METHOD OF CONTINUOUS ENZYMATIC DEGRADATION, ENRICHMENT, MODIFICATION AND PURIFICATION OF XANTHOHUMIN-LIKE FLAVONOIDS

Claims

1. A method for continuous enzymatic cleavage, enrichment, modification and purification of xanthohumol-like flavonoids, comprising the following steps: S1 - continuous enzymatic degradation: brown coal, weathered coal, Mongolian dandelion, sugarcane bagasse, tea tree twigs and corn cobs are crushed and mixed, laccase, esterase, pectinase, chitinase, tannase and thioisomerase are added to the mixture, with a liquid:solid phase ratio of 3-5:1, fermentation is carried out at a temperature of 25-55 °C, direct enzymatic degradation - 1-3 hours, stirring speed 30-60 rpm, pH of the medium - 4.5-8.5; S2 - regeneration of the enzyme complex: by passing through a column system filled with macroporous resin, with a dielectric constant of ε=2.3-2.9, the adsorption rate on the upper column is 5.0 ml / min and the elution rate on the lower column is 5.0 ml / min, the diluted enzyme complex is selectively isolated from an aqueous solution containing xanthohumol-like flavonoids and returned back to the fermentation medium S1 to increase the concentration of enzymes, after 5 cycles the fermentation medium S1 is centrifuged in a horizontal centrifuge at a speed of 4500-5000 rpm, which ensures the regeneration of 75% to 85% of the enzyme complex; S3 - heterologous expression: based on the cloning of the enzyme complex genes into the yeast plasmid P0463 / pRS316, by amplifying the homologous plasmid, heterologous expression is naturally carried out in both forward and reverse directions in the yeast host cells, obtaining a complex engineered enzyme, which is added to the mixture of the S1 step; also, the enzyme complex, regenerated using a horizontal centrifuge, is analyzed by agarose gel electrophoresis for further optimization of gene cloning, after which the complex engineered enzyme is added to the mixture of the S1 fermentation medium; S4 - purification by electrodialysis-reverse osmosis: with a feed solution capacity of 0.15 m 3 / h and a concentration of xanthohumol-like flavonoids of 20-25%, separation is carried out at an isoelectric point of 4.5-4.8 with an electrical conductivity of 18 mS / cm, then desalination is carried out with a purity of 95-98% by electrodialysis through anion-exchange membranes at a voltage of 5.0 V, the electrical conductivity of the desalinated solution is <700 μS / cm, in the process of reverse osmosis, a water recirculation degree of 80-85% is achieved with a current efficiency of 93.5-96.8%; S5 - structural modification: polyphenolcarboxylic acids, flavonoids, sesquiterpenes and methoxybenzofurans contained in the fermentation medium obtained from brown coal, weathered coal, dandelion root, sugarcane bagasse, tea tree twigs and corn cobs undergo structural modification towards xanthohumol-like flavonoids, this process is carried out through a condensation (depsoid) reaction catalyzed by tannase, a spatial rearrangement reaction catalyzed by thioisomerase and a hydrolysis reaction catalyzed by esterase at a temperature of 50-55 °C, the reaction time of structural modification is controlled by HPLC with an internal standard and is from 0.5 to 1.0 hour.

2. A method for continuous enzymatic cleavage, enrichment, modification and purification of xanthohumol-like flavonoids according to claim 1, characterized in that it further includes step S6, in which a methylation analysis is performed: a qualitative analysis is carried out on a UV fluorescence analyzer at wavelengths of 254 nm and 365 nm, quantitative determination is carried out by HPLC, and the overall purity of the final pharmaceutical product is from 99.00% to 99.99%.

3. The method for continuous enzymatic cleavage, enrichment, modification and purification of xanthohumol-like flavonoids according to claim 2, characterized in that at step S6, using a reagent kit for determining methylation of the Septin9 gene, a concentration test is carried out on a microplate reader, checking for an increase in the DNA methylation index compared to the control, this method is used for early diagnosis and confirmation of optic nerve atrophy with an efficiency of 90-95%.

4. A method for continuous enzymatic cleavage, enrichment, modification and purification of xanthohumol-like flavonoids according to claim 2, characterized in that at said step S6, using reagent kits for determining methylation of the SHOX2 and RASSF1A genes, an assessment of prognostic indicators is carried out in the treatment of chronic hepatitis, gastric ulcer, diabetes and other chronic diseases using stem cells, with an efficiency of 90-98%.

5. The method for continuous enzymatic cleavage, enrichment, modification and purification of xanthohumol-like flavonoids according to claim 1, characterized in that at said stage S1 the mass percentage ratios of brown coal, weathered coal, Mongolian dandelion, sugarcane bagasse, tea tree twigs and corn cobs are, respectively, 25.0-30.0: 60.0-65.0: 0.5-1.5: 1.0-3.5: 3.0-7.5: 0.5-3.5; wherein the mass percentage ratios of the said mixture and laccase, esterase, tannase, pectinase, chitinase and thioisomerase are respectively 0.01: 0.02: 0.01: 0.02: 0.03: 0.

01.

6. A method for continuous enzymatic cleavage, enrichment, modification and purification of xanthohumol-like flavonoids according to claim 1 or 3, characterized in that the particle size of brown coal, weathered coal, dandelion root, sugarcane bagasse, tea tree twigs and corn cobs is 30-300 mesh, the liquid:solid phase ratio is 3-5:1, the extraction temperature is 25-55°C, and the pH value is 4.5-8.

5.

7. A method for continuous enzymatic cleavage, enrichment, modification and purification of xanthohumol-like flavonoids according to claim 1, characterized in that baker's yeast (Saccharomyces cerevisiae) is used as yeast.

8. The method for continuous enzymatic cleavage, enrichment, modification and purification of xanthohumol-like flavonoids according to claim 1, characterized in that at said step S3 the electric field velocity is 2-3 V / cm; 0.5×TBE is used as an electrophoretic buffer, according to the results of electrophoresis in 0.7% agarose gel of complex plasmids of the engineered enzyme complex, compared to the original enzyme complex, an increase in the concentration of enzymatic cleavage by 3-4 times, an increase in the rate of the enzymatic reaction by 3.5-5.0 times, as well as a decrease in the costs of carrying out the enzymatic cleavage by 1.5-3.0 times are achieved.

9. The method for continuous enzymatic cleavage, enrichment, modification and purification of xanthohumol-like flavonoids according to claim 1, characterized in that at said stage S1, laccase, esterase, chitinase and pectinase function in the mode of parallel first-order reactions, which ensures an increase in the rate of enzymatic cleavage; while tannase and thioisomerase act in the oxidation-reduction mode, carrying out a structural modification of xanthohumol-like flavonoids, as a result of which unification of the molecular weight and composition of the structure of the product is achieved.