Method for increasing zinc levels in rats

Abstract

FIELD: pharmaceuticals; biotechnology.SUBSTANCE: method for increasing the zinc content in the body of rats, comprising orally administering a suspension of a chelated zinc metal complex in a volume of 1 ml per head, characterized in that the chelated zinc metal complex is obtained from the destructured biomass of the Bacillus subtilis VKPM B-10641 or Bacillus amyloliquefaciens VKPM B-10643 strain, cultured on an agar substrate with the addition of ZnSO4 at a concentration of 0.031 M, followed by destruction of bacterial cells by double freezing and thawing, filtration through membrane filters with a pore size of 0.45 μm and centrifugation at 10,000 rpm for 30 minutes, the particles precipitated by centrifugation are diluted with distilled water to an optical density of 0.1 at 625 nm to obtain suspensions, and the administration is carried out on an empty stomach once a day for 7 days with an interval of 24 hours.EFFECT: increasing the concentration of zinc ions in the body of rats using chelated zinc metal complexes obtained by bacterial synthesis.1 cl, 1 dwg, 1 ex

Description

[0001] The invention relates to biotechnology and can be used in biological and medical laboratories that address issues of using probiotic preparations in the system of delivering essential elements to the body of animals.

[0002] A method is known for increasing the iron content in the body of animals by introducing the biomass of inactivated microorganisms included in the probiotics Vetom 1.1 Bacillus subtilis 10641 and Bactisubtil Bacillus cereus IP 5832 after their cultivation in a liquid nutrient medium with the addition of FeSO4 at a concentration of 0.05 M / ml, which will ultimately increase the iron level in the body of laboratory animals. (RU 2 676 687 C1).

[0003] The disadvantage is that the method is only applicable to increasing the iron content in the body of animals.

[0004] The applicant's analysis of sources of information on a technical solution identical to the present invention revealed no results. This allows us to conclude that it meets the "novelty" criterion.

[0005] The technical result of the invention is achieved by increasing the concentration of zinc ions in the body of animals by using chelate zinc metal complexes obtained by bacterial synthesis by bacterial strains included in the probiotic preparations Vetom 1.1 - Bacillus subtilis VKPM B-10641 and Vetom 4 - Bacillus amyloliquefaciens VKPM B-10643 (DSM 24615) pre-cultivated on agar substrates with the addition of maximum non-inhibitory concentrations of ZnSO4 at a concentration of 0.031 M corresponding to a zinc cation content of 2043 mg, which made it possible to increase the level of zinc ions in animal tissues under conditions of polyvalent mineral deficiency.

[0006] The solution of the set problem is achieved by creating a mineral-deficient state of animals caused by keeping them on a diet including boiled, three times washed rice and distilled water for 30 days before the experiment, followed by individual nasal administration of 1.0 ml of chelated zinc metal complexes isolated from the destructured biomass of pure cultures of bacterial cells Bacillus subtilis VKPM B-10641 (DSM 24613) and Bacillus amyloliquefaciens VKPM B-10643 (DSM 24615) per head. Cultivation of strains was carried out on dense nutrient media - GRM-agar with the addition of ZnSO4 at a concentration of 0.031 mmol / ml. The course of administration of chelated metal complexes was 7 days with an interval of administration of 24 hours, on an empty stomach.The destruction of the structure of bacterial cells washed from agar substrates with sterile distilled water was carried out by double freezing and thawing in a low-temperature refrigerator at -70°C for 8 hours with intermediate thawing in a thermostat at a temperature of 37°C. At the end of this stage, the biomass was kept in an ultrasonic washer for 6 hours at a temperature of 70°C, followed by sedimentation of the biomass in a high-speed centrifuge at 10,000 rpm for 30 minutes. The supernatant was removed using an automatic pipette, sterile distilled water was added to the sediment, the contents were carefully pipetted and filtered through polytetrafluoroethylene membrane filters with a pore size of 0.45 μm to remove ballast proteins and spores of the microorganisms used.After filtration, the contents were centrifuged again at 10,000 rpm for 30 minutes. The supernatant was removed using an automatic pipette. Sterile distilled water was added to the sediment to achieve an OD value of 0.1 at a spectrophotometer wavelength of 625 nm. The resulting suspension was used as a dosage form for the correction of zinc deficiency conditions.

[0007] The cumulative properties of heavy metals in Bacillus spp. bacteria are determined by their high physiological and adaptive mechanisms associated with the chelation of excess concentrations of essential elements, followed by their biotransformation into biologically active forms. These characteristics of Bacillus spp. are widely used in various fields of human activity aimed at the bioremediation of environmental objects.

[0008] Vetom 1.1 - Bacillus subtilis VKPM B10641. 1 capsule contains: corn extract - 133 mg; potato starch - 100 mg; powdered sugar - 67 mg; dry culture of probiotic microorganisms Bacillus subtilis strain VKPM B-10641 (DSM 24613), 1x109 CFU / g - 30 mg. The healing effect is provided by the properties of Bacillus subtilis bacteria, which, multiplying mainly in the large intestine, secrete biologically active substances that suppress the growth and development of pathogenic and opportunistic microflora. As a result of this process, the microbial composition of the intestine is transformed to correspond to the evolutionarily established norm, its walls are cleared of indigestible food residues, which promotes the active removal of toxins from the body, unhindered delivery of biologically active substances and nutrients. Under the influence of Vetom 1.1, metabolism, digestion and absorption of biologically active food components in the gastrointestinal tract are normalized.Manufacturer: Research and Production Company Research Center, Novosibirsk Region, Koltsovo village (Russia).

[0009] Vetom 4 - 1 capsule contains: corn extract - 2000 mg; potato starch - 1500 mg; sucrose (powdered sugar) - 1000 mg; dry culture of probiotic microorganisms Bacillus amyloliquefaciens VKPM B-10643 (DSM 24615), 1×108 CFU / g - 500 mg. Bacillus amyloliquefaciens is highly resistant to digestive juices and enzymes of the gastrointestinal tract and has the ability to quickly colonize it. In the animal intestine, bacterial spores transform into vegetative forms and secrete antibiotic-like substances, enzymes, and other biologically active substances. These substances normalize intestinal biocenosis, pH, digestion, absorption, and metabolism of iron, calcium, fats, proteins, carbohydrates, triglycerides, amino acids, dipeptides, sugars, and bile salts. Manufacturer: Research Center, LLC, Novosibirsk Region, Koltsovo, Russia.

[0010] The method is implemented as follows:

[0011] Eighty mature Wistar rats were divided into four experimental groups (males in sexual maturity weighing 220.0 ± 10.0 g): two control groups, and two experimental groups. The intact group (K0) was fed a complete diet ensuring growth and development. The zinc-deficient control group (K1) and the experimental groups were switched to a diet containing boiled, three-times-washed rice and distilled water 30 days before the experiment, and this diet was maintained throughout the experiment. In the experimental groups, chelated zinc metal complexes based on the destructured biomass of pure bacterial cell cultures Bacillus subtilis VKPM B-10641 (DSM 24613) - group O1 and Bacillus amyloliquefaciens VKPM B-10643 (DSM 24615) - group O2 were used as preparations correcting mineral deficiency.The preparation of the preparations was carried out as follows: bacterial strains were cultivated on GRM agar with the addition of ZnSO4 at a concentration of 0.031 mmol / ml. The destruction of bacterial cells, washed from the agar substrates with sterile distilled water, was carried out by double freezing and thawing in a low-temperature refrigerator at -70°C for 8 hours with intermediate thawing in a thermostat at a temperature of 37°C. After this stage, the biomass was kept in an ultrasonic washer for 6 hours at a temperature of 70°C, followed by sedimentation of the biomass in a high-speed centrifuge at 10,000 rpm for 30 minutes, the supernatant was removed with an automatic pipette, sterile distilled water was added to the sediment in a ratio of 1 g.The sediment was added to 10 ml of distilled water, the contents were carefully pipetted and filtered through polytetrafluoroethylene membrane filters with a pore size of 0.45 μm to remove ballast proteins and spores of the used microorganisms. After filtration, the contents were centrifuged again at 10,000 rpm for 30 minutes, the supernatant was removed using an automatic pipette, and sterile distilled water was added to the sediment to an OD value of 0.1 at a spectrophotometer wavelength of 625 nm. The resulting suspension was used as a dosage form for the correction of zinc deficiency. In the first experimental group, a chelated zinc metal complex based on Bacillus subtilis VKPM B-10641 was used, in the second - based on Bacillus amyloliquefaciens VKPM B-10643, respectively. The suspended complexes were administered to animals per os individually on an empty stomach in a volume of 1 ml per head; the course of therapy was 7 days with an administration interval of 24 hours.

[0012] Experimental studies on animals were carried out in accordance with the instructions recommended by the Russian Regulations, 1987 and “The Guide for the Care and Use of Laboratory Animals (National Academy Press Washington, DC 1996)”. The experimental animals were kept under identical conditions.

[0013] Biological samples from Wistar laboratory rats were analyzed immediately before administration of synthesized zinc chelate metal complexes (baseline study), and after administration of zinc chelate metal complexes (day 7), as well as days 10 and 14, to assess tissue zinc concentrations under conditions of an ongoing mineral-deficient diet. The ability of Bacillus spp. to deposit zinc ions from substrates with an increased cationic load, followed by their transformation into biologically active forms, ensuring high rates of zinc absorption by the animals, was analyzed. Biological samples, including skin, muscle, and bone tissue, were analyzed at each stage of the study. Samples were collected from five animals in each study group.The data obtained were statistically processed at each stage of the study to establish differences and the reliability of the results (Student's t-test). Zinc content in the biological samples is presented in Fig. 1.

[0014] Analysis of the studied rat biosubstrates was performed in the testing laboratory of the Federal Scientific Center of Bioorganic Structural Sciences, Russian Academy of Sciences, Orenburg. Sample preparation was carried out in accordance with recommendations 4.1.1482-03 and 4.1.1483-03, using microwave digestion on a Multiwave 3000 instrument (A. Paar).

[0015] The results of the conducted studies aimed at assessing the effectiveness of the use of chelated zinc metal complexes obtained on the basis of probiotic strains of the genus Bacillus spp. with high resistance rates to chemical compounds of zinc, with a high level of dissociation in aqueous solutions allowing for the creation of a high level of cationic load on the substrate, as a means of treating zinc mineral deficiency states, indicate a high level of their biological activity when introduced into the body of animals.Thus, upon completion of the course of introducing suspended zinc metal complexes into the animals’ bodies, the indicators of the experimental groups exceeded the indicators of the control group of the zinc mineral deficiency state by 33.98% when using Bacillus subtilis VKPM B-10641 as a reducing agent and by 35.24% (p<0.05 in muscle tissue and skin and p<0.01 in bone tissue) when using Bacillus amyloliquefaciens VKPM B-10643 (p<0.05 in muscle tissue and skin and p<0.001 in bone tissue).

[0016] The dynamic indices of zinc distribution and accumulation 3 days after the end of zinc replacement therapy indicate the presence of positive dynamics of zinc cumulation in the studied biological samples (Figure 1) with the excess of the control values ​​of the zinc mineral deficiency group by 41.74% in the first experimental group and 45.97% in the second experimental group, while it should be noted that the use of a chelate zinc metal complex synthesized using Bacillus amyloliquefaciens VKPM B-10643 provided an increase not only in relation to the control of the deficiency state but also in the intact group by 4.84%. The dynamics of zinc distribution in the studied samples maintains the general trend of distribution in tissues with maximum accumulation in the skin (p <0.01) and muscle tissue (p <0.01). In the control groups, no significant changes in the concentration of zinc ions were recorded throughout the study.

[0017] 7 days after the end of the course of replacement therapy using chelated metal complexes based on the probiotic strains Bacillus subtilis VKPM B-10641 and Bacillus amyloliquefaciens VKPM B-10643 against the background of a continued mineral-deficient diet, a slight decrease in zinc concentration was recorded in all studied samples with a total decrease in concentration in relation to day 10 of the study by 8.70% in the first experimental group and 8.87% in the second group. It should be noted that the maximum bioavailability of zinc is recorded when using a chelated zinc metal complex obtained on the basis of Bacillus amyloliquefaciens VKPM B-10643 with a reliably significant increase in zinc at the final stage of the study by 43.59% (p<0.05) in the skin, 44.18% (p<0.01) in muscle tissue and 38.71% (p<0.01) in bone tissue in relation to the control group of zinc mineral deficiency.

Claims

A method for increasing the zinc content in the body of rats, comprising orally administering a suspension of a chelated zinc metal complex in a volume of 1 ml per head, characterized in that the chelated zinc metal complex is obtained from the destructured biomass of the Bacillus subtilis VKPM B-10641 or Bacillus amyloliquefaciens VKPM B-10643 strain, cultured on an agar substrate with the addition of ZnSO4 at a concentration of 0.031 M, followed by destruction of bacterial cells by double freezing and thawing, filtration through membrane filters with a pore size of 0.45 μm and centrifugation at 10,000 rpm for 30 minutes, the particles precipitated by centrifugation are diluted with distilled water to an optical density of 0.1 at 625 nm to obtain a suspension, and the administration is carried out on an empty stomach once a day for 7 days with an interval of 24 hours.

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