RU2782196C9 - Synbiotic composition for suppressing microbial pathogens of intestinal infections and method for production thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to biotechnology, the pharmaceutical and food industries, and pertains to a synbiotic composition for suppressing pathogens of intestinal infections based on bifidobacteria and a fraction of burdock root fructans, as well as to a method for production thereof. Content of components in 1 g of the composition: lyophilised biomass of bifidobacteria 10⁸ to 10¹⁰ colony-forming units; fraction of burdock root fructans 0.80 to 0.98 g. A fraction of burdock root fructans is herein used, produced by crushing burdock roots, performing water extraction at a temperature of 60 to 90°C for 30 min, separating the extract from the solid residue by filtration, removing the high molecular weight impurities by ultrafiltration, removing the low molecular weight impurities by sorption on activated carbon, concentrating the extract by vacuum evaporation, adding ethanol to the extract as calculated for the final content thereof at 20 vol.%, holding in cold, separating and drying the resulting deposited fraction of fructans. The composition can be made in the form of a powder or capsules. Method for producing the composition consists in producing a fraction of burdock root fructans by the above method, mixing said fraction with lyophilised biomass of bifidobacteria, packing in packets or encapsulating.

EFFECT: possibility of producing a synbiotic composition containing a fraction of burdock root fructans, with a greatest stimulating effect on the growth of bifidobacteria, ensuring the maximum level of suppression of pathogens of intestinal infections thereby.

3 cl, 3 tbl, 3 ex

Description

The field of technology to which the invention belongs.

SUBSTANCE: invention relates to biotechnology, pharmaceutical and food industries and is a synbiotic composition based on bifidobacteria and burdock root fructans. The claimed composition can be used to suppress pathogens of intestinal infections in pharmaceuticals, specialized foodstuffs and dietary supplements, and also as a feed additive.

The level of technology.

Synbiotics are a combination of probiotic microorganisms and prebiotic substances that synergistically enhance the action of each other. One of the features of the action of probiotics is their suppression of the growth of microorganisms that cause infectious diseases. Prebiotics are substances that selectively stimulate the growth and/or activity of beneficial members of the gut microbial community. In this vein, the synergistic effect of the prebiotic can be correlated with an increase in the antagonism of probiotic microorganisms against pathogens, as well as a decrease in the growth rate of pathogens as a result of a deficiency of an easily digestible substrate.

The prebiotic properties of fructans (inulin and fructooligosaccharides) are known, which are widely represented as reserve substances in plants mainly of the Compositae family: chicory, Jerusalem artichoke, echinacea, garlic, burdock, etc. At the same time, the production and use of fructans accumulated in burdock roots is currently not has found wide application and has not actually been implemented on an industrial scale.

For example, a method is described (RU 2360927 C1), which provides for the production of inulin from crushed burdock roots using exhaustive extraction for 3÷5 days, precipitation and recrystallization when treated with 96% ethanol at a temperature below minus 15°C. The disadvantage of this method is the high duration of the process, which reduces the yield of the product from a piece of equipment and increases the risk of contamination.

Another method (RU 2254138 C1) consists in preparing a fine powder of one or more than one plant, in particular burdock roots, and extracting with an aqueous solution of ethanol. However, it is known that with an increase in the degree of polymerization of fructans, their solubility in aqueous ethanol solutions decreases. On the other hand, the solubility of phenolic plant compounds in aqueous ethanol solutions can be high. Therefore, the use of this approach for the isolation of fructans is not rational.

There is a known method (RU 2604934 C2), according to which the crushed roots of common burdock are subjected to triple extraction at a ratio of raw material: extractant 1:30, at a temperature of 80°C in an ultrasonic bath for 30 minutes. Plant material is separated by filtration, and water-soluble polysaccharides are precipitated with three times the amount of 95% ethanol with stirring, cooling in a freezer at a temperature of -18 ° C for 1 hour, filtered under vacuum, washed and dried. Although the yield of fructans in this case is high , and the process is intensive, ballast substances such as proteins are not soluble in water-alcohol solutions and will contaminate the target product.

It should be noted that in these methods, the prebiotic potential of the obtained substances was not evaluated, which limits the possibility of their use in synbiotic compositions. It has been previously shown that fructans of various degrees of polymerization can be obtained by fractional precipitation with water-organic precipitant solutions of various concentrations (Wack M., Blaschek W., 2006), and are capable of having different effects on the growth of probiotic bacteria (Rossi et al., 2005). However, none of the above methods allows you to get a certain fraction of burdock with the desired properties.

Compositions are known that include probiotic microorganisms, including bifidobacteria, as well as extracts of plant materials, in particular burdock roots (EP 1281403 A1). However, this composition does not show the prebiotic activity of fructans. Burdock extracts also contain a significant amount of phenolic substances that can have an inhibitory effect on the growth of microorganisms, including probiotics.

Closest to the invention is a composition comprising lactobacillus strain Lactobacillus rhamnosus HN001 and fructans, which, as the authors indicate, can be obtained, including from burdock roots (EP 2525811 B2). However, the effectiveness of this composition has not been evaluated in terms of the possibilities of using different fractions of the resulting fructans. Also for this composition is not considered the possibility of using bifidobacteria.

Thus, a large number of both methods for obtaining burdock fructans and compositions of probiotic bacteria with plant extracts, including those from burdock roots, as well as fructans from them, are presented. However, the rationale for the effectiveness of these compositions against microbial food contaminants and foodborne pathogens, which cause the risk of developing foodborne infections, is not given, and the relationship between the technology for obtaining fructans, which determines their fractional composition, and this effectiveness has not been demonstrated.

Disclosure of the essence of the invention.

The objective of this invention is to create such a synbiotic composition that would be experimentally substantiated, the most effective against various pathogens of intestinal infections.

The technical result of the invention is a synbiotic composition of bifidobacteria and a fructan fraction of burdock roots obtained by extraction, purification and subsequent precipitation with water-alcohol solutions of a certain concentration, i.e. obtained by a certain method and under certain process parameters, which determines its effectiveness against test strains, as well as the specified method for obtaining the claimed composition. The greatest stimulating effect of the selected fraction on the growth of bifidobacteria and the highest efficiency against pathogens of intestinal infections, expressed in the suppression of the growth of test strains in a mixed culture, were experimentally confirmed.

Implementation of the invention.

To obtain fructans from burdock roots, dry burdock roots (medicinal plant material) are washed, crushed to particles 0.5-1 mm in size, mixed with water in a ratio of 1:8-1:12, and extraction is carried out at elevated temperature. Extract I is separated by vacuum filtration, the precipitate is washed with warm water. The precipitate is subjected to repeated extraction under the same conditions, obtaining extract II. The resulting combined extract (I and II) contains ballast substances of protein and phenolic nature. At the same time, the former can become food for pathogenic microorganisms, which will cause an effect opposite to the desired one, and the latter can inhibit the growth of probiotic bifidobacteria. Separation from macromolecular compounds (peptides and polyphenols) is carried out by ultrafiltration through membranes with a retention threshold of 20 kDa. The resulting permeate is treated with activated charcoal to remove low molecular weight impurities (such as oligopeptides, furfural) by passing through a column with an adsorbent. A clear aqueous solution of fructans is obtained.

Fractionation of fructans is carried out by precipitation from water-alcohol solutions containing from 20 to 80% vol. ethanol. For this purpose, the resulting aqueous solutions are evaporated under vacuum at a temperature not exceeding 45°C, which prevents the hydrolysis of fructans, an estimated amount of 95% ethyl alcohol is added, and placed in the cold for 48 hours. The resulting precipitate of fructans is filtered and dried under vacuum. The dry precipitate is crushed on a roller or knife mill to particles with a size of 100-300 microns.

Dried fractions of fructans are mixed with lyophilized biomass of bifidobacteria pre-crushed on a knife mill at the rate of 10 ⁸ to 10 ¹⁰ CFU per 1 g of the resulting synbiotic composition. The finished mixture is packaged in the form of a powder in bags or encapsulated.

The effectiveness of the synbiotic composition was tested by co-cultivating it with test strains of the causative agents of intestinal infections Staphylococcus aureus and Bacillus cereus in a nutrient medium of a suitable composition, in which the only carbohydrate substrate is the corresponding fraction of burdock root fructans. The most pronounced stimulating effect on the growth of bifidobacteria and an inhibitory effect (up to 1.8 times) on test strains (up to 26 times) compared with the control was shown when using fractions of fructans of burdock roots obtained by precipitation with 20% ethanol.

Example 1

Obtaining dried fractions of fructans of burdock roots.

Burdock roots (medicinal plant raw material, FS.2.5.0025.15) in the amount of 70 g were washed with cold tap water until the contaminants were completely removed, dried in air and ground in a knife mill to particles 0.5-1 mm in size. The crushed raw materials were placed in a container and filled with distilled water in a ratio of 1:8-1:12 by dry weight of the roots. Extraction was carried out, for which the container was placed in a water thermostat, a stirrer was placed on top, and extraction was carried out at a temperature of 60 to 90°C for 30 min. The yields of fructans depending on the extraction parameters are presented in Table 1.

The extract was separated from the solid residue by vacuum filtration, the precipitate was washed with warm extractant in an amount of approximately 20% of that taken for extraction. Extract I was obtained. The precipitate was returned to the container and the extraction process was repeated with the same parameters. After filtration and washing, extract II was obtained. Both extracts were combined.

To remove high-molecular impurities, ultrafiltration through a UPM-20 membrane in a tangential flow was used. Concentration was carried out to about 10 times the original volume, obtaining 1.45 l of permeate (filtrate). The resulting filtrate was passed through a column containing activated carbon brand OU-B, in a ratio of 10 g per 1 liter of the purified solution for 3-5 cycles (until a colorless solution was obtained).

The purified solution of fructans was placed in a rotary-film evaporator at the rate of approximately 54 of the flask volume, the vacuum was 0.94-0.98 kgf/cm ² , the temperature was gradually increased from room temperature to maintain an equal rate of water evaporation, but not higher than 45°C . Ethyl alcohol 95% was added to the resulting concentrate based on its final content of 20, 40, 60 or 80% vol. The concentrates were kept at a temperature of 3-6°C for 48 hours, before the formation of a precipitate. The supernatant was decanted, the precipitate was dried in a vacuum oven to a residual moisture content of not more than 5% for 12-15 hours. The yields of fructan fractions from the dry mass of raw materials, depending on the concentration of the precipitant, are given in Table 2

Example 2

Preparation of synbiotic compositions

Lyophilized biomass of bifidobacteria Bifidobacterium bifidum 8 VKPM Ac-2136, obtained by previously known methods, was ground in a knife mill in an inert gas atmosphere (argon, nitrogen). The weights of biomass samples were calculated based on the number of bifidobacteria in 1 g of the finished composition 1⋅10 ⁸ or 1⋅10 ⁹ or 1⋅10 ¹⁰ CFU, which corresponds to approximately from 0.2 to 20% of the mass, and weights of fructans. The compositions were obtained by mixing weighed fractions of fructans with lyophilized biomass in a laboratory mixer for bulk products. The finished mixture was packaged in bags or encapsulated.

Example 3

Verification of the effectiveness of compositions against test strains

Samples of the compositions, the preparation of which is described above, were added at a rate of 1 g per 100 ml to a carbohydrate-free nutrient medium containing casein trypton, yeast extract, meat extract, cysteine, and mineral components and pre-autoclaved at 115°C for 30 min. Incubated for 30 minutes in an atmosphere of 2% carbon dioxide and 98% nitrogen at 37°C and shaking at a frequency of 120 rpm./min for complete dissolution of the components and activation of the culture of bifidobacteria. Next, the inoculum of the test strain was added at the rate of the initial number of 1⋅10 ⁷ CFU/ml. Incubated under the same conditions for 10 hours. The final number of bifidobacteria and microorganisms of the test strain was determined by seeding on selective media. As a control, mixtures of lyophilized biomass of bifidobacteria and commercial fructooligosaccharides were used, in the same proportions as the studied compositions. As can be seen from the results obtained (table 3), the composition effectively inhibits the growth of the test strain, especially when using burdock root fructan fractions obtained by precipitation with 20% ethanol.

Bibliography

1. Wack M., Blaschek W. Determination of the structure and degree of polymerization of fructans from Echinacea purpurea roots. Carbohydr Res. 2006, Vol.341, No 9, p 1147-53. doi: 10.1016/j.carres.2006.03.034.

2 Rossi M. et al. Fermentation of fructooligosaccharides and inulin by bifidobacteria: a comparative study of pure and fecal cultures //Appl. Environ. microbiol. - 2005. - T. 71. - No. 10. - C. 6150-6158.

Claims (5)

1. Synbiotic composition for the suppression of microbial pathogens of intestinal infections, containing bifidobacteria and a fraction of fructans of burdock roots, obtained by grinding burdock roots, water extraction at a temperature of 60 to 90°C for 30 min, separating the extract from the solid residue by filtration, removing high-molecular impurities ultrafiltration, removal of low molecular weight impurities by sorption on activated carbon, concentration of the extract by vacuum evaporation, addition of ethanol to the extract based on its final content of 20% vol., keeping in the cold, separating and drying the precipitate of the resulting fructan fraction, with the following ratio of components in 1 g: lyophilized biomass of bifidobacteria - from 10 ⁸ to 10 ¹⁰ colony-forming units; fraction of fructans of burdock roots from 0.80 to 0.98 g. 2. The composition according to claim 1 is in the form of a powder or capsules. 3. The method of obtaining the composition according to claim 1, which consists in obtaining crushed burdock roots, water extraction at a temperature of 60 to 90 ° C for 30 minutes, separating the extract from the solid residue by filtration, removing high-molecular impurities by ultrafiltration, removing low-molecular impurities by sorption on an activated charcoal, concentration of the extract by vacuum evaporation, addition of ethanol to the extract at the rate of its final content of 20% vol., keeping in the cold, separating and drying the precipitate of the resulting fructan fraction, mixing it with bifidobacteria lyophilisate, packing in bags or encapsulation.