Continuous enzymolysis enrichment modification and purification utilization method for xanthohumol flavonoid

Abstract

The present invention relates to the field of biochemical and pharmaceutical technologies, and in particular to a continuous enzymolysis enrichment modification and purification utilization method for xanthohumol flavonoid. The method comprises the following steps: S1, continuous enzymolysis; S2, recovery of complex enzymes; S3, heterologous expression; S4, electrodialysis-reverse osmosis purification; and S5, structural modification. Compared with the prior art, on the basis of lignite humic acid and fulvic acid gap enzymolysis, the use of continuous enzymolysis enrichment modification and purification utilization to obtain a dietetic and medical product xanthohumol flavonoid having a clear composition structure and capable of symptomatic treatment has remarkable progress and substantive effects in the aspects of product quality, low-carbon emission and environmental friendliness compared with chemical extraction.

Description

Continuous enzymatic hydrolysis, enrichment, modification, purification and utilization method of xanthohumol flavonoids Technical Field

[0001] The invention relates to the technical field of biochemical engineering and medicine, and in particular to a method for continuous enzymatic hydrolysis, enrichment, modification, purification and utilization of xanthohumol flavonoids. Background Art

[0002] The pharmaceutical application of xanthohumol flavonoids as typical isopentenyl flavonoid compounds has been seen in: Qingdao National Laboratory for Marine Science and Technology Development Center, Qingdao Institute of Marine Biotechnology Co., Ltd.'s invention patent CN202011310476.7, a use of xanthohumol in the preparation of new coronavirus inhibitors; Shenzhen Xiaxiwan Pharmaceutical Technology Co., Ltd.'s invention patent CN201711472048.2, the use of xanthohumol and its derivatives in the prevention or treatment of neurodegenerative diseases, which can be further developed and utilized as potential therapeutic drugs for diseases such as Alzheimer's disease; the Xinjiang Institute of Physics and Chemistry, Chinese Academy of Sciences, and the Shanghai Institute of Materia Medica, Chinese Academy of Sciences' invention patent CN 202010957790.8, a xanthohumol derivative, and its preparation method and use; Yumen Tuopu Technology Development Co., Ltd.'s invention patent CN201010167119.X, an industrial production method for high-purity xanthohumol using CO2 extraction of hop residue and re-extraction with dichloroethane, dichloromethane, or chloroform; it shows that the utilization of xanthohumol flavonoids is being continuously developed and valued, while most remain in the research and development stage, and the raw materials are basically hops and residues. As a small-scale industrial production method of high-purity xanthohumol, it is also based on CO2 extraction and re-purified with volatile and toxic organic solvents such as dichloroethane, dichloromethane, or chloroform, and the production cost is relatively high. Zhejiang Huijia Biotechnology Co., Ltd.'s invention patent CN202221732993.8 describes a multifunctional enzymatic hydrolysis system that combines stirring and enzymatic hydrolysis with a rinsing process, resulting in a utility model device. Yishuiyuan Biotechnology (Xi'an) Co., Ltd.'s invention patent CN202210403024.6 describes a high-efficiency enzymatic hydrolysis device for crop straw. This device improves enzymatic hydrolysis efficiency through temperature sensors, heat exchangers, and signal connections between the controller and the distribution box within the enzymatic hydrolysis tank. Mingyisheng Biotechnology (Shanghai) Co., Ltd.'s invention patent CN202111683728.5 describes a simple, high-throughput DNA methylation detection method, an important method for epigenetic modification of genes and suitable for early screening of major diseases. Studies have shown that DNA methylation markers used for tumors, such as the GSTPI gene, are hypermethylated in 80-90% of prostate cancers. DNA methylation methods can also detect early-stage lung cancer. DNA methylation methods are also effective in conditions such as atherosclerosis, coronary heart disease, hypertension, heart failure, and diabetes, where hypermethylation is also detected. Earlier, EPI Genomics Co., Ltd.'s invention patent, CN98812738.5, also disclosed a method for preparing complex DNA methylation fingerprints, demonstrating the importance of DNA methylation detection for RNA preparation. Stem cells possess powerful regenerative and repair capabilities and are known as "universal cells."Mesenchymal stem cells have good therapeutic effects on leukemia, diabetes, antiviral treatment, recovery of burned skin, and pulmonary fibrosis (Yang Hongju et al. Mesenchymal Stem Cells 2021 Yunnan Publishing Group), and fulvic acid also has these functions to varying degrees (Zhou Xiaping et al. New Progress in In Vitro and In Vivo Experimental Pharmacological Research of Fulvic Acid. Humic Acid, 2019 (6): 3-9). Due to the different mineral resource locations and the uncertainty of the composition and structure of humic acid and fulvic acid, clinical research on nearly 10,000 people in my country alone has been stagnant. A review paper from the Russian Medical Academy, "Fulvic acid is a kind of food additive or medicine" (2020) and a Canadian literature: A toxicological evaluation of a fulvic and humic acids preparation. Toxicology Reports, 2020, 7: 1242-1254. The exact composition and structure of fulvic flavonoids and the exact peaks of HPLC-MS can break this deadlock and enable the medicinal resources of natural products such as lignite, weathered coal, and peat (peat) to be better used as medicine. For example, the Brazilian National Publishing House published: Chemical Composition of Oral Flavonoids (COMPOSIÇÕES PARA TRATAMENTO BUCAL QUE COMPREENDEM FLAVONOIDES PRENILADOS.2023.5.2); European Patent, OPHTHALMIC COMPOSITION COMPRISING XANTHOHUMOL (21.12.2022); PCT International Patent: Xanthohumol-Based Protein Kinase Modulation Cancer Treatment (XANTHOHUMOL BASED PROTEIN KINASE MODULATION CANCER TREATMENT.PCT US 2007 / 014374); Xanthohumol Cosmetics and Uses, Including Skin Lightening and Reducing Skin Redness (PCT IB 2009 / 054555); and even the PCT International Patent: A METHOD FOR SYNTHESIZING XANTHOHUMOL.WO 2022 / 233428 Al). This invention synthesizes xanthohumol (XN) from naringenin, including acylation of the hydroxyl group of the flavonoid portion of naringenin to convert the flavonoid portion into bupleurone, followed by hydrolysis of the ester group and substitution reaction of the olefinic portion to obtain xanthohumol.Yuan Hongli, Chen Wenxin, Kimura Masatoshi, et al., China Agricultural University. Acta Microbiologica Sinica, 1998, 38(6): 411-416. The succession of microbial communities during lignite weathering was reported. Yuan Hongli, Chen Wenxin, et al., Study on the chemical characteristics of humic acid produced by microbial degradation of lignite. Environmental Chemistry, 2000, 19(3): 240-243. Yang Jinshui, Yuan Hongli, Chen Wenxin. Lignite degradation activity and cDNA cloning of Penicillium decumbens P6 lignin peroxidase. Journal of Peking University, 2005, 41(6): 833-839. Hong Li Yuan, Jin Shui Yang, Feng Qin Wang, et al. Degradation and solubilization of chinese lignite by Penicillium sp. P6. Applied Biochemistry and Microbiology, 2006, 42: 52-55; The mechanism of Penicillium degradation of lignite Yuan Hongli et al. used a mixed fermentation of one strain of actinomycetes, one fungus, and one bacteria to study the degradation of lignite in the laboratory and produced fulvic acid, which was used in broccoli cultivation and improved the quality of broccoli in terms of soluble sugar, protein, vitamin C and other parameters. (Humic Acid 2009 (5) 4~), focusing on the mechanism of microorganisms, did not specify the scale of the experiment, such as shake bottles or other forms. Tao Xiuxiang of China University of Mining and Technology pointed out: The characteristics and technical bottlenecks of coal microbial conversion: (1) The lack of efficient coal bioconversion bacteria, with a conversion rate of about 30% and a time of one week to several months. The solution is to domesticate and induce mutation, or genetically engineer and construct engineered bacteria. (2) There is a lack of research on coal structure and depolymerization mechanism; (3) There is a lack of research on the relationship between coal and microorganisms; (4) The cost of coal bioconversion process is relatively high (more expensive nutrients such as glucose, vitamins and other culture media; the difficulty and cost of separating water-soluble products compared to oil-soluble mixtures are higher; Anhui Provincial Key Laboratory of Modern Mining Engineering, Anhui University of Science and Technology Materials Science, through the UV mutagenesis breeding of the protoplasts of Psoralea chrysosporium and the degradation and transformation of Yima lignite, to screen efficient coal degrading bacteria. The experiment showed that: UV radiation, nitric acid pretreatment of lignite Psoralea chrysosporium The water-soluble products of lignite biodegradation by Pseudomonas aeruginosa are a complex mixture. Compared to the original lignite, the functional group content has also changed, with an increase in phenolic, hydroxyl, and carbonyl substances, and a large amount of aromatic polymers degraded into low-molecular-weight substances and other substances (Journal of Safety and Environment, 2015, 15(4):227-230). Therefore, in light of the shortcomings of the prior art in terms of raw materials and methods, the present invention, based on the enzymatic hydrolysis of humic acid and fulvic acid, produces a product that can be used for symptomatic medicinal and health care, representing a significant and substantial advancement in low-carbon and environmental protection. Summary of the Invention

[0003] In order to overcome the deficiencies of the prior art, the present invention provides a method for continuous enzymatic hydrolysis, enrichment, modification, purification and utilization of xanthohumol flavonoids.

[0004] To achieve the above objectives, a method for continuous enzymatic hydrolysis, enrichment, modification, purification and utilization of xanthohumol flavonoids was designed, which is characterized by comprising the following steps:

[0005] S1, continuous enzymatic hydrolysis: grind lignite, weathered coal, dandelion, bagasse peel, tea branches, and corn cobs, mix them, and add laccase, esterase, pectinase, chitinase, tannase, and sulfhydryl isomerase to the mixture, respectively. The enzymatic hydrolysis is carried out at a liquid-solid ratio of 3-5:1, a hydrolysis temperature of 25-55°C, a forward enzymatic hydrolysis time of 1-3 h, a stirring speed of 30-60 r / min, and a pH of 4.5-8.5;

[0006] S2, recovery of complex enzyme: selectively separate the dilute complex enzyme from the xanthohumol flavonoid aqueous solution through the macroporous resin filled in the pipeline, with a dielectric constant of ε2.3-2.9, an upper column adsorption rate of 5.0 ml / min, and an elution rate of 5.0 ml / min from the lower column, and reversely return the complex enzyme to the S1 enzymatic solution to increase the concentration of the complex enzyme. After 5 cycles, the enzymatic solution of step S1 is recovered in a horizontal centrifuge at a speed of 4500-5000 r / min, with a ratio of 75-85%;

[0007] S3, heterologous expression: Based on the cloning of the complex enzyme into the yeast plasmid P0463 / pRS316, heterologous expression is naturally achieved in yeast host cells in the forward and reverse directions through amplification of the homologous plasmids, and the resulting complex engineered enzyme is added to the mixture of step S1; the complex enzyme is also recovered by horizontal centrifugation and analyzed by agarose gel electrophoresis to further optimize the gene clone, and the complex engineered enzyme is then added to the enzymatic hydrolyzate mixture of step S1;

[0008] S4, electrodialysis-reverse osmosis purification: at a feed liquid processing capacity of 0.15 m3 / h, a xanthohumol flavonoid concentration of 20-25%, separation at an isoelectric point of 4.5-4.8 and a conductivity of 18 ms / cm, and desalination of 95-98% through an electrodialysis anion membrane at a voltage of 5.0 V, with a desalted liquid conductivity of < 700 us / cm, 80-85% of the reverse osmosis reclaimed water reuse, and a current efficiency of 93.5-96.8%;

[0009] S5, Structural modification: The structural modification of polyphenols, flavonoids, sesquiterpenes, and methoxybenzofurans in the enzymatic hydrolysates of lignite, weathered coal, dandelion, bagasse peel, tea branches, and corn cobs to xanthohumol flavonoids was carried out through condensation reaction (phenol-degrading) by tannase, spatial arrangement reaction by sulfhydryl isomerase, and hydrolysis reaction by esterase at 50°C-55°C, and the structural modification reaction time was regulated to 0.5-1.0 h by HPLC internal standard analysis.

[0010] The method further includes step S6, methylation test: qualitative analysis using a 254nmh and 365nm ultraviolet fluorescence analyzer, and quantitative analysis using HPLC. The final total purity of the drug is 99.00-99.99.

[0011] In step S6, a concentration test is performed on a microplate reader under the action of a Septin 9 methylation kit to examine the increase in DNA methylation compared with the control, which is effective for early diagnosis and verification of optic neurasthenia with an efficiency of 90-95%.

[0012] In the step S6, under the action of the SHOX2 and RASSF1A methylation kit, the prognostic evaluation indicators of terminal diseases such as chronic hepatitis, gastric ulcer, and diabetes are assisted by stem cells, and the efficiency is 90-98%.

[0013] In step S1, the mass percentages of lignite, weathered coal, dandelion, bagasse peel, tea branches, and corn cobs are 25.0-30.0:60.0-65.0:0.5-1.5:1.0-3.5:3.0-7.5:0.5-3.5, and the mass percentages of the aforementioned mixture and laccase, esterase, tannase, pectinase, chitinase, and sulfhydryl isomerase are 0.01:0.02:0.01:0.02:0.03:0.01.

[0014] The particle size of the lignite, weathered coal, dandelion, bagasse peel, tea branch and corn cob is 30-300 mesh, the liquid-solid ratio is 3-5:1, the extraction temperature is 25-55 DEG C and the pH is 4.5-8.5.

[0015] The yeast is selected from saccharomyces cerevisiae.

[0016] In step S3, the electric field velocity is 2-3 V / cm, and 0.5 x TBE electrophoresis buffer is used. The results of 0.7% agarose gel electrophoresis of the engineered complex enzyme and complex plasmid show that compared with the initial complex enzyme, the enzymatic concentration is increased by 3-4 times, the enzymatic reaction rate is increased by 3.5-5.0 times, and the enzymatic cost is saved by 1.5-3.0 times.

[0017] In the step S1, laccase, esterase, chitinase and pectinase are used in a first-order parallel reaction mode to increase the enzymatic hydrolysis rate, and tannase and sulfhydryl isomerase are used in a redox mode to modify the structure of xanthohumol flavonoids to make the composition structure and molecular weight of the product consistent.

[0018] Compared with the existing technology, the present invention, based on the intermittent enzymatic hydrolysis of lignite humic acid and fulvic acid, continuously performs enzymatic hydrolysis enrichment, modification and purification to obtain xanthohumol flavonoids, a food therapy and medical product with a clear composition structure and symptomatic treatment. Compared with chemical extraction, it has significant progress and substantial effects in product quality, low carbon and environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG1 is a schematic diagram of the process of continuous enzymatic hydrolysis, enrichment, purification and utilization of structure-modified xanthohumol flavonoids according to the present invention.

[0020] Figure 2 shows the structural modifications that can be achieved through coupling (condensation) esterification of polyphenol structures in lignite and weathered coal.

[0021] Figure 3 shows the flavonoid structure before lignite structure modification.

[0022] Figure 4 shows the sesquiterpenoid structures of lignite and biomass straw before structural modification.

[0023] Figure 5 shows that the number average molecular weight Mw of xanthohumol flavonoids is 468.

[0024] Figure 6 Structures of methoxybenzofuran before structural modification of lignite, weathered coal, and biomass straw.

[0025] FIG7 is a standardized structural formula of xanthohumol flavonoids.

[0026] FIG8 is an HPLC composition analysis of xanthohumol flavonoids according to Example 1 of the present invention.

[0027] FIG9 is an HPLC composition analysis of xanthohumol flavonoids according to Example 2 of the present invention.

[0028] FIG10 is an HPLC composition analysis of xanthohumol flavonoids according to Example 3 of the present invention.

[0029] FIG11 is an HPLC composition analysis of xanthohumol flavonoids according to Example 4 of the present invention. Implementation Method

[0030] The present invention will be further described below with reference to the accompanying drawings.

[0031] As shown in FIG1 , the method for continuous enzymatic hydrolysis, enrichment, modification, purification and utilization of xanthohumol flavonoids in this embodiment includes the following steps:

[0032] S1, continuous enzymatic hydrolysis: grind lignite, weathered coal, dandelion, bagasse peel, tea branch, and corn cob, mix them, and add laccase, esterase, pectinase, chitinase, tannase, and sulfhydryl isomerase to the mixture respectively. The enzymatic hydrolysis is carried out at a liquid-solid ratio of 3-5:1, an enzymatic hydrolysis temperature of 25-55°C, a forward enzymatic hydrolysis time of 1-3 h, a stirring speed of 30-60 r / min, and a pH of 4.5-8.5.

[0033] S2, recovery of complex enzyme: through the macroporous resin filled in the pipeline, with a dielectric constant of ε2.3-2.9, an upper column adsorption rate of 5.0ml / min; a lower column elution rate of 5.0ml / min, the dilute complex enzyme is selectively separated from the xanthohumol flavonoid aqueous solution and reversely returned to the S1 enzymatic solution to increase the concentration of the complex enzyme. After 5 cycles, the enzymatic solution of S1 is sent to a horizontal centrifuge at a speed of 4500-5000r / min to recover the complex enzyme at a ratio of 75-85%.

[0034] S3, based on the cloning of the complex enzyme into the yeast plasmid P0463 / pRS316, the heterologous expression in the forward and reverse yeast host cells is naturally completed through the amplification of the homologous plasmid, and the complex engineered enzyme is returned to S1; the complex enzyme is also recovered by a horizontal centrifuge and analyzed by agarose gel electrophoresis to further optimize the gene cloning, and then the complex engineered enzyme is added to the enzymatic solution mixture of step S1.

[0035] S4, electrodialysis-reverse osmosis purification: At a feed liquid processing capacity of 0.15m3 / h, a xanthohumol flavonoid concentration of 20-25%, separation is performed at an isoelectric point of 4.5-4.8 and a conductivity of 18ms / cm. The product is then desalted to 95-98% purity at a voltage of 5.0V using an electrodialysis anion membrane. The desalted liquid conductivity is <700us / cm, and the reverse osmosis water reuse rate is 80-85%. The current efficiency is 93.5-96.8%.

[0036] S5, Structural modification: The structural modification of polyphenols, flavonoids, sesquiterpenes, and methoxybenzofurans in the enzymatic hydrolysates of lignite, weathered coal, dandelion, bagasse peel, tea branches, and corn cobs to xanthohumol flavonoids was carried out through condensation (phenol-degrading) reaction by tannase, spatial arrangement reaction by sulfhydryl isomerase, and hydrolysis reaction by esterase at 50℃-55℃, and the structural modification reaction time was regulated to 0.5-1.0 h by HPLC internal standard analysis.

[0037] The method further includes step S6, methylation test: qualitative analysis using a 254nmh and 365nm ultraviolet fluorescence analyzer, and quantitative analysis using HPLC. The final total purity of the drug is 99.00-99.99.

[0038] In step S6, a concentration test is performed on a microplate reader under the action of a Septin 9 methylation kit to examine the increase in DNA methylation compared with the control, which is effective for early diagnosis and verification of optic neurasthenia with an efficiency of 90-95%.

[0039] In step S6, under the action of the SHOX2 and RASSF1A methylation kit, the prognostic evaluation indicators of terminal diseases such as chronic hepatitis, gastric ulcer, and diabetes are assisted by stem cells, with an efficiency of 90-98%.

[0040] In step S1, the mass percentages of lignite, weathered coal, dandelion, bagasse husk, tea branch, and corn cob are 25.0-30.0:60.0-65.0:0.5-1.5:1.0-3.5:3.0-7.5:0.5-3.5, and the mass percentages of the aforementioned mixture and laccase, esterase, tannase, pectinase, chitinase, and sulfhydryl isomerase are 0.01:0.02:0.01:0.02:0.03:0.01.

[0041] The particle size of lignite, weathered coal, dandelion, bagasse peel, tea branch, and corn cob is 30-300 mesh, the liquid-solid ratio is 3-5:1, the extraction temperature is 25-55℃, and the pH is 4.5-8.5.

[0042] The yeast is selected from saccharomyces cerevisiae, which can be purchased.

[0043] In step S3, the electric field velocity was set at 2-3 V / cm, 0.5x TBE electrophoresis buffer was used, and EB staining was performed to obtain DNA agarose gel electrophoresis results. DNA agarose gel electrophoresis results obtained at an electric field velocity of 2-3 V / cm, 0.5x TBE electrophoresis buffer, and EB staining confirmed that the engineered enzymes evolved for efficient production reduced the steric and interfacial hindrance between the hydrophobic and hydrophilic groups of the original laccase, esterase, pectinase, and chitinase molecules on lignite and weathered coal macromolecules, thereby increasing enzyme activity and improving enzymatic hydrolysis efficiency.

[0044] In step S1, laccase, esterase, chitinase, and pectinase react in a first-order parallel mode to increase the enzymatic hydrolysis rate, while tannase and sulfisomerase act in a redox mode to modify the structure of xanthohumol flavonoids. As shown in Figure 2, under the action of tannase and sulfisomerase, the monomeric phenol (thiophenol) undergoes polycondensation and the structure of catechin is modified, aligning the product's composition and molecular weight with those of fulvic acid flavonoids.

[0045] In this example, the enzyme activity was determined by HPLC. On the premise that flavonoids were detected by GC-MS, xanthohumol flavonoids from hops were used as standard samples. The peak height and half-peak height of xanthohumol flavonoids in the structurally modified lignite-weathered coal, dandelion, pomegranate peel, and tea branch samples were qualitatively measured in a mobile phase of methanol:water = 7.5:2.5, UV detection λ = 290 nm, column temperature 30 ° C, and retention time 7.5 min. The correlation coefficient was 0.998. The content of the enzymatic reaction product was calculated by the single-point control method using the UV detection intensity (concentration) of the reference standard sample, and U = [∆A / (0.01×t)] × D, where: U is the enzyme activity; ∆A is the UV detection intensity value of different samples at different reaction times; t is the different reaction time (min); and D is the dilution factor to convert the enzyme activity value. As shown in Figure 11, after integrating the advantages of complex enzymes such as tannase, sulfhydryl isomerase, and esterase, the HOLC analysis determined that the content of xanthohumol flavonoids increased, the total enzyme activity increased, and the gel chromatography molecular weight determination assisted in verifying the molecular size of xanthohumol flavonoids. Example 1:

[0046] In this example, the mass percentage of lignite, weathered coal, dandelion, bagasse husk, tea branch, and corn cob is 35.0:55.0:0.5:1.5:2.5:5.5, and the mass percentage of laccase, esterase, tannase, pectinase, phytase, and sulfhydryl isomerase is 0.01:0.02:0.01:0.02:0.03:0.01.

[0047] The raw materials of lignite, weathered coal, dandelion, bagasse peel, tea branch, and corn cob have a particle size of 300 mesh, a liquid-solid ratio of 5:1, an extraction temperature of 50°C, a pH of 4.5, and an enzymatic hydrolysis time of 1.5 h.

[0048] Under the optimization of a complex enzyme-engineered bacterial strain, enzymatic enrichment and structurally modified xanthohumol flavonoids were obtained. Desalination and purification were achieved through an electrodialysis anion membrane at a voltage of 5.0V, with reverse osmosis water reuse, achieving a current efficiency of 96.8%. The power consumption per kg of xanthohumol flavonoids was 0.97 kWh. The xanthohumol flavonoids were qualitatively analyzed by UV fluorescence at 254 nm and 365 nm, and quantitatively analyzed by an Agilent 1260 HPLC. The final product had an overall purity of 99.50%.

[0049] Optic atrophy is the ultimate outcome of various severe retinal and optic nerve diseases and is a common and difficult eye disease in ophthalmology. The xanthohumol flavonoids enriched and purified by continuous enzymatic hydrolysis in this example were used as an adjunct agent in mitochondrial DNA methylation analysis of patients with autosomal dominant optic atrophy. The septin9 methylation kit (fluorescence PCR) from Suzhou Weizhen Biopharmaceutical Co., Ltd., registration number: 20183400103, was used to detect optic nerve atrophy. Compared to controls, the DNA count was reduced, indicating genetic heterogeneity and autosomal dominant optic neuropathy. The validation efficiency was 90.00-95.00%, providing direct evidence for the pathological mechanism of optic nerve gene mutations in patients. Example 2:

[0050] In this embodiment, the mass percentage of lignite, weathered coal, dandelion, bagasse peel, tea branch, and corn cob is 25.0:65.0:0.5:1.0:3.0:0.5, and the mass percentage of laccase, esterase, tannase, pectinase, phytase, and sulfhydryl isomerase is 0.01:0.02:0.01:0.02:0.03:0.01; the particle size of the raw materials lignite, weathered coal, dandelion, bagasse peel, tea branch, and corn cob is 100 mesh, the liquid-solid ratio is 3:1, the extraction temperature is 35°C, the pH is 4.5, and the enzymatic hydrolysis time is 2.0 h. Under the optimization of the composite enzyme engineering bacteria, the enzymatic hydrolysis enrichment and structurally modified xanthohumol flavonoids are obtained, which are desalted and purified at a voltage of 5.0 V on an electrodialysis anion membrane, and the reverse osmosis water is reused with a current efficiency of 96.8%. The power consumption for purifying 1 kg of xanthohumol flavonoids was 0.97 / kWh. The xanthohumol flavonoids were qualitatively analyzed by 365 nm UV fluorescence analyzer and quantitatively analyzed by Agilent 1260 HPLC. The final total purity of the drug was 99.90%.

[0051] Stem cells are a type of multipotent cell with the ability to self-replicate. Under certain conditions, they can differentiate into a variety of functional cells and treat diseases such as neurological diseases, Parkinson's disease, diabetes, and pulmonary nodules. The xanthohumol flavonoids enriched and purified by continuous enzymatic hydrolysis in this example were used as an auxiliary drug and verified by DNA methylation testing. Sputum cells from patients with pulmonary nodules were sampled and tested using the SHOX2 and RASSF1A Methylation Kit (Fluorescence PCR) (Registration No.: 20173403354) from Shanghai Toujing Life Science Co., Ltd. Methylation levels before and after stem cell treatment were compared, and the efficacy was 95.00-98.00%. Example 3:

[0052] In this example, the weight percentages of lignite, weathered coal, dandelion, bagasse husk, tea branch, and corn cob were 25.0:65.0:0.5:1.0:3.0:0.5, and the weight percentages of laccase, esterase, tannase, pectinase, phytase, and sulfhydryl isomerase were 0.01:0.02:0.01:0.02:0.03:0.01. The raw materials, lignite, weathered coal, dandelion, bagasse husk, tea branch, and corn cob, had a particle size of 200 mesh, a liquid-to-solid ratio of 3:1, an extraction temperature of 55°C, a pH of 4.5, and an enzymatic hydrolysis time of 3.0 h. Under the optimization of the composite enzyme engineering bacteria, enzymatic hydrolysis enriched and structurally modified xanthohumol flavonoids were obtained. The products were then desalted and purified using an electrodialysis anion membrane at a voltage of 5.0 V, and the reverse osmosis water was reused with a current efficiency of 96.8%. The power consumption for purifying 1 kg of xanthohumol flavonoids was 0.97 / kWh. The xanthohumol flavonoids were qualitatively analyzed by 254 nm UV fluorescence analyzer and quantitatively analyzed by Agilent 1260 HPLC. The final product had an overall purity of 99.97%. Example 4:

[0053] In this example, the weight ratio of lignite, weathered coal, dandelion, bagasse husk, tea branch, and corn cob was 25.0:65.0:0.5:1.0:3.0:0.5, and the weight ratio of laccase, esterase, tannase, pectinase, phytase, and sulfhydryl isomerase was 0.01:0.02:0.01:0.02:0.03:0.01. The raw materials had a particle size of 200 mesh, a liquid-solid ratio of 3:1, an extraction temperature of 55°C, a pH of 4.5, and an enzymatic hydrolysis time of 3.5 h. Under the optimization of the composite enzyme engineering bacteria, enzymatic hydrolysis enriched and structurally modified xanthohumol flavonoids were obtained. The products were desalted and purified at a voltage of 5.0 V using an electrodialysis anion membrane, and the reverse osmosis water was reused with a current efficiency of 96.8%. The power consumption for purifying 1 kg of xanthohumol flavonoids was 0.97 / kWh. The xanthohumol flavonoids were qualitatively analyzed by 254 nm UV fluorescence analyzer and quantitatively analyzed by Agilent 1260 HPLC. The final product had an overall purity of 99.99%.

Claims

1. A method for continuous enzymatic enrichment, modification and purification of xanthohumol flavonoids, characterized in that: The steps include: S1, continuous enzymatic hydrolysis: grind lignite, weathered coal, dandelion, bagasse peel, tea branch, corn cob and mix them, add laccase, esterase, pectinase, chitinase, tannase, sulfhydryl isomerase to the mixture respectively, and carry out the enzymatic hydrolysis at a liquid-solid ratio of 3-5:1, enzymatic hydrolysis temperature of 25-55°C, forward enzymatic hydrolysis of 1-3 h, stirring speed of 30-60 r / min, and pH of 4.5-8.5; S2, recovery of complex enzyme: selectively separate the dilute complex enzyme from the xanthohumol flavonoid aqueous solution through the macroporous resin filled in the pipeline, with a dielectric constant of ε2.3-2.9, an upper column adsorption rate of 5.0 ml / min, and a lower column elution rate of 5.0 ml / min, and reversely return the complex enzyme to the S1 enzymatic solution to increase the concentration of the complex enzyme. After 5 cycles, the enzymatic solution of step S1 is recovered in a horizontal centrifuge at a speed of 4500-5000 r / min, and the ratio is 75-85%; S3, heterologous expression: on the basis of cloning the complex enzyme into the yeast plasmid P0463 / pRS316, heterologous expression in the forward and reverse yeast host cells is naturally completed by amplification of the homologous plasmid, and the complex engineered enzyme is added to the mixture of step S1; the complex enzyme is also recovered by a horizontal centrifuge and analyzed by agarose gel electrophoresis, and the gene clone is further optimized, and then the complex engineered enzyme is added to the enzymatic solution mixture of step S1; S4, electrodialysis-reverse osmosis purification: at a feed liquid processing volume of 0.15m 3 / h, xanthohumol flavonoids concentration 20-25%, separated at isoelectric point 4.5-4.8, conductivity 18ms / cm, and desalted 95-98% purified by electrodialysis anion membrane voltage 5.0V, desalted liquid conductivity < 700us / cm, reverse osmosis water reuse 80-85%, current efficiency 93.5-96.8%; S5, structural modification: The structural modification of polyphenols, flavonoids, sesquiterpenes and methoxybenzofurans in the enzymatic hydrolysates of lignite, weathered coal, dandelion, bagasse peel, tea branches and corn cobs to xanthohumol flavonoids was carried out through the condensation reaction of tannase (phenol decomposition), the spatial arrangement reaction of sulfhydryl isomerase and the hydrolysis reaction of esterase at 50℃-55℃, and the structural modification reaction time was regulated to 0.5-1.0 h by HPLC internal standard analysis.

2. The method for continuous enzymatic hydrolysis, enrichment, modification and purification of xanthohumol flavonoids according to claim 1, characterized in that: The method also includes step S6, methylation test: qualitative analysis using 254nmh and 365nm ultraviolet fluorescence analyzers, quantitative analysis using HPLC, and the final total purity of the drug is 99.00-99.

99.

3. The method for continuous enzymatic hydrolysis, enrichment, modification and purification of xanthohumol flavonoids according to claim 2, characterized in that: In the step S6, under the action of the Septin9 methylation kit, a concentration test is performed on an ELISA instrument to check the increase in the DNA methylation compared with the control, which is effective for early diagnosis and verification of optic neurasthenia, with an efficiency of 90-95%.

4. The method for continuous enzymatic enrichment, modification and purification of xanthohumol flavonoids according to claim 2, characterized in that: In the step S6, under the action of the SHOX2 and RASSF1A methylation kit, the prognosis evaluation indicators of terminal diseases such as chronic hepatitis, gastric ulcer, and diabetes are assisted by stem cells, and the effective rate is 90-98%.

5. The method for continuous enzymatic hydrolysis, enrichment, modification and purification of xanthohumol flavonoids according to claim 1, characterized in that: In the step S1, the mass percentages of lignite, weathered coal, dandelion, bagasse peel, tea branch and corn cob are 25.0-30.0:60.0-65.0:0.5-1.5:1.0-3.5:3.0-7.5:0.5-3.5, and the mass percentages of the above mixture and laccase, esterase, tannase, pectinase, chitinase and sulfhydryl isomerase are 0.01:0.02:0.01:0.02:0.03:0.

01.

6. The method for continuous enzymatic enrichment, modification and purification of xanthohumol flavonoids according to claim 1 or 3, characterized in that: The particle size of the lignite, weathered coal, dandelion, bagasse skin, tea branch and corn cob is 30-300 meshes, the liquid-solid ratio is 3-5:1, the extraction temperature is 25-55°C, and the pH is 4.5-8.

5.

7. The method for continuous enzymatic hydrolysis, enrichment, modification and purification of xanthohumol flavonoids according to claim 1, characterized in that: The yeast is selected from Saccharomyces cerevisiae.

8. The method for continuous enzymatic hydrolysis, enrichment, modification and purification of xanthohumol flavonoids according to claim 1, characterized in that: In the step S3, the electric field speed is 2-3v / cm, and 0.5 x TBE electrophoresis buffer is selected to perform 0.7% agarose gel electrophoresis of the engineered complex enzyme and complex plasmid. Compared with the initial complex enzyme, the enzymatic concentration is increased by 3-4 times, the enzymatic reaction speed is increased by 3.5-5.0 times, and the enzymatic cost is saved by 1.5-3.0 times.

9. The method for continuous enzymatic hydrolysis, enrichment, modification and purification of xanthohumol flavonoids according to claim 1, characterized in that: In the step S1, laccase, esterase, chitinase and pectinase are used in a first-order parallel reaction mode to increase the enzymatic hydrolysis rate, and tannase and sulfhydryl isomerase are used in a redox mode to modify the structure of xanthohumol flavonoids to make the composition structure and molecular weight of the product consistent.

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