阅读说明：本技术 人参皂苷的生物制备方法 (Biological preparation method of ginsenoside ) 是由 王宇 李玉花 吴昊 曹领改 张贺 张旸 于 2020-04-03 设计创作，主要内容包括：本发明公开了人参皂苷的生物制备方法,包括：(1)通过组织培养方式诱导生产人参不定根；(2)将诱导的人参不定根转到旋转摇床中的液体培养基中培养；(3)将人参不定根从摇瓶转移到生物反应器中培养一段时间后添加茉莉酸甲酯诱导人参皂苷含量增加；(4)从诱导培养后的不定根中提取人参皂苷；(5)用β-葡萄糖苷酶作为转化酶以人参皂苷混合液为转化底物进行酶促转化反应。本发明提供了组合的人参皂苷的生物制备方法,包括组织培养、酶固定化和水解方法来获得稀有人参皂苷。本发明筛选到6种β-糖苷酶进行酶促水解并将它们组合使用,转化产物中稀有人参皂苷的含量明显提高。本发明生物制备方法可替代直接从人参中提取人参皂苷的方法。(The invention discloses a biological preparation method of ginsenoside, which comprises the following steps: (1) inducing and producing the ginseng adventitious root by a tissue culture mode; (2) transferring the induced ginseng adventitious root into a liquid culture medium in a rotary shaking table for culture; (3) transferring the ginseng adventitious roots from the shake flask to a bioreactor to culture for a period of time, and adding methyl jasmonate to induce the increase of the content of ginsenoside; (4) extracting ginsenoside from adventitious roots after induction culture; (5) and (3) carrying out enzymatic conversion reaction by taking beta-glucosidase as converting enzyme and ginsenoside mixed liquor as a conversion substrate. The invention provides a biological preparation method of combined ginsenoside, which comprises tissue culture, enzyme immobilization and hydrolysis methods to obtain rare ginsenoside. 6 kinds of beta-glycosidases are screened out for enzymatic hydrolysis and are combined for use, and the content of rare ginsenoside in the converted product is obviously improved. The biological preparation method of the invention can replace the method for directly extracting ginsenoside from ginseng.)

1. A biological preparation method of ginsenoside is characterized by comprising the following steps: (1) inducing and producing the ginseng adventitious root by a tissue culture mode; (2) transferring the induced ginseng adventitious root into a liquid culture medium in a rotary shaking table for culture; (3) transferring the ginseng adventitious roots from the shake flask to a bioreactor to culture for a period of time, and adding methyl jasmonate to induce the increase of the content of ginsenoside; (4) extracting ginsenoside from adventitious root after induction culture.

2. The bioproduction method according to claim 1, wherein the method for inducing production of ginseng adventitious roots by tissue culture in step (1) comprises: (a) inoculating the ginseng root slice to the solution containing 1.0mg L^-12,4-D+0.1mg L^-1kinetin+30g L^-1Induction on sucrose Induction MediumCallus tissue; (b) callus was transferred to 5.0mg L^-1Carrying out callus propagation on an MS culture medium of IBA; (c) in the presence of 3.0mg L^-1IBA and 30g L^-1The callus was cultured on a sucrose MS solid medium, and the generation of adventitious roots was induced from the callus.

3. The biological preparation method according to claim 1, wherein the ginseng adventitious roots in the step (3) are transferred from the shake flask to the bioreactor to be cultured for a period of time, and then 200 μ M methyl jasmonate is added to induce the increase of the ginsenoside content; preferably, the ginseng adventitious roots are transferred from the shake flask to the bioreactor and cultured for 50 days, and 200 μ M methyl jasmonate is added to induce the increase of the ginsenoside content.

4. The biological preparation method according to claim 1, wherein the method for extracting ginsenoside from adventitious roots of ginseng in the step (4) comprises: (a) adding ginseng root powder to the methanol/water mixture overnight; (b) extracting with methanol as solvent by ultrasonic instrument: (c) filtering the supernatant of the extractive solution for sterilization, evaporating to remove methanol, and dissolving the residue in water; preferably, the methanol/water mixture in step (a) consists of methanol and water in a volume ratio of 4: 1; in the step (b), 80% methanol is used as a solvent to extract for 3 times at room temperature of an ultrasonic instrument, and the extraction time is 0.5h each time.

5. The bioprocess of claim 1 further comprising: carrying out enzymatic conversion reaction by using beta-glucosidase as converting enzyme and ginsenoside mixture extracted from adventitious root as converting substrate.

6. The process of claim 5, wherein the β -glucosidase is selected from any one or any combination of more than one of BglSk, BglPm, bgpp1, bglb10, Tpebgl3, or Abf 22-3.

7. The bioproduction process of claim 6 wherein the β -glucosidase is selected from the group consisting of BglSk, BglPm, a combination of BglSk and bgpp1, a combination of BglSk and BglBX10, a combination of BglSk and Tpebgl3, a combination of BglSk and Abf22-3, a combination of BglPm and bgpp1, a combination of BglPm and BglBX10, a combination of BglPm and Tpebgl3, and a combination of BglPm and Abf 22-3.

8. The biological preparation method according to claim 5, wherein the enzymatic conversion reaction is carried out by reacting a β -glucosidase solution with the extracted ginsenoside mixture in 50mM sodium phosphate buffer at 37 ℃.

9. The biological process of claim 5, wherein the β -glucosidase is an immobilized β -glucosidase.

10. The biological preparation method according to claim 9, wherein the immobilized β -glucosidase is immobilized by polysulfone hollow fiber membrane, preferably by equilibrating polysulfone hollow fiber membrane in phosphate buffer of pH8.0 for 30min, and immobilizing enzyme by pressure-driven filtration, and by peristaltic pump for 10mL min of β -glucosidase solution^-1Is pumped through the lumen of the membrane and circulated for 2h, β -glucosidase is embedded in an asymmetric, aqueous porous membrane and immobilized by a 30kDa fibrous membrane on the inner surface.

Technical Field

The invention relates to a method for preparing ginsenoside, in particular to a method for preparing ginsenoside through biotransformation, belonging to the field of biological preparation of ginsenoside.

Background

Ginsenoside is the main pharmacological component of ginseng, especially rare ginsenoside is the molecule which plays the functions of resisting tumor, resisting inflammation, improving immunity and the like. Because wild ginseng has low content of saponin, the source of rare ginsenoside is very limited, and the application of the wild ginseng in functional foods and medicines is limited.

Therefore, there is a great need in the production practice to provide a method for preparing ginsenosides with high efficiency and yield.

Disclosure of Invention

The invention mainly aims to provide a biological preparation method of ginsenoside with high yield;

the above object of the present invention is achieved by the following technical solutions:

a biological preparation method of ginsenoside comprises: (1) inducing and producing the ginseng adventitious root by a tissue culture mode; (2) transferring the induced ginseng adventitious root into a liquid culture medium in a rotary shaking table for culture; (3) transferring the ginseng adventitious roots from the shake flask to a bioreactor to culture for a period of time, and adding methyl jasmonate to induce the increase of the content of ginsenoside; (4) extracting ginsenoside from adventitious root after induction culture.

Wherein the method for inducing and producing the ginseng adventitious roots in the step (1) by the tissue culture mode comprises the following steps: (a) inoculating the ginseng root slice to the solution containing 1.0mg L^-12,4-D+0.1mg L^-1kinetin+30g L^-1Inducing callus on the inducing culture medium of cane sugar; (b) callus was transferred to 5.0mg L^-1Carrying out callus propagation on an MS culture medium of IBA; (c) in the presence of 3.0mg L^-1IBA and 30g L^-1The callus was cultured on a sucrose MS solid medium, and the generation of adventitious roots was induced from the callus.

The step (3) of adding methyl jasmonate is to add 200 mu M of methyl jasmonate to induce the content of ginsenoside to be increased; preferably, the ginseng adventitious roots are transferred from the shake flask to the bioreactor and cultured for 50 days, and 200 μ M methyl jasmonate is added to induce the increase of the ginsenoside content.

The method for extracting ginsenoside from the adventitious roots of ginseng in the step (4) comprises the following steps: (a) adding Ginseng radix powder into methanol/water mixture at 37 deg.C overnight; (b) extracting with methanol as solvent by ultrasonic instrument: (c) filtering the supernatant of the extractive solution for sterilization, evaporating to remove methanol, and dissolving the residue in water; wherein the methanol/water mixture of step (a) consists of methanol and water in a volume ratio of 4: 1; in the step (b), 80% methanol is used as a solvent to extract for 3 times at room temperature of an ultrasonic instrument, and the extraction time is 0.5h each time.

In order to further improve the content of rare ginsenoside in the adventitious root, the beta-glucosidase is adopted as the invertase, the ginsenoside mixed solution extracted from the adventitious root is used as the conversion substrate to carry out enzymatic conversion reaction, and the content of rare ginsenoside in the conversion product can be obviously improved.

The invention discovers that the content of rare ginsenoside in the converted product can be effectively improved by adding beta-glucosidase into a PPD type ginsenoside mixture for enzymatic reaction; wherein, the beta-glucosidase is preferably any one or any combination of more than one of BglSk, BglPm, Bgp1, BglBX10, Tpebgl3 or Abf 22-3; wherein the enzymatic reaction may be a reaction of a beta-glucosidase solution with a PPD-type ginsenoside mixture in 50mM sodium phosphate buffer (pH8.0) at 37 ℃.

UPLC analysis after the enzymatic reaction is completed detects the relative content of ginsenoside, and compared with untreated control, the results show that Bgpl, BglBX10 and Abf22-3 convert Rd into Rg3, and Tpebgl3 converts Rb1 and Rd into Rg 3. The generation amount of Rg3 in the four reactions is about 15-30% of the PPD type ginsenoside mixture, and the peak value of Tpebgl3 is reached when the concentration is 149.74 +/-5.67 mg. All combined enzyme treatments of BglPm with the other four enzymes with C-20 hydrolytic activity (Bgpl, BglBX10, Tpebgl3 and Abf22-3) produced over 40% Rh2, and over 10% CMc1 and CO. The combination of BglSk with these four enzymes can yield the right amount of Rg3, Gyp75, CMc, CY and Rh2, with a ratio between 10% and 20%. BglPm and BglSk both produce high proportions of CK (277.61 + -4.27 mg, 34.22 + -1.91%). In all transformation processes, 3.0g of PPD-type ginsenoside mixture was hydrolyzed by BglPm + Bgp1 combination to obtain Rh2 with highest yield (326.61 + -7.04 mg) accounting for 10.89 + -0.23% of the PPD-type ginsenoside mixture

The beta-glucosidase BglSk, BglPm, Bgp1, BglBX10, Tpebgl3 or Abf22-3 can be purchased from commercial sources or prepared into recombinant beta-glucosidase by adopting conventional genetic engineering means in the field, and can be applied to the invention.

In order to improve the utilization rate of the enzyme and the separation efficiency of the beta-glucosidase and the product, the invention further immobilizes the beta-glucosidase to obtain the immobilized beta-glucosidase, and the immobilized beta-glucosidase is adopted to carry out enzymatic reaction, so that the utilization rate of the enzyme and the separation efficiency of the enzyme and the product can be effectively improved.

As a reference, the β -glucosidase immobilization method comprises immobilizing enzyme with polysulfone hollow fiber Membrane (MWCO) with molecular weight of 30kDa, equilibrating the membrane in phosphate buffer (pH8.0) for 30min, immobilizing enzyme with pressure-driven filtration, and immobilizing β -glucosidase solution (0.5mg mL)^-1) Using peristaltic pump at 10mL min^-1Is pumped through the lumen of the membrane, and the circulating 2h β -glucosidase is embedded in an asymmetric, aqueous porous membrane and is encapsulated by 30kDa fibers on the inside surfaceFixing the membrane to obtain the product.

The invention takes the hollow fiber membrane immobilized recombinant His-Bgp1 and His-BglPm as examples to carry out the experiment of converting the PPD type ginsenoside mixture into Rh2, and verifies the effectiveness of the method. The immobilized beta-glucosidase has high repeated utilization rate, and the relative activity of the immobilized beta-glucosidase is kept at about 85% after 9 cycles of repeated catalytic reaction on day 9. The immobilized β -glucosidase maintained 40% of the initial activity even after 15 consecutive cycles of repeated catalytic reactions.

The invention provides a biological preparation method of combined ginsenoside, which comprises tissue culture, enzyme immobilization and hydrolysis methods to obtain rare ginsenoside, wherein 6 β -glycosidases are screened according to glycosidase hydrolysis route for enzymatic hydrolysis and are combined for use, and the yield range of the rare ginsenoside Rg3, Rh2, CK, Gyp75, CMc and CY in the converted product is 5.54-32.66mg L^-1. Under the optimized conditions of pH and temperature, the immobilized BglPm and Bgp1 can improve the yield of Rh2 by 7 percent, and the maximum yield can reach 51.17mg L^-1(17.06% of the mixture of PPD-type ginsenosides). The biological preparation method provides an efficient way for obtaining various rare ginsenosides, and can replace a method for directly extracting the ginsenosides from ginseng.

Drawings

FIG. 1 is a schematic diagram of a rare ginsenoside biotransformation system.

FIG. 2 biotransformation pathway for glycosidase conversion of PPD-type ginsenosides.

FIG. 3 culturing ginseng adventitious roots in a bioreactor for efficient production of ginsenosides; (A) inoculating adventitious roots of ginseng to a solid multiplication culture medium for growth; (B) proliferation of adventitious roots on solid medium for 1 month; (C) further culturing the adventitious roots in a liquid culture medium for 7 days; (D) adventitious roots were expanded to a 10L bioreactor and grown for 2 months; (E) the content of a large amount of ginsenoside in adventitious roots and 5-year-old wild ginseng roots.

FIG. 4 is a graph showing SDS-PAGE results of purified glycosidase.

FIG. 5 is a plot of optimum temperature vs. pH contours for glycosidases.

FIG. 6 UPLC analysis of the conversion of a mixture of glycosidase combination treated PPD-type ginsenosides for 24 hours.

FIG. 7 heatmap of the relative content of PPD-type ginsenosides in glycosidase conversion reactions.

Figure 8 yield of each rare ginsenoside in all glycosidase combinations.

FIG. 9 Loading efficiency and thermostability of immobilized β -glucosidase. (A) Fixing Bglpm and Bgp1 on a hollow fiber column; (B) storage stability determination of immobilized Bgp1 and BglPm.

Fig. 10 UPLC results of combined conversion of PPD-type ginsenoside mixtures by β -glucosidase Bgp1 and BglPm.

Detailed Description

The invention is further described below in conjunction with specific embodiments, the advantages and features of which will become apparent from the description. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.