilus 887. Although the last three strains take the last place according to the mathematical surface, they belong to the “middle” group of adhesion, and the L. Acidophilus AST-44 strain showed not the lowest ability to lower cholesterol concentration 16 ± 0.41% . This strain of acidophilic lactic acid bacteria is valuable from the point of view of lactic acid production, antagonism of conditionally pathogenic and pathogenic test cultures, therefore, it is advisable to use the L. Acidophilus AST-44 strain in a probiotic composition.

Based on the processing and analysis of the data on the study of adhesion properties and the ability to lower cholesterol concentration, strains were selected to create a probiotic composition: B. bifidum GG-72; L. Fermentum LFM-2; L. Plantarum GVI-1, L. Rhamnosus LC-52GV, L. Acidophilus ACT-44.

Next, the possibility of enhancing the target activity - lowering the concentration of cholesterol in probiotic strains was studied. It is known that the composition of the nutrient medium and cultivation conditions, the phase of development of microorganisms affect their metabolism. However, information in the prior art about the influence of these factors on the ability of probiotic cells to lower cholesterol concentrations was not found.

At the first stage, the effect of various nutrient media was compared.

With regard to varying cultivation conditions in line with the study, the possibility of enhancing the properties of probiotic bacteria, the very first approach was the choice of nutrient media of various compositions (table 2, table 3).

For lactobacilli, common culture media are MRS and lactobacillus, a comparative composition of which is given in table 2.

It turned out that the strains in question showed the greatest ability to lower cholesterol concentration on MRS broth. The strain L. Fermentum LFM-2 showed almost the same ability to lower cholesterol concentration both on MRS broth and on the medium for lactobacilli (table 3).

When studying the influence of the composition of nutrient media on lowering cholesterol by B. bifidum GG-72 strain, nutrient media were used: MRS broth with the addition of 0.05% L-cysteine, GMK-2, Blaurock, which are most widely used in microbiological practice. The comparative composition of these culture media is discussed below (table 4).

An analysis of the results showed that the ability to lower cholesterol concentration in the studied strains of bifidobacteria was manifested to a greater extent during their development on Blaurock medium (table 5).

Next, the intensity of lowering the concentration of cholesterol by probiotic strains at different stages of their development on selected media was studied. The results of the study of the dependence of the ability of the selected strains to lower cholesterol concentration on the growth stage are shown in Figures 3-7.

An analysis of the data obtained indicates that during the development of cultures of probiotic microorganisms, there is a change in their ability to lower cholesterol concentration in the culture medium. It was revealed that the greatest ability of the studied probiotic bacteria strains to lower cholesterol concentration in the cultivation medium is manifested at the end of the logarithmic growth phase - at the very beginning of the stationary growth phase.

The number of cells at the time of the greatest ability to lower cholesterol concentration in different studied probiotic strains was different (table 6).

Thus, the dependence of the ability to lower cholesterol concentration on the stage of growth of probiotic strains was established: the greatest ability was noted at the end of the logarithmic growth phase - the beginning of the stationary phase, which was 12-14 hours after the onset of development, depending on the strain. Different ability to lower cholesterol concentration, depending on the age of the culture, may be associated with differences in metabolic rate and different adsorbing capacity of cell walls.

Thus, on the basis of the conducted studies, it can be concluded that the rational conditions for biotechnology of the studied strains of probiotic bacteria that show the ability to lower cholesterol concentrations are: a nutrient medium for bifidobacteria - Blaurock; for lactobacilli - MRS-broth; cultivation at 37 ± 1 ° C until the stationary phase of development.

The compatibility of the selected strains of probiotic bacteria was investigated, the data obtained are shown in Figure 8. Colonies of probiotic strains in the form of strokes in the zone of their intersection were uniform without obvious zones of growth inhibition. It was established that the strains do not exhibit antagonism with respect to each other and, thus, the possibility of their joint presence in the probiotic composition is proved.

The proposed method for producing a probiotic composition that reduces cholesterol, is as follows:

1. Each strain is cultivated separately under optimal conditions that provide the maximum reduction in cholesterol concentration: B. bifidum GG-72 strain is cultured in Blaurock medium at a temperature of 37 ° C for 14 hours before the onset of the stationary phase; L. rhamnosus LC-52GV strain is cultured on MRS broth at 37 ° C for 13 hours before the onset of the stationary phase; strain L. plantarum GVI-1 is cultured on MRS broth at a temperature of 37 ° C for 12.5 hours before the onset of the stationary phase; L. fermentum LFM-2 strain is cultured on MRS broth at a temperature of 37 ° C for 13.5 hours before the onset of the stationary phase; the strain L. acidophilus AST-44 is cultured on MRS broth at 37 ° C for 12 hours before the onset of the stationary phase.

2. After cultivation, the resulting culture fluid of each individual strain is concentrated, mixed with a protective medium in a ratio of 1: 2, and thoroughly mixed.

3. Biomass mixed with a protective medium is poured onto sterile pallets under aseptic conditions, placed in a freeze-drying unit, in which it is dried to obtain dry biomass with a mass fraction of moisture of not more than 4.0%.

4. To obtain a probiotic composition, an equal amount of dry biomass of each strain is introduced into a sterile container and mixed thoroughly. Then the dry probiotic composition is Packed.

It was revealed that in the composition of the composition the strains of probiotics showed an increase in the ability to lower cholesterol concentration. The composition showed the ability to reduce the concentration of cholesterol in vitro by 47.6%, which is more than the ability to reduce the concentration of cholesterol in any individual strain in the phase of logarithmic growth:

Claims (2)

1. A method of obtaining a probiotic composition for lowering cholesterol containing dry biomass of probiotic strains of bacteria B. bifidum GG-72 VKPM Ac-1884, L. fermentum LFM-2 VKPM B-10368, L. rhamnosus LC-52GV VKPM B-9475, L. plantarum GVI-1 VKPM B-8556, L. acidophilus AST-44 VKPM - B-9647, including the separate cultivation of B. bifidum GG-72 VKPM As-1884 strains on Blaurock medium, L. fermentum LFM-2 VKPM B strains -10368, L. rhamnosus LC-52GV VKPM B-9475, L. plantarum GVI-1 VKPM B-8556 and L. acidophilus AST-44 VKPM B-9647 on MRS broth at 37 ° С ± 1 ° С to the stationary phase development, concentration, mixing with a protective environment, drying and mixing of dry biomass of probiotic strains in equal amounts. 2. The probiotic composition obtained by the method according to claim 1, containing dry biomass of probiotic bacterial strains of B. bifidum GG-72 VKPM Ac-1884, L. fermentum LFM-2 VKPM B-10368, L. rhamnosus LC-52GV VKPM B-9475 , L. plantarum GVI-1 VKPM B-8556, L. acidophilus AST-44 VKPM B-9647 in equal amounts.

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