RU2222997C1 - Biologically active food additive - Google Patents

Abstract

FIELD: food industry. SUBSTANCE: additive contains nucleoprotein complex (Tinrostim) isolated from soft roe of salmon fish and containing low-molecular RNA with molecular weight 100 to 300 kDa in amounts 75-80% with specified proportions of components. Following specific components are further included: ascorbic acid, microcrystalline cellulose and/or starch, and also tableting or encapsulation promoting ingredients. EFFECT: enlarged assortment of biologically active additives possessing general healing, immunostimulating, and antistress effect as well as capacity of activating mental and muscular performances. 3 cl, 2 tbl, 7 ex

Description

 The invention relates to the food industry and can be used as a dietary supplement for food. When using the proposed supplement, the immune system is activated, increasing mental and physical performance, stress resistance.

Known nutritional dietary supplement that enhances the resistance of the human body (US Patent 5292538, IPC 5 A 23 L 1/305, publ. 08.03.94). The additive is a mixture of substances and contains 25-100 parts of polymer glucose, 5-25 parts of a source of hydrolyzed protein, consisting of dipeptides and peptides with a longer chain, 0-20 parts of a lipid source, 50-500 (• 10 ³ ) parts of Mg, 0.03-6 parts of the source of amino acid ligands. The source of hydrolyzed protein is a partially hydrolyzed lactoalbumin. Anabolic substances such as vitamins, antioxidants and lipotropic substances are included in the nutritional supplement in order to give it optimal energy value. A distinctive feature of this supplement is that the source of peptides is milk protein.

 Known food supplement (French Patent 2676648, IPC 5 A 61 K 35/407), which includes extracts of the heart and liver of duck Barbari., Which is used for the prevention and treatment of infectious diseases.

 A known composition for promoting performance during sports, the method of its manufacture and use as a dietary supplement (German Patent 19903560, IPC A 23 L 1/29). The proposed composition for promoting performance during sports contains MCT, carbohydrates, alanine, lysine, phenylalanine, glutamic acid, methionine, serine, glycine, ornithine and taurine. A method for the manufacture and use of this composition as a dietary supplement is proposed.

 Known drug from the tissues of marine hydrobionts "Orbitar" (RF patent 2053779, IPC 6 A 61 K 35/56) with a nitrogen content of amino groups of 0.8-1.0% of the total mass, with a ratio of free amino acids to peptide groups of 6: 7, mass solids 25-35 wt.% and pH 4.5-6.0, used to increase human resistance to adverse factors.

 Known therapeutic and preventive balm "Vitagen" (TU 9169-010-20785204-94), containing 37 components of plant and animal origin, including ginseng, eleutherococcus, preparations from kelp, salmon milk, mummy. This tool is used in the rehabilitation of patients after various diseases, a decrease in the functions of the central nervous system, and a decrease in muscle and mental performance.

A biologically active additive is known (RF Patent 2164765, IPC7 A 23 L 1/30, A 23 D 9/00, A 23 L 1/24, A 61 K 9/00) containing an immunobiological preparation (biomass of non-pathogenic microorganisms in an amount of 10 ³ -10 ⁹ CFU and / or autolysate of microorganisms in an amount of 0.1-20.0 wt.%, And / or lysozyme in an amount of 0.1-20.0 wt.%), As well as target additives in an amount of 0.5- 90.0 wt. % The tread contains the following ratio of its components, wt.%: Ascorbic acid - 1.0-94.0; and / or lactose 5.0-98.0; and / or glucose 1.0-10.0; and / or sugar 1.0-20.0; and / or gelatin 0.5-3.0; and / or polyglykin 1.0-94.0; and / or albumin 1.0-94.0; and / or immunoglobulins 3.0-98.0. A dry tread, and / or dry target additives, and / or a dry immunobiological preparation, and / or a dry mixture of immunobiological preparations, the dietary supplement contains in a crushed state with a particle size of not more than 250 microns. Also, as targeted additives, dietary supplements can contain components for tabletting or encapsulation and can be presented in the form of tablets or capsules.

 Known biologically active food supplement (RF Patent 2165719, IPC A 23 L 1/30, 1/39, 1/06), containing soluble dietary fiber, biologically active substances of marine and vegetable origin, citric acid, potassium sorbate, sugar, water , and additionally contains an amino acid - taurine in an amount of not less than 0.03%, while astragalus densely colored or woolly, arnica mountain (inflorescences), frangipani (rhizomes), and hawthorn red (fruits) are contained as a source of biologically active substances of plant origin be drooping droplets (buds), medicinal valerian (roots and rhizomes), dried grapes (fruits), hibiscus (flower tea petals), St. John's wort perforated (grass), wild strawberries (leaves), common viburnum (fruits), meadowsweet (flowers) , woodpecker penny (aerial part), dye boiling (rhizomes and roots), medicinal lemon balm (underground part), peppermint (leaves), dodging peony (roots and rhizomes), motherwort (grass), licorice (roots and rhizomes) , blackcurrant (leaves), common hop (fruit) or "female cones", Chinese (green) tea, and as a source of biologically active substances of marine origin - scallop food hydrolyzate, squid food hydrolyzate, salmon fish milk food hydrolyzate, mussel food hydrolyzate, crab food hydrolyzate, sea urchins, ice cream, caviar sea urchins are ravine, as soluble fiber - zosterol, with a certain ratio of ingredients. Biologically active food additives have a directed effect on the cardiovascular and nervous systems of the body, and also strengthens capillaries and artery walls, normalizes pressure and weight.

 Known biologically active food supplement "Russian Island" (RF Patent 2130738, IPC 6 A 23 L 1/30, 1/29, A 61 K 35/78, C 12 G 3/100, A 23 L 2/52) containing potassium, iron and ascorbic acid, additionally contains carotene, amino acids and flavonoids, and the ratio of carotene, amino acids, flavonoids is (0.02-3.0) :( 0.05-3.8) :( 1.01-6, 25). Dietary supplement can be used in soft drinks, balms, tinctures, etc. This allows you to get a biologically active supplement with antidepressant, tonic, antioxidant, anti-inflammatory, immunocorrective and general strengthening properties, increase mental and physical performance, has mental and physical performance, has a beneficial effect on the gastrointestinal tract due to the optimal ratio of components due to synergism of action components, and also this will expand the range of well-known biologically active additives .

 Known biologically active food supplement (RF Patent 2165161, IPC7 A 23 L 1/30, 1/39, 1/06), which consists of a mixture of organic food substances (in the following ratio of components, wt.%) Amino acids: flavonoids: carotenoids : polyunsaturated fatty acids are (8.34-21.45): (0.14-8.34) :( 0.001-0.53) :( 0.009-1.54) :( 0.0001-0.009). The specified mixture of components allows you to get a biologically active additive with antioxidant, anti-inflammatory, immunocorrective and general strengthening properties, have a beneficial effect on the gastrointestinal tract, liver and kidney function, support the central nervous system and cardiovascular system, regulate lipid peroxidation in the body for due to the optimal ratio of components due to the synergistic action of the components.

 Known food product (RF Patent 2061389, IPC6 A 23 L 1/08), containing, wt.%: Honey 92-94, mummy 1-2, water 3-4, oil extract of California worms. This product is recommended as a prophylactic for various diseases, because it normalizes cholesterol metabolism and completely covers an insufficient amount of proteins in the body.

 Known natural multivitamin complex "Astenoton" (RF Patent 2149566, IPC 7 A 23 L 1/076, 1/30, 1/302, A 61 K 35/64). The invention relates to the food industry. The natural multivitamin complex "Astenoton" contains, wt.%: Flower pollen 48.0-52.0, pink rhodiola 8.0-12.0, vitamin C 6.0-7.0, vitamin E 1.0-2, 0, propolis 1.0-3.0, calcium stearate 0.9-1.0, talc 2.7-3.2, the rest is lactose. The combination of components provides enhanced adaptogenic and antioxidant properties.

A wide range of therapeutic and prophylactic agents is known (RF patent 2083218, MKI ⁶ A 61 K 35/78, 31/66), which includes pollen and DNA at a ratio of 60-95: 5-40.

The closest technical solution is a biologically active food supplement "Tinrostim-S" (RF Patent 2105504, IPC6 A 23 L 1/305, A 23 L 1/29, A 61 K 37/02,) which includes,% :
Gangliin (trade name Tinrostim) - 0.2-0.4
Ascorbic acid - 9-11
Glucose - Else
Gangliin - peptides from squid ganglia, is a high activity immunostimulant, the technology for which was developed at the TINRO Center.

 Tinrostim-C is recommended to be taken as a general strengthening and immunostimulating biologically active food supplement of 550 mg per day for 3-10 days.

 The problem solved by the invention, the expansion of the range of dietary supplements with tonic, immunostimulating, anti-stress effect and the ability to activate mental and physical performance.

The essence of the invention lies in the fact that the inventive biologically active additive contains in its composition, wt.%:
Tinrostim - 0.1-0.3
Nucleoprotein Complex Isolated from Salmon Milk - Rest
Targeted additives may also be included in the dietary supplement: ascorbic acid; microcrystalline cellulose (MCC) and / or starch in an amount of 10-35 wt.%. As target additives, the biologically active additive may also contain components for tabletting or encapsulation in an amount of 0.5-1.5 wt.%.

 The inventive biologically active food supplement is obtained by simple mixing of the components.

 The novelty of the invention lies in the joint previously unknown use of two biologically active food additives Tinrostim and Nucleoprotein complex isolated from salmon milk.

 The nucleoprotein complex of salmon milk contains 75-80% of low molecular weight DNA with a molecular weight of 100 to 300 kDa, 15-20% RNA and 5% protein.

 Tinrostim is a complex of low molecular weight peptides isolated from squid nervous tissue with a molecular weight of 1 to 15 kDa. With the combined use of Tinrostim and the Nucleoprotein complex from salmon milk, in the indicated amounts, a synergy of action is manifested, expressed in a decrease in the effective dose of the components by 15-20% than with the separate use of Tinrostim and the Nucleoprotein complex from salmon milk with the same biological effect.

 The inventive dietary supplement exhibits a wider spectrum of biological action than the Nucleoprotein complex of salmon milk and Tinrostim, used separately. The inventive dietary supplement exhibits a general strengthening effect, high immunostimulating activity, increases stress resistance, mental and physical performance, improves memory processes.

 A dietary supplement in the form of powders, tablets or capsules of 250 or 500 mg is used prophylactically in the amount of 500 mg 1-2 times a day for 3-15 days at the same time as food.

 The invention is illustrated by examples.

Example 1
The components are mixed in the following ratio, wt.%:
Tinrostim - 0.1
Nucleoprotein complex isolated from salmon milk - 99.9
The ingredients are mixed in a mixer, ensuring a uniform distribution of components, and packaged in powders of 500 mg. The dietary supplement is a light cream powder, reminiscent of dried seafood in taste. It is recommended to use one powder 1-2 times a day at a time with food.

 The inventive biologically active food supplement has been investigated.

 The mass fraction of deoxyribonucleic acid in the nucleoprotein complex of salmon milk was determined on a spectrophotometer according to the Spirin method (Spirin A.S. Spectrophotometric determination of the total number of nucleic acids. // Biochemistry, 1958. - T.23, 5, - p. 566-662) . The method is based on the extraction and standard hydrolysis of low molecular weight DNA with 0.5 M perchloric acid to soluble fragments and the measurement of optical density at a wavelength of 270 and 290 nm. Mass fraction of DNA is determined by the difference in optical densities. The content of low molecular weight DNA from salmon fish milk in the powder was 75.8%.

 The impact of the proposed application on stress resistance, mental and physical performance and memory processes was determined at the Institute of Physiology. I.P. Pavlova RAS. Studies were performed on monozygotic twins (5 pairs) and a group of children with mental retardation (moderate debility).

The following indicators were evaluated:
Bioelectric activity of the brain - EEG, 16 leads with subsequent computer processing of data according to a specially developed program;
The current psychological state of the subjects, their stress resistance, the presence of anxiety, etc. - Lusher test and SAN test of well-being, activity, mood by Doskin V.A., Lavrentieva N.A., Miroshennikov M.P., Shary V.B. Test differentiated self-assessment of the functional state. // Questions of psychology, 1973, 6, - p.141-145;
The level of neurotization and psychopathization (UNP test) - according to methodological recommendations. L., 1980, Leningrad Research Institute. V.M. Bekhterev, min. health USSR;
The volume of short-term memory, the process of memorization, retention - according to Woodwards F. “Experimental Psychology”, M .: I.L., 1950;
Physical Performance Index (Harvard Step Test) by - Boykov Yu.G. and other Pharmacological correction of fatigue. M .: Medicine, 1984;
Intelligence Level (WISC Test) by Agafonov, Kolechenko. Methods of studying intelligence. Part 1. Guidelines. Publishing House of the Teacher Improvement Institute, St. Petersburg, 1991, pp. 4-106.

 The above studies of the claimed dietary supplements found that introduced into the composition of the food supplement Nucleoprotein complex, the main component of which is low molecular weight DNA, increases physical performance by 34%, reduces the level of neurotization, anxiety, asthenic symptoms of emotional lability by 20%, improves selective attention by 25% . The analysis of bioelectric activity showed that the basis of the observed mental improvements is the optimization of the cortical-subcortical relationships of the central nervous system, which was expressed in an increase in the alpha index and a decrease in beta activity and slow waves. The data obtained allow us to conclude that the stimulating effect of low molecular weight DNA from salmon milk on mental and physical performance is based not on the depletion of body reserves, but on the optimization of the interaction of the functional systems of the body.

The immunostimulating effect of the drug was determined at the Research Institute of Epidemiology and Microbiology SB RAMS of Vladivostok and was evaluated in the following areas:
I. Determination of the effect of the claimed additives on the humoral immune response of immunodeficient mice in tests: the effect on the level of antibodies in serum; the number of antibody-forming (AOK) cells in the spleens of mice immunized with sheep erythrocytes (EB); and the number of immune rosette-forming cells (ROS) in the spleen of mice. Immunodeficiency in mice was caused by intraperitoneal administration of cyclophosphamide at a dose of 100 mg per 1 kg of animal weight.

Studies to determine the level of antibodies and the number of AOK were carried out according to the method of WK Yerne and A.A. Nordin (1963). Sheep erythrocytes were used as a thymus-dependent antigen, which, entering the body, are quickly fixed by phagocytic cells, processed into highly immunogenic products, and then the latter are expressed on the cell membrane of macrophages. The immune response of B-lymphocytes to this antigen implies mandatory participation in cooperative processes of T-helpers and (or) substances secreted by them. The level of antibody formation was determined in a hemagglutination reaction (RGA). For the tests, 4 groups of mice were created - two control (1st — healthy mice that did not receive dietary supplements; 2nd — mice with immunodeficiency that did not receive dietary supplements; 3rd group — healthy mice that received dietary supplements; 4th group — immunodeficient mice treated with dietary supplements). 10 mice were taken in each group, experiments were repeated twice on mice (males) weighing 16-18 g. The dietary supplement was fed to mice of the experimental group along with food at a dose of 0.5 μg per day per mouse for 3 days. Sheep red blood cells were administered intraperitoneally at a dose of 2 • 10 ⁸ . On the fourth day after immunization, the number of antibody-forming cells (AOK) and the hemagglutinin titer were determined.

 Studies of the influence of dietary supplements on the number of ROCKs were performed according to the method of J.S. Haskill et.al. (1972).

 The results of the study showed a statistically significant increase in the humoral immune response: hemagglutinin titer, the number of AOKs and the number of ROCKs in the spleens of mice compared to the control (immunodeficient mice). The results of these experiments are presented in table 1.

 As follows from the table, after the introduction of cyclophosphamide, the immunological parameters of mice decreased by half (group 2). Adding the inventive dietary supplement to the food of immunodeficient mice deduces the mice from the state of immunodeficiency, their indices increased significantly (group 4) and approached those of healthy mice that did not take dietary supplements (group 1). In healthy mice that took dietary supplements, the humoral factor was highest.

 II. The effect of the claimed dietary supplement on cellular factors of immunity.

The experiments were performed on CBA mice according to the method of R.N. Lagrange et.al. (1974) - a change in the level of delayed-type hypersensitivity (HRT). HRT was induced in mice by administering a sensitizing dose of EB (5 x 10 ⁸ in 0.5 ml of a 0.85% sodium chloride solution intraperitoneally). On the fourth day, a resolving dose of EB (1 × 10 ⁸ in 20 μl of a 0.85% sodium chloride solution) was injected into the paw pad of mice. A foot was served as a control, into which an equal volume (20 μl) of a 0.85% sodium chloride solution was introduced. The dietary supplement was fed to mice daily during the experiment and one day before the experiment at a dose of 0.25 mg per mouse. 24 hours after the introduction of the permissive dose, the mice were euthanized, the legs were cut off along the protrusion of the bone below the junction of the tibia and above the heel joint and limbs were weighed on an analytical balance. The percentage of weight gain of the paw, characterizing the intensity of the HRT reaction, was determined by the formula

where X is the% increase in paw weight.

 III. The influence of dietary supplements on the phagocytic activity of macrophages of the abdominal cavity of mice.

 It has been established that the main point of application of tinrostim (gangliin) are cells of the mononuclear phagocyte system. Of particular importance is phagocytosis in infectious processes caused by facultative intracellular microorganisms (salmonellosis, pseudotuberculosis, etc.). In these diseases, pathogens can persist for a long time in the cytoplasm of macrophages, remaining unattainable for humoral immunity factors and drugs. As a rule, phagocytosis in this case is incomplete. There is evidence of the leading role of phagocytosis in protecting the body against staphylococcal infection, the causative agents of which are phagocytized mainly by polymorphonuclear leukocytes (PMNL). Therefore, for the complete elimination of such pathogens from the body, it is necessary to significantly activate the absorption and digestion function of macrophages.

The study of phagocytic processes in macrophages of the peritoneal cavity of non-inbred mice was carried out according to the generally accepted method (Braude NI, 1964). To form a monolayer, macrophages were cultured in test tubes with floating glasses (2 × 10 ⁶ cells per 1 ml) in medium 199 with the addition of 20% serum of cattle and antibiotics (100 IU / ml penicillin and 100 IU / ml streptomycin) for 1 hour at 37 ^o C. Observation of the culture of macrophages was carried out for 6 hours. Glasses with a monolayer of cells were stained with azure P-eosin. 300 cells were counted in each preparation: they gave a qualitative description of the state of macrophages and a quantitative assessment determining the phagocytic index in% (AF) —the number of phagocytic cells from 100 and phagocytic numbers (PS) —the average number of microbes absorbed by one macrophage. Before taking macrophages, experimental groups of mice were fed the claimed dietary supplement at a dose of 0.25 mg per day for 3 days. Y. pseudotuberculosis and E. coli were taken as objects of phagocytosis. The materials of this series of experiments are presented in table 2.

 As follows from the table, feeding mice of the claimed BAS stimulated the absorption and digestion activity of macrophages in relation to microorganisms. This is expressed in the involvement in the phagocytic process, already in the first hours of observation, of a greater number of phagocytic cells compared to macrophages of animals that did not receive dietary supplements and in an increase in the number of microorganisms absorbed by one macrophage. In addition, under the action of biologically active substances, a more complete and earlier completion of the phagocytic process in macrophages is noted.

 IV. The influence of dietary supplements on the stimulation of nonspecific resistance to experimental infections.

 One of the main criteria for the biological activity of immunity stimulants is the total effect, consisting of the action of numerous factors that protect the body from pathogens, which is expressed in general in the body's ability to withstand the development of the infectious process. It is shown that tinrostim induces a multicomponent protective reaction in the body, thereby providing increased resistance to infectious processes. In a model of generalized infection that develops as a result of infection with a large dose of E. coli (million microbial cells), the ability of the proposed dietary supplement to increase the level of immunobiological protection under these conditions was studied.

 E. coli was administered intraperitoneally to mice. Mice in the control group that did not receive dietary supplements died within 3 days. The survival of the experimental group of mice receiving dietary supplements at a dose of 0.5 μg per mouse for five days was 79.7%. In the control group, the death of mice was 100%.

 Thus, studies have found that the claimed dietary supplement has a wide range of biological activity.

Example 2
The components are mixed in the following ratio, wt.%:
Tinrostim - 0.3
Nucleoprotein complex isolated from salmon milk - 99.7
The ingredients are mixed in a mixer, ensuring a uniform distribution of components, and Packed in 250 mg in capsules. It is recommended to take two capsules 1-2 times a day. The biological effect of the food additive obtained in accordance with the recipe specified in example 2 corresponds to the action of the additive obtained according to the recipe specified in example 1.

Example 3
The components are mixed in the following ratio, wt.%:
Tinrostim - 0.2
Nucleoprotein complex isolated from salmon milk - 99.8
The ingredients were mixed in a mixer, ensuring a uniform distribution of components, and dosed in powders of 500 mg. It is recommended to use one powder 1-2 times a day. The dietary supplement is a light cream powder with the taste of dried seafood. The biological effect of the obtained additives corresponds to the action of dietary supplements specified in example 1.

Example 4
The components are mixed in the following ratio, wt.%:
Tinrostim - 0.1
Ascorbic acid - 5
Starch - 20
Nucleoprotein complex isolated from salmon milk - 74.9
The ingredients are mixed in a mixer, ensuring a uniform distribution of components, and Packed in powders of 500 mg. It is recommended to use one powder 1-2 times a day. The dietary supplement is a light cream powder. The taste resembles dried seafood, sour. The biological effect of the obtained additive is close to the action indicated for the additive obtained according to the recipe specified in example 1.

Example 5
The components are mixed in the following ratio, wt.%:
Tinrostim - 0.3
Ascorbic acid - 7
Starch - 15
Calcium Stearate - 1
Nucleoprotein complex isolated from salmon milk - 76.7
The ingredients are mixed in a mixer, ensuring a uniform distribution of components, and tableted. The tablet is strong, whole with a smooth surface, the disintegration time of the tablet is 10 minutes. The color of the tablet is light cream, the taste is slightly acidic with a touch of dry seafood. The weight of the tablet is 250 mg. It is recommended to take 2 tablets 1-2 times a day. The biological effect of the tablets of dietary supplements specified in example 5, corresponds to the action of the additives obtained by the formulation specified in example 1
Example 6
The components are mixed in the following ratio, wt.%:
Tinrostim - 0.2
Ascorbic acid - 10.0
MCC - 25
Calcium Stearate - 1
Nucleoprotein complex isolated from salmon milk - 63.8
The ingredients are mixed in a mixer, ensuring a uniform distribution of the components and tableted. The tablet is a light cream color, durable, whole. The taste is sour with a touch of seafood. Disintegration time of the tablet is 8 minutes. The weight of the tablet is 250 mg. It is recommended to take 2 tablets 1-2 times a day. The biological effect of the tablets corresponds to the action of the additive specified in example 1.

Example 7
The components are prepared in the following ratio, wt.%:
Tinrostim - 0.1
Ascorbic acid - 5
MCC - 15
Starch - 15
Calcium Stearate - 1
Nucleoprotein complex isolated from salmon milk - 63.9
The ingredients are mixed in a mixer, ensuring a uniform distribution of components, and tableted. The tablet is white, durable, intact, disintegration time 9 min. The taste is sour with a touch of dried seafood. The weight of the tablet is 250 mg. It is recommended to take two tablets 1-2 times a day. The biological effect of the tablets corresponds to the additive obtained in accordance with the formulation indicated in example 1.

 Thus, a biologically active food supplement, including Tinrostim and DNA, has a wide range of biological effects that exceed the effects of these substances used separately.

Claims (3)

1. A biologically active food supplement, including Tinrostim (gangliin), characterized in that it additionally contains a nucleoprotein complex isolated from salmon milk, containing low molecular weight DNA with a molecular weight of from 100 to 300 kDa in an amount of 75-80%, in the following the ratio of components, wt.%: Tinrostim (gangliin) 0.1-0.3 Nucleoprotein complex isolated from salmon milk containing low molecular weight DNA with a molecular weight of 100 to 300 kDa in an amount of 75-80% 2. The biologically active food supplement according to claim 1, characterized in that it further comprises ascorbic acid, microcrystalline cellulose (MCC) and / or starch in the following proportions, wt.%: Tinrostim 0.1-0.3 Ascorbic acid 5-10 MCC and / or starch 15-30 Nucleoprotein complex isolated from salmon milk containing low molecular weight DNA with a molecular weight of 100 to 300 kDa in an amount of 75-80% 3. The biologically active food supplement according to claim 1, characterized in that it further comprises ascorbic acid, MCC and / or starch and a tabletting substance in the following proportions, wt.%: Tinrostim 0.1-0.3 Ascorbic acid 5-10 MCC and / or starch 15-30 Substance for tableting 0.5-1.5 Nucleoprotein complex isolated from salmon milk containing low molecular weight DNA with a molecular weight of 100 to 300 kDa in an amount of 75-80%

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