BG2481U1 - A probiotic food supplement - Google Patents

Abstract

The utility model relates to a probiotic food supplement which has an immunomodulatory property, which finds use for prevention of dysbacteriosis with a varying ethnology, improvement of general gut health, and decrease of the irritation processes in the intestinal mucosa and for complex therapy of any illnesses of the gastrointestinal tract. It includes lyophilized active culture Bifidobacterium longum subsp. longum GT15, VKPM AC-1928 with a content of active live cells of 108 up to 109 CFU, arabinosic vitamin D3 and magnesium stearate. An advantage of the probiotic food supplement is the high quantity of active cells of a selective probiotic marker, which has a high survival rate and a high life viability, during the manufacturing process as well as during the storage process.

Description

Field of technology

The utility model refers to a probiotic food supplement with immunomodulatory action, which is used for the prevention of dysbacteriosis of various etiologies, improving the general condition of the intestine, reducing inflammatory processes in the intestinal mucosa and complex therapy of diseases of the gastrointestinal tract.

BACKGROUND OF THE INVENTION

The body of modern man, especially people living in megacities, is negatively affected by various factors related to poor ecology, constant stress, unbalanced diet, use of various drugs and antibiotics, which negatively affects his immune status. This largely determines the desire of physicians to use means to correct the damaged immune system to heal the human body. Promising in this regard are biologically active compositions, including probiotic bacteria with immunomodulatory properties, which combine the beneficial effect on the body's immune system with an inhibitory effect on infectious agents.

Bifidobacteria are known to have a beneficial effect on human health through their antagonism to pathogenic bacteria and the ability to modulate the local and systemic immune response. They are involved in the stabilization and preservation of the intestinal barrier, are able to convert various nutrients into biologically active molecules, regulate intestinal function and maintain microbial homeostasis. (Bermudez-Brito M, Plaza-DHaz J, Mucoz-Quezada S, Gymez-Llorente C, Gil A Probiotic Mechanisms of Action. Ann Nutr Metab. 2012, 61, 160-174; Lyte M. The microbial organ in the gut as a driver of homeostasis and disease.Med Hypotheses. 2010,74 (4), 634-8). Among the known species of bifidobacteria, the representatives of the species Bifidobacterium longum are the most common bacteria among the bifidoflora, both in adults and children (Turroni F., Peano C., Pass D.P. et al. Diversity of Bifidobacteria

UI within the Infant Gut Microbiota PLoS ONE. 2012, 7 (5), 36957). Bifidobacteria are considered safe and are widely used in functional foods, dietary supplements and probiotic medicines.

International application WO 2010055499 describes a group of isolated probiotic bacteria of the species Bifidobacterium longum and the use of their viable cells in combination with a pharmaceutically acceptable carrier in the form of capsules, tablets or powder to improve the intestinal microflora and facilitate gastrointestinal transit.

International application WO 2008141240 discloses probiotic foods and compositions of food supplements with immunological activity. The compositions comprise viable microbial cells of at least one probiotic in combination with at least one natural polysaccharide in the form of capsules, gel capsules, tablets or effervescent tablets. In one embodiment, the probiotic is Bufidobacterium longum.

The task of the utility model is to create a probiotic food supplement with a strong immunomodulatory and anti-inflammatory effect, applicable for the prevention and elimination of dysbiotic disorders of the intestine, conjugated with manifestations of various diseases of the gastrointestinal tract, which should include a selection strain Bifidobacterium longum, having good viability during production and storage, with a high content of active cells with high survival.

Technical essence of the utility model

The problem is solved by a probiotic food supplement with immunomodulatory properties, applicable for the prevention of dysbacteriosis and inflammatory bowel diseases in the form of an intestinal-soluble capsule, including a lyophilized viable culture of Bifidobacterium longum subsp. longum GT15, VKPM AC-1928 with content of active viable cells from 10 ⁸ to 10 ⁹ CFU, the natural polysaccharide arabinogalactan, vitamin D ₃ and magnesium stearate. The components of the probiotic food supplement are in the following quantitative ratios in the composition of the capsule: from 200 to 250 mg

2481 UI

Bifidobacterium longum subsp. longum GT15, 200 to 250 mg arabinogalactan, 10 to 15 microg vitamin D ₃ and 2.5 to 3.5 mg magnesium stearate.

The strain Bifidobacterium longum subsp. longum GT15, VKPM AC-1928 is a probiotic selection strain isolated from the gastrointestinal tract of a healthy adult. The strain is characterized in accordance with biosafety requirements. The strain gene was sequenced using next-generation technology (Roche 454) and the resulting contigs were pooled. The DNA genome of the strain Bifidobacterium longum subsp. longum GT15 is deposited in GenBank under № CP006741. The strain Bifidobacterium longum subsp. longum GT15, VKPM AC-1928 exhibits immunomodulatory activity manifested in the induction of in vitro cell expression of both the anti-inflammatory cytokine 1L-10 and the proinflammatory cytokines IL-8 and FHO-alpha in THP-1.

Arabinogalactan is an indigestible polysaccharide that serves as a nutrient medium for the probiotic Bifidobacterium longum subsp. longum GT15 in the body. Arabinogalactan selectively stimulates the growth of the beneficial intestinal microflora, increases the formation of extremely important for their normal functioning intestinal fatty acids with short chains and exhibits immunomodulatory properties. Arabinogalactan is a prebiotic recognized as promising in medicine, pharmacology and biotechnology, which increases the therapeutic effect when used concomitantly with probiotics containing live lacto- and bifidobacteria.

Vitamin D ₃ (cholecalciferol) regulates specific and nonspecific immunity through various mechanisms. Receptors to hormonally active forms of vitamin D ₃ have been identified in activated proliferating T and B lymphocytes, monocytes and macrophages.

The use of intestinal-soluble capsules allows to exclude the aggressive effect of gastric juice and bile on the components of the developed probiotic composition and increases its effectiveness.

For the preparation of the probiotic food supplement, the probiotic selection strain Bifidobacterium longum subsp. longum GT15, VKPM AC-1928 was cultured on liquid and agar Mann-Rogoza-Sharp medium (MRS) with the addition of 0.05% sterile L-cysteine hydrochloride. Cultivation was performed under anaerobic conditions for 24-48 hours. The incubation temperature is 37 ° C. The probiotic strain Bifidobacterium longum subsp. longum GT15 is grown in the fermenter to a concentration of 10 ⁸ - 10 ⁹ CFU, transferred to a protective drying medium containing 1% gelatin and 10% sucrose, lyophilized and mixed with arabinogalactan, vitamin D ₃ and magnesium stearate. The resulting homogeneous mixture is sterilely packaged in intestinal soluble capsules.

The advantage of the probiotic food supplement is its strong immunomodulatory and anti-inflammatory action, its effectiveness in the prevention and elimination of supra-dysbiotic disorders of the intestine, conjugated with manifestations of various diseases of the gastrointestinal tract, as well as the high content of active cells in selection samples. strain with high survival and good viability, both during production and during storage of the additive.

Examples of implementation of the utility model

The utility model is illustrated by the following embodiments.

Example 1.

For the preparation of the food supplement, the strain Bifidobacterium longum subsp. longum GT15, VKPM AC-1928, was cultured on Mann-Rogoza-Sharp liquid and agar medium (MRS, Himedia, India) to which 0.05% sterile L-cysteine hydrochloride was added. The composition of 1 1 MRS medium in grams is: proteozopepton - 10.0; meat extract - 10.0; yeast extract - 5.0; glucose - 20.0; polysorbate 80 -1.0; ammonium citrate - 2.0; sodium acetate - 5.0; magnesium sulfate - 0.1; manganese sulfate - 0.05; disubstituted potassium phosphate - 2.0. The pH of the medium at 25 ° C is 6.5. Cultivation was performed under anaerobic conditions for 40 h using the HiAnaerobic System-Mark III anaerostat and the Himedia rasnaKerAnaeroHiGas Pack. The incubation temperature is 37 ° C. The concentration of the probiotic strain in the culture medium at the end of the process is 10 ⁹ CFU / ml. The culture is transferred to a protective drying medium containing 1% gelatin and 10%

2481 IU sucrose and lyophilized.

The lyophilized viable selection strain of Bifidobacterium longum subsp. longum

GT 15, VKPM AC-1928 is used for the preparation of a biologically active food supplement with the following composition:

Components of the composition Quantity per capsule Lyophilized viable culture of Bifidobacterium longum subsp. longum GT 15 containing active viable cells 10 ⁹ CFU 250 mg arabinogalactan 250 mg vitamin D ₃ 15 pg / 600 IU / magnesium stearate 3.5 mg

The components of the probiotic food supplement are dosed in a mixer, mixed, 2θ after which the resulting mixture is packaged under sterile conditions in enteric-coated capsules of 500 mg.

The viability of the strain remains unchanged throughout the year. 25

Example 2. Study of the specific activity of a probiotic food supplement

The study was performed on a model of experimental enteropathy induced by 5-fluorouracil in the intestines of Wistar rats. The change in the intestinal microbiota, the morpho-functional characteristics of the intestine and the state of immunity of the animals to which the composition described in Example 1 was applied were studied in comparison with the control group and with groups receiving the preparations used for comparison. ) and Linex (Slovenia). Observed: rapid normalization of the microbiota (disappearance of signs of excessive growth of Klebsiella, Proteus, staphylococci and other opportunistic bacteria and fungi), increase in the content of butyrate produced Faecalibacterium prausnitzii; stimulation of the synthesis of the anti-inflammatory cytokines IL-10 and TCPbeta, tendency to decrease the synthesis of the pro-inflammatory cytokine MCP1; increase in the phagocytic activity of leukocytes; thickening of the layer of mucus secreted on the intestinal microbiota from the surface of the epithelium; increase in the production of basic phosphatase cells of the small intestinal mucosa; rapid compensation after the toxic effect of antimicrobials on biochemical parameters of blood serum. The data obtained from the study of the composition described in Example 1 show that it is not inferior to the preparations used for comparison - Bifiform and Linex.

Example 3. Toxicity study of a probiotic food supplement

The determination of the acute toxicity of the composition described in Example 1 was performed using different doses of it on outbred white mice (10 ⁷ -10 ¹⁰ CFU) and Wistar rats (10 ⁸ -10 ”CFU). The test preparation is administered once in the stomach to all animals. In the maximum 35 doses used, it did not cause animal death or intoxication. Intraperitoneal administration of the probiotic at doses: 10 ¹⁰ CFU for mice (mean weight 20 g) and 10 ¹¹ CFU for rats (mean weight 70 g) also showed no deaths or intoxication. The therapeutic dose of the probiotic for humans at an average weight of 50 kg is 10 ⁸ -10 ¹⁰ CFU. Taking into account the ratio of human and rodent body weight, it is seen that the 45 doses of the preparation obtained in mice and rats exceed the therapeutic doses many times over.

The study of subacute toxicity of the food supplement on mice when administered intragastrically did not lead to significant deviations in the dynamics of weight gain. Inoculation on faeces showed no signs of intestinal dysbiosis in mice. Research4

2481 UI of chronic toxicity to both species of animals (mice and rats) when administered intragastrically did not show significant deviations in the dynamics of body weight accumulation, food and water consumption, haematological and biochemical parameters, morphology of the internal organs of the animals and did not lead to a rise in body temperature.

The obtained results allow the probiotic food supplement based on a strain of Bifidobacterium longum subsp. longum GT15, arabinogalactan and vitamin D ₃ when administered in therapeutic doses, refer to non-toxic drugs.

Example 4. Study of the specific types of toxicity of the probiotic food supplement

The results obtained in the study of immunotoxicity allow us to conclude that in the range of studies at doses (10 ⁷ and 10 ⁸ CFU / mouse) the food supplement does not show immunotoxic effects, but exhibits immunostimulatory (phagocytic) and suppressive (hypersensitivity reaction) of delayed type) action and does not cancel the formation of antibodies, and the initiation of protein antigen.

In a study of mutagenicity, it was found that the probiotic food supplement, both at a dose of 10 ° CFU when administered orally and 24 h exposure, and at a five-fold dose of 10 ⁸ CFU, did not cause a statistically significant increase in the number of cells with chromosomal aberrations. bone marrow of mouse lines 10 CBA. Therefore, the probiotic food supplement does not have mutagenic activity against somatic cells of warm-blooded animals.

When examining the teratogenicity of the probiotic food supplement, from Example 1, on Wistar rats (preparations are administered to pregnant females inside the stomach), the embryo analysis / fetal action did not show an excess of pre- and post-implantation death of the fetal total embryo (feto) lethal effect, compared to control indicators. Also, no fetal macropathology was observed in any of the experimental groups.

Claims (1)

Claims A probiotic food supplement comprising viable cells of a probiotic bacterium of the species Bifidobacterium longum and a natural polysaccharide contained in a capsule, characterized in that the food supplement also includes vitamin D ₃ and magnesium stearate, the probiotic bacterium being Bifidobac subsp. longum GT15, VKPM AC-1928 in lyophilized form with content of active viable cells from 10 ⁸ to 10 ⁹ CFU, the natural polysaccharide is arabinogalactan, and the capsule is an intestinal-soluble capsule, the components are in the following quantitative ratios: from 200 to 250 mg Bifidobacterium longum subsp. longum GT15, 200 to 250 mg arabinogalactan, 10 to 15 microg vitamin D ₃ and 2.5 to 3.5 mg magnesium stearate.