Preparation method for improving red ginseng saponin RG3

Abstract

The invention discloses a preparation method for improving radix ginseng rubra saponin RG3, and relates to the technical field of pharmacy, according to the method, by establishing a double-enzyme synergistic catalysis system of beta-glucosidase and alpha-L-arabinosidase, stereoselective hydrolysis of C-20 glucosidic bonds is achieved, and the selectivity reaches 95% or above; according to the present invention, by using the directionally screened strain, optimizing the fermentation condition and adding the 0.1% ginsenoside Rb1 inducer, the target enzyme gene expression quantity is increased by 2.5 times, and the enzyme activity is increased by 50%; by adding a compound stabilizer system containing 5% of glycerol and 0.1% of Tween-80, the enzyme activity half-life period is prolonged by 1.8 times, and the 48-hour conversion rate is 90% or above under the temperature control of 38-42 DEG C; and an ultrafiltration-resin-crystallization multiple separation strategy is adopted, so that the purity of the final product is ensured to be greater than or equal to 98%, and a reliable process basis is provided for large-scale production.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to a method for preparing red ginsenoside RG3. Background Art

[0002] Ginsenoside RG3 is an important secondary metabolite of ginseng, belonging to the protopanaxadiol type saponin, with significant anti-tumor, antioxidant, anti-inflammatory, immunomodulatory and neuroprotective pharmacological activities. Studies have shown that RG3 can exert anti-cancer effects through multiple mechanisms such as inducing tumor cell apoptosis, inhibiting angiogenesis and metastasis, and has shown good application prospects in the treatment of various malignant tumors such as breast cancer, lung cancer, and gastric cancer. At the same time, RG3 also has the effects of improving cognitive function, protecting the cardiovascular system and regulating blood sugar, and is a functional active ingredient with important development value.

[0003] However, the content of RG3 in natural ginseng is extremely low (usually less than 0.001%), which is difficult to meet the needs of clinical applications. At present, RG3 is mainly obtained by chemical or biological transformation of ginsenosides (such as Rb1, Rb2, Rc, etc.) in industrial production. Among them, the traditional acid hydrolysis method is the most commonly used process route, that is, the specific glycosidic bonds in the main ginsenoside molecules of ginseng are hydrolyzed under strong acid conditions (pH 1.8-2.2) and high temperature (80-90℃). However, this method has several serious problems: first, the harsh reaction conditions lead to serious product degradation and low selectivity (usually between 65-75%), and a variety of by-products such as Rg5 and Rk1 will be produced; second, the reaction process is difficult to accurately control, and it is easy to over-hydrolyze or incompletely hydrolyze, produce non-target products, and significantly increase the difficulty and cost of subsequent separation and purification; third, the strong acid conditions not only cause serious corrosion to the equipment, but also produce a large amount of waste acid, and the environmental pollution problem is prominent, which does not meet the current development requirements of green manufacturing.

[0004] As a new preparation technology, biotransformation has attracted wide attention for its mild reaction conditions, high selectivity, and environmental friendliness. This method mainly uses biological enzymes such as glycosidase to selectively cut glycosidic bonds to achieve directional conversion of RG3. However, the existing biotransformation technology still faces many challenges: first, there are few highly active strains that can be used for industrial production, and the enzyme yield and activity are generally not high; second, the recognition and hydrolysis efficiency of a single enzyme for glycosidic bonds is limited, and it is easy to be inactivated during the reaction process, resulting in a generally low RG3 conversion rate (usually between 60-70%); third, the stability of enzyme preparations is poor, and the activity decreases rapidly during a long reaction process, affecting the conversion efficiency; fourth, there are many technical bottlenecks in the amplification of the biotransformation process, such as optimization of fermentation conditions, maintenance of enzyme activity, separation and purification, etc., which have not been well solved. These problems have seriously restricted the industrial application of RG3 biotransformation technology. Summary of the invention

[0005] In order to solve or partially solve the problems existing in the related art, the present invention provides a method for preparing red ginsenoside RG3, comprising the following steps: S1 reagent preparation Prepare 0.15-0.25M phosphate buffer and adjust the pH to 5.2-5.8; weigh 2-4g of the ginsenoside Rb1 / Rb2 / Rc mixture extracted from red ginseng and dissolve it in 100mL buffer to make a 2-4% substrate solution; prepare the enzyme solution according to the ratio of 80-100U / g of β-glucosidase, 40-60U / g of α-L-arabinosidase, and 20-30U / g of auxiliary enzyme system; prepare a stabilizer mixed solution containing 4-6% glycerol and 0.05-0.15% Tween-80. At the same time, prepare the basal culture medium, each liter of culture medium contains: 15-25g of glucose, 3-7g of yeast extract, 8-12g of peptone, 1.5-2.5g of KH2PO4, and 0.3-0.7g of MgSO4, sterilize at 120-122℃ for 15-20 minutes for use; S2 bacterial culture The strain activation starts with Bacillus licheniformis stored at -80℃. First, it is inoculated into the slant medium. After culturing at 26-30℃ for 45-50 hours, a single colony is picked and inoculated into the seed medium. It is cultured at 26-30℃ and 160-200rpm for 22-26 hours. In the fermentation culture stage, the seed liquid is transferred into the fermentation medium at an inoculation rate of 4-6%. The fermentation temperature is controlled at 26-30℃, the stirring speed is 160-200rpm, and the dissolved oxygen level is maintained at 35-65%. After 22-26 hours of fermentation, 0.08-0.12% ginsenoside Rb1 is added as an inducer; S3 enzymatic conversion During the enzymatic conversion stage, add the pre-prepared substrate solution and stabilizer mixture to the fermentation broth, adjust the reaction temperature to 38-42°C, and maintain stirring at 160-200rpm. During the conversion process, samples are taken every 1.5-2.5 hours to monitor the progress of the conversion, and the reaction lasts for 45-50 hours. After the conversion is completed, enter the separation and purification stage: first, centrifuge the fermentation broth at 3800-4200rpm for 12-18 minutes, filter the supernatant through a 0.45μm filter membrane, and use an 8-12kDa ultrafiltration membrane for membrane separation; S4 separation and purification The separated filtrate is purified by D101 macroporous resin column, and polar impurities are removed by elution with pure water in turn, and then gradient elution is performed with 25-35%, 45-55%, and 65-75% ethanol to collect the target elution segment. The collected target component is concentrated under reduced pressure to a suitable concentration, and an appropriate amount of crystallization aid is added, and crystallization is allowed to stand at 2-6°C for 22-26 hours. After the crystallization is completed, the crystals are collected by suction filtration and vacuum dried at 38-42°C to obtain the finished product.

[0006] Furthermore, the pH value of the phosphate buffer is preferably 5.5±0.1, and the concentration is preferably 0.2 M; and the concentration of the substrate solution is preferably 3%.

[0007] Furthermore, in the enzyme solution, the activity unit ratio of β-glucosidase, α-L-arabinosidase, and auxiliary enzyme system is preferably 90:50:25 U / g.

[0008] Furthermore, the preferred dosage of each component in the basic culture medium is: glucose 20 g / L, yeast extract 5 g / L, peptone 10 g / L, KH2PO4 2 g / L, MgSO4 0.5 g / L.

[0009] Furthermore, the temperature during the bacterial culture stage is preferably 28° C., the stirring speed is preferably 180 rpm, and the fermentation time is preferably 24 hours.

[0010] Furthermore, the temperature of the enzymatic conversion stage is preferably 40° C., the stirring speed is preferably 180 rpm, and the reaction time is preferably 48 hours.

[0011] Furthermore, during the ethanol gradient elution process, 30%, 50%, and 70% ethanol aqueous solutions are preferably used in sequence for elution.

[0012] Furthermore, the temperature of the crystallization process is preferably 4° C., and the crystallization time is preferably 24 hours.

[0013] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present invention.

[0014] Beneficial technical effects of the present invention: The method of the present invention achieves stereoselective hydrolysis of the C-20 glycosidic bond with a selectivity of more than 95% by establishing a dual-enzyme synergistic catalytic system of β-glucosidase and α-L-arabinosidase; adopts a strain that has been screened, optimizes fermentation conditions and adds 0.1% ginsenoside Rb1 inducer, so that the target enzyme gene expression amount is increased by 2.5 times and the enzyme activity is increased by 50%; by adding a composite stabilizer system of 5% glycerol and 0.1% Tween-80, the enzyme activity half-life is extended by 1.8 times, and a 48-hour conversion rate of more than 90% is achieved under a temperature control of 38-42°C; and adopts a multiple separation strategy of ultrafiltration-resin-crystallization to ensure that the purity of the final product is ≥98%, providing a reliable process basis for large-scale production. DETAILED DESCRIPTION

[0015] The optional embodiments of the present invention will be described in more detail below. Although the optional embodiments of the present invention are described, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to make the present invention more thorough and complete, and to fully convey the scope of the present invention to those skilled in the art.

[0016] The terms used in this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The singular forms of "a", "said" and "the" used in this application and the appended claims are also intended to include plural forms unless the context clearly indicates other meanings. It should also be understood that the term "and / or" used herein refers to and includes any or all possible combinations of one or more associated listed items.

[0017] The present invention provides a method for preparing red ginsenoside RG3, comprising the following steps: S1 reagent preparation Prepare 0.15-0.25M phosphate buffer and adjust the pH to 5.2-5.8 with hydrochloric acid and sodium hydroxide; weigh 2-4g of a mixture of ginsenosides Rb1 / Rb2 / Rc extracted from red ginseng (the mass ratio of Rb1, Rb2 and Rc is 3:1:1) and dissolve it in 100mL of buffer to prepare a 2-4% substrate solution; prepare an enzyme solution according to the ratio of 80-100 U / g of β-glucosidase, 40-60 U / g of α-L-arabinosidase, and 20-30 U / g of an auxiliary enzyme system (the auxiliary enzyme system consists of cellulase, pectinase and xylanase, and the activity unit ratio of the three enzymes is 2:1:1); and prepare a stabilizer mixed solution containing 4-6% glycerol and 0.05-0.15% Tween-80. Meanwhile, prepare the basic culture medium, each liter of which contains: 15-25 g glucose, 3-7 g yeast extract, 8-12 g peptone, 1.5-2.5 g KH2PO4, 0.3-0.7 g MgSO4, sterilize at 120-122°C for 15-20 minutes for later use; S2 bacterial culture The strain activation starts with Bacillus licheniformis stored at -80°C (the Bacillus licheniformis used in this application was purchased from Zhengzhou Best Food Additive Co., Ltd.), which is first inoculated into a slant culture medium, cultured at 26-30°C for 45-50 hours, and a single colony is picked and inoculated into a seed culture medium, and cultured at 26-30°C and 160-200rpm for 22-26 hours. During the fermentation culture stage, the seed liquid is transferred into the fermentation culture medium at an inoculation rate of 4-6%, the fermentation temperature is controlled at 26-30°C, the stirring speed is 160-200rpm, the dissolved oxygen level is maintained at 35-65%, and after 22-26 hours of fermentation, 0.08-0.12% ginsenoside Rb1 is added as an inducer; S3 enzymatic conversion During the enzymatic conversion stage, add the pre-prepared substrate solution and stabilizer mixture to the fermentation broth, adjust the reaction temperature to 38-42°C, and maintain stirring at 160-200rpm. During the conversion process, samples are taken every 1.5-2.5 hours to monitor the progress of the conversion, and the reaction lasts for 45-50 hours. After the conversion is completed, enter the separation and purification stage: first, centrifuge the fermentation broth at 3800-4200rpm for 12-18 minutes, filter the supernatant through a 0.45μm filter membrane, and use an 8-12kDa ultrafiltration membrane for membrane separation; S4 separation and purification The separated filtrate is purified by D101 macroporous resin column, and polar impurities are removed by elution with pure water in turn, and then gradient elution is performed with 25-35%, 45-55%, and 65-75% ethanol to collect the target elution segment. The collected target component is concentrated under reduced pressure to a suitable concentration, and an appropriate amount of crystallization aid is added (with anhydrous ethanol as a crystallization aid, and slowly added at a ratio of 1:2 (V / V) of the concentrate to anhydrous ethanol), and crystallization is allowed to stand at 2-6°C for 22-26 hours. After the crystallization is completed, the crystals are collected by suction filtration and vacuum dried at 38-42°C to obtain the finished product.

[0018] This approach achieves systematic innovation at the molecular, cellular and process levels, as follows: 1. Molecular Mechanism This method designs a specific dual enzyme catalytic system. β-glucosidase can specifically recognize and hydrolyze the β-D-glucosidic bond at the C-20 position, while α-L-arabinosidase selectively acts on the α-L-arabinosidic bond. The synergistic action of the two enzymes establishes a stereoselective hydrolysis network. Experimental data show that the selective hydrolysis efficiency of the target glycosidic bond of this system is more than 95%, which is significantly higher than the 65-75% of the traditional chemical hydrolysis method.

[0019] The ionic strength (0.15-0.25M) and pH value (5.2-5.8) of the buffer system optimize the formation of the enzyme-substrate complex by affecting the surface charge distribution of the enzyme molecule. Experiments have confirmed that at pH 5.5, the enzyme activity reaches its maximum value and the conformational stability of the enzyme molecule increases by 30%. The addition of nonionic surfactant Tween-80 (0.05-0.15%) reduces interfacial tension, making it easier for substrate molecules to enter the enzyme active center, thereby improving the accessibility of the substrate.

[0020] 2. Cellular Metabolism Regulation Mechanism During the fermentation process, a metabolic environment conducive to the expression of the enzyme system is established by precisely controlling the dissolved oxygen level (35-65%) and the stirring speed (160-200rpm). The control of the dissolved oxygen level directly affects the respiratory metabolism and energy supply of the cells, thereby regulating the synthesis of the target enzyme system.

[0021] Adding 0.08-0.12% ginsenoside Rb1 as an inducer can activate the transcription of related operons and promote the expression of the target enzyme system. Experimental data show that the enzyme activity increased by 50-60% after induction. The optimized culture medium formula (glucose 15-25g / L, yeast extract 3-7g / L, etc.) is adjusted by the C / N ratio to make the metabolic flow conducive to the accumulation of target products.

[0022] 3. Process Control Mechanism 4-6% glycerol is added to the reaction system as an osmotic protectant to stabilize the enzyme molecular conformation by forming a hydrogen bond network. Experiments show that glycerol in this concentration range can extend the half-life of enzyme activity by 1.8 times. The temperature is controlled in the range of 38-42°C, which not only ensures sufficient reaction rate but also avoids enzyme inactivation. After 48 hours of reaction, the conversion rate can reach more than 90%.

[0023] The separation and purification adopted multiple separation mechanisms based on molecular size and polarity differences. The ultrafiltration membrane with a molecular weight cutoff of 8-12kDa can effectively remove large molecular impurities; the D101 macroporous resin selectively adsorbs the target product through hydrophobic interaction, and uses 25-35%, 45-55%, and 65-75% ethanol for gradient elution, and uses polarity differences to achieve precise separation. The final crystallization process is carried out at 2-6°C. By controlling the temperature and concentration gradient, the product molecules are promoted to be arranged in an orderly manner, and a crystalline product with a purity of ≥98% is obtained.

[0024] 4. Summary of Method Advantages Selectivity: The dual-enzyme synergistic catalytic system achieves precise recognition and hydrolysis of specific glycosidic bonds.

[0025] Efficiency: Optimized fermentation conditions and inducible expression strategies significantly increased enzyme production and activity.

[0026] Stability: Multiple protection mechanisms ensure the stability of enzyme molecules and the controllability of reactions.

[0027] Purity: Multi-level separation strategy ensures high purity of the product.

[0028] In one embodiment of the present application, the pH value of the phosphate buffer is preferably 5.5±0.1, and the concentration is preferably 0.2 M; the concentration of the substrate solution is preferably 3%.

[0029] In one embodiment of the present application, in the enzyme solution ratio, the activity unit ratio of β-glucosidase, α-L-arabinosidase, and auxiliary enzyme system is preferably 90:50:25 U / g.

[0030] In one embodiment of the present application, the preferred dosage of each component in the basic culture medium is: glucose 20g / L, yeast extract 5g / L, peptone 10g / L, KH2PO4 2g / L, MgSO4 0.5g / L.

[0031] In one embodiment of the present invention, the temperature during the bacterial culture stage is preferably 28°C, the stirring speed is preferably 180 rpm, and the fermentation time is preferably 24 hours.

[0032] In one embodiment of the present application, the temperature of the enzymatic conversion stage is preferably 40° C., the stirring speed is preferably 180 rpm, and the reaction time is preferably 48 hours.

[0033] In one embodiment of the present application, during the ethanol gradient elution, preferably 30%, 50%, and 70% ethanol aqueous solutions are used in sequence for elution.

[0034] In one embodiment of the present invention, the temperature of the crystallization process is preferably 4°C, and the crystallization time is preferably 24 hours.

[0035] For the purpose of greater clarity, the invention is described in detail through the following examples.

[0036] Example 1: Preparation of S1 reagent: prepare 0.15M phosphate buffer and adjust the pH to 5.2 with hydrochloric acid and sodium hydroxide; weigh 2g of the ginsenoside Rb1 / Rb2 / Rc mixture extracted from red ginseng (the mass ratio of Rb1, Rb2 and Rc is 3:1:1) and dissolve it in 100mL buffer to prepare a 2% substrate solution; prepare enzyme solution according to the ratio of 80 U / g of β-glucosidase, 40 U / g of α-L-arabinosidase, and 20 U / g of auxiliary enzyme system (the auxiliary enzyme system consists of cellulase, pectinase and xylanase, and the activity unit ratio of the three enzymes is 2:1:1); prepare a stabilizer mixed solution containing 4% glycerol and 0.05% Tween-80. At the same time, prepare a basal culture medium, each liter of culture medium contains: 15g glucose, 3g yeast extract, 8g peptone, 1.5g KH2PO4, 0.3g MgSO4, sterilize at 120℃ for 15 minutes for use; S2 strain culture: strain activation starts with Bacillus licheniformis stored at -80°C. First, it is inoculated into a slant medium. After culturing at 26°C for 45 hours, a single colony is picked and inoculated into a seed medium, and then cultured at 26°C and 160rpm for 22 hours. During the fermentation culture stage, the seed liquid is transferred into the fermentation medium at a 4% inoculation rate. The fermentation temperature is controlled at 26°C, the stirring speed is 160rpm, and the dissolved oxygen level is maintained at 35%. After 22 hours of fermentation, 0.08% ginsenoside Rb1 is added as an inducer; S3 enzymatic conversion: During the enzymatic conversion stage, the pre-prepared substrate solution and stabilizer mixture are added to the fermentation broth, the reaction temperature is adjusted to 38°C, and the stirring is maintained at 160rpm. During the conversion process, samples are taken every 1.5 hours to monitor the progress of the conversion, and the reaction duration is 45 hours. After the conversion is completed, the separation and purification stage begins: first, the fermentation broth is centrifuged at 3800rpm for 12 minutes, and the supernatant is filtered through a 0.45μm filter membrane and then separated using an 8kDa ultrafiltration membrane; S4 separation and purification: The separated filtrate is purified by D101 macroporous resin column, and pure water is used to remove polar impurities in turn, and then gradient elution is performed with 25%, 45%, and 65% ethanol, and the target elution segment is collected (5 times the column volume of pure water is used to remove polar impurities, and then 3 times the column volume of 25% ethanol, 5 times the column volume of 45% ethanol, and 3 times the column volume of 65% ethanol are used for gradient elution, and the 45% ethanol eluate is collected). The collected target component is concentrated under reduced pressure to an appropriate concentration (50 mg / mL), and an appropriate amount of crystallization aid is added (absolute ethanol is used as a crystallization aid, and is slowly added at a ratio of 1:2 (V / V) of the concentrate to anhydrous ethanol), and crystallization is allowed to stand at 2°C for 22 hours. After the crystallization is completed, the crystals are collected by suction filtration and vacuum dried at 38°C to obtain the finished product, and the purity of the finished product is >98.8%.

[0037] Example 2: Preparation of S1 reagent: Prepare 0.2M phosphate buffer and adjust the pH to 5.5 with hydrochloric acid and sodium hydroxide; weigh 3g of the ginsenoside Rb1 / Rb2 / Rc mixture extracted from red ginseng (the mass ratio of Rb1, Rb2 and Rc is 3:1:1) and dissolve it in 100mL buffer to prepare a 3% substrate solution; prepare enzyme solution according to the ratio of 90 U / g of β-glucosidase, 50 U / g of α-L-arabinosidase, and 25U / g of auxiliary enzyme system (the auxiliary enzyme system consists of cellulase, pectinase and xylanase, and the activity unit ratio of the three enzymes is 2:1:1); prepare a stabilizer mixed solution containing 5% glycerol and 0.1% Tween-80. At the same time, prepare a basal culture medium, each liter of culture medium contains: 20g of glucose, 5g of yeast extract, 10g of peptone, 2g of KH2PO4, and 0.5g of MgSO4, sterilize at 121℃ for 17.5 minutes for use; S2 strain culture: strain activation starts with Bacillus licheniformis stored at -80℃. First, it is inoculated into a slant medium. After culturing at 28℃ for 47.5 hours, a single colony is picked and inoculated into a seed medium. It is cultured at 28℃ and 180rpm for 24 hours. During the fermentation culture stage, the seed liquid is transferred into the fermentation medium at a 5% inoculation rate. The fermentation temperature is controlled at 28℃, the stirring speed is 180rpm, and the dissolved oxygen level is maintained at 50%. After 24 hours of fermentation, 0.1% ginsenoside Rb1 is added as an inducer; S3 enzymatic conversion: During the enzymatic conversion stage, the pre-prepared substrate solution and stabilizer mixture are added to the fermentation broth, the reaction temperature is adjusted to 40°C, and the stirring is maintained at 180rpm. During the conversion process, samples are taken every 2 hours to monitor the progress of the conversion, and the reaction duration is 47.5 hours. After the conversion is completed, the separation and purification stage begins: first, the fermentation broth is centrifuged at 4000rpm for 15 minutes, and the supernatant is filtered through a 0.45μm filter membrane and then separated using a 10kDa ultrafiltration membrane; S4 separation and purification: The separated filtrate is purified by D101 macroporous resin column, and pure water is used to elute to remove polar impurities, and then gradient elution is performed with 30%, 50%, and 70% ethanol, and the target elution segment is collected (5 times the column volume of pure water is used to elute to remove polar impurities, and then 3 times the column volume of 30% ethanol, 5 times the column volume of 50% ethanol, and 3 times the column volume of 70% ethanol are used for gradient elution, and 45% ethanol eluate is collected). The collected target component is concentrated under reduced pressure to an appropriate concentration (55 mg / mL), and an appropriate amount of crystallization aid is added (absolute ethanol is used as a crystallization aid, and is slowly added at a ratio of 1:2 (V / V) of the concentrate to anhydrous ethanol), and crystallization is allowed to stand at 4°C for 24 hours. After the crystallization is completed, the crystals are collected by suction filtration and vacuum dried at 40°C to obtain the finished product, and the purity of the finished product is >99.7%.

[0038] Example 3: Preparation of S1 reagent: Prepare 0.25M phosphate buffer and adjust the pH to 5.8 with hydrochloric acid and sodium hydroxide; weigh 4g of the mixture of ginsenosides Rb1 / Rb2 / Rc extracted from red ginseng (the mass ratio of Rb1, Rb2 and Rc is 3:1:1) and dissolve it in 100mL buffer to prepare a 4% substrate solution; prepare enzyme solution according to the ratio of 100 U / g of β-glucosidase, 60 U / g of α-L-arabinosidase, and 30 U / g of auxiliary enzyme system (the auxiliary enzyme system consists of cellulase, pectinase and xylanase, and the activity unit ratio of the three enzymes is 2:1:1); prepare a stabilizer mixed solution containing 6% glycerol and 0.15% Tween-80. At the same time, prepare a basal culture medium, each liter of culture medium contains: 25g glucose, 7g yeast extract, 12g peptone, 2.5g KH2PO4, 0.7g MgSO4, sterilize at 122℃ for 20 minutes for use; S2 strain culture: strain activation starts with Bacillus licheniformis stored at -80℃. First, it is inoculated into a slant medium. After culturing at 30℃ for 50 hours, a single colony is picked and inoculated into a seed medium. It is cultured at 30℃ and 200rpm for 26 hours. During the fermentation culture stage, the seed liquid is transferred into the fermentation medium at a 6% inoculation rate. The fermentation temperature is controlled at 30℃, the stirring speed is 200rpm, and the dissolved oxygen level is maintained at 65%. After 26 hours of fermentation, 0.12% ginsenoside Rb1 is added as an inducer; S3 enzymatic conversion: During the enzymatic conversion stage, the pre-prepared substrate solution and stabilizer mixture are added to the fermentation broth, the reaction temperature is adjusted to 42°C, and the stirring is maintained at 200rpm. During the conversion process, samples are taken every 2.5 hours to monitor the progress of the conversion, and the reaction duration is 50 hours. After the conversion is completed, the separation and purification stage begins: first, the fermentation broth is centrifuged at 4200rpm for 18 minutes, and the supernatant is filtered through a 0.45μm filter membrane and then separated using a 12kDa ultrafiltration membrane; S4 separation and purification: The separated filtrate is purified by D101 macroporous resin column, and pure water is used to elute to remove polar impurities, and then gradient elution is performed with 35%, 55%, and 75% ethanol, and the target elution segment is collected (5 column volumes of pure water are used to elute to remove polar impurities, and then 3 column volumes of 35% ethanol, 5 column volumes of 55% ethanol, and 3 column volumes of 75% ethanol are used for gradient elution, and 45% ethanol eluate is collected). The collected target component is concentrated under reduced pressure to an appropriate concentration (60 mg / mL), and an appropriate amount of crystallization aid is added (absolute ethanol is used as a crystallization aid, and is slowly added at a ratio of 1:2 (V / V) of the concentrate to anhydrous ethanol), and crystallization is allowed to stand at 6°C for 26 hours. After the crystallization is completed, the crystals are collected by suction filtration and vacuum dried at 42°C to obtain the finished product, and the purity of the finished product is >99.1%.

[0039] Experimental example 1 Experimental plan: Experimental groups (3 parallels per group): Group A: Chemical hydrolysis (control group) 2M hydrochloric acid solution 80℃ water bath Reaction 4 hours Group B: Biotransformation method (method of Example 2) Reaction system (100 mL): Substrate: 3g ginsenoside Rb1 / Rb2 / Rc mixture extracted from red ginseng (3%) Stabilizer: 5% glycerol + 0.1% Tween-80 Sampling time: 0h, 12h, 24h, 36h, 48h Detection indicators: HPLC determination of RG3 content Calculation of conversion and selectivity LC-MS analysis of by-product composition Experimental results: Dynamic changes of RG3 content Table 1. Changes in RG3 content in different transformation methods (mg / mL) 48h endpoint test results Table 2. Comparison of conversion effects of the two methods Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made based on the present invention. Therefore, these modifications and improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing red ginsenoside RG3, characterized in that: The steps include: S1 reagent preparation: Prepare 0.15-0.25M phosphate buffer and adjust the pH to 5.2-5.8; weigh 2-4g of the ginsenoside Rb1 / Rb2 / Rc mixture extracted from red ginseng and dissolve it in 100mL buffer to prepare a 2-4% substrate solution; prepare enzyme solution according to the ratio of 80-100 U / g of β-glucosidase, 40-60 U / g of α-L-arabinosidase, and 20-30 U / g of auxiliary enzyme system; separately prepare a stabilizer mixed solution containing 4-6% glycerol and 0.05-0.15% Tween-80, and prepare a basal culture medium at the same time, each liter of culture medium contains: 15-25g of glucose, 3-7g of yeast extract, 8-12g of peptone, 1.5-2.5g of KH2PO4, and 0.3-0.7g of MgSO4, and sterilize at 120-122℃ for 15-20 minutes for use; S2 bacterial culture: The strain activation starts with Bacillus licheniformis stored at -80°C. First, it is inoculated into a slant medium. After culturing at 26-30°C for 45-50 hours, a single colony is picked and inoculated into a seed medium. The culture is carried out at 26-30°C and 160-200rpm for 22-26 hours. During the fermentation culture stage, the seed liquid is transferred into the fermentation medium at an inoculation rate of 4-6%. The fermentation temperature is controlled at 26-30°C, the stirring speed is 160-200rpm, and the dissolved oxygen level is maintained at 35-65%. After 22-26 hours of fermentation, 0.08-0.12% ginsenoside Rb1 is added as an inducer. S3 enzymatic conversion: In the enzymatic conversion stage, the pre-prepared substrate solution and stabilizer mixture are added to the fermentation broth, the reaction temperature is adjusted to 38-42°C, and the stirring is maintained at 160-200rpm. During the conversion process, samples are taken every 1.5-2.5 hours to monitor the progress of the conversion. The reaction lasts for 45-50 hours. After the conversion is completed, the separation and purification stage is entered: first, the fermentation broth is centrifuged at 3800-4200rpm for 12-18 minutes, and the supernatant is filtered through a 0.45μm filter membrane and then membrane separated using an 8-12kDa ultrafiltration membrane; S4 separation and purification: The separated filtrate is purified by a D101 macroporous resin column, and polar impurities are removed by elution with pure water in turn, and then gradient elution is performed with 25-35%, 45-55%, and 65-75% ethanol, and the target elution segment is collected. The collected target component is concentrated under reduced pressure to a suitable concentration, and an appropriate amount of crystallization aid is added. Crystallization is allowed to stand at 2-6°C for 22-26 hours. After the crystallization is completed, the crystals are collected by suction filtration and vacuum dried at 38-42°C to obtain the finished product.

2. The preparation method according to claim 1, characterized in that: The pH value of the phosphate buffer is 5.5±0.1, and the concentration is 0.2M; the concentration of the substrate solution is 3%.

3. The preparation method according to claim 1, characterized in that: In the enzyme solution, the activity unit ratio of β-glucosidase, α-L-arabinosidase and auxiliary enzyme system is 90:50:25 U / g.

4. The preparation method according to claim 1, characterized in that: The dosage of each component in the basic culture medium is: glucose 20g / L, yeast extract 5g / L, peptone 10g / L, KH2PO4 2g / L, MgSO4 0.5g / L.

5. The preparation method according to claim 1, characterized in that: The temperature during the bacterial culture stage was 28° C., the stirring speed was 180 rpm, and the fermentation time was 24 hours.

6. The preparation method according to claim 1, characterized in that: The temperature of the enzymatic conversion stage was 40° C., the stirring speed was 180 rpm, and the reaction time was 48 hours.

7. The preparation method according to claim 1, characterized in that: During the ethanol gradient elution, 30%, 50%, and 70% ethanol aqueous solutions are used in sequence for elution.

8. The preparation method according to claim 1, characterized in that: The temperature of the crystallization process is 4° C. and the crystallization time is 24 hours.

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