A preparation method for improving ginsenoside RG3

Abstract

The method of the present invention discloses a preparation method for improving ginsenoside RG3. The present invention application relates to the field of pharmaceutical technology. By establishing a dual-enzyme synergistic catalytic system of β-glucosidase and α-L-arabinofuranosidase, the method of the present invention realizes the stereoselective hydrolysis of the glycosidic bond at the C-20 position, with a selectivity of more than 95%. Using strains screened by orientation, by optimizing the fermentation conditions and adding 0.1% of the inducer ginsenoside Rb1, the expression level of the target enzyme gene 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 half-life of the enzyme activity is extended by 1.8 times, and a conversion rate of more than 90% is achieved in 48 hours under the temperature control of 38-42°C. Adopting a multiple separation strategy of ultrafiltration-resin-crystallization ensures that the purity of the final product is ≥98%, providing a reliable process basis for large-scale production.

Description

Technical Field

[0001] This invention application relates to the field of pharmaceutical technology, and particularly relates to a preparation method for improving ginsenoside RG3. Background Art

[0002] Ginsenoside RG3 is an important secondary metabolite in ginseng, belonging to the protopanaxadiol type saponin, and has various pharmacological activities such as significant anti-tumor, antioxidant, anti-inflammatory, immunomodulatory and neuroprotective effects. Research shows that RG3 can exert anti-cancer effects through multiple mechanisms such as inducing apoptosis of tumor cells, inhibiting angiogenesis and metastasis, and shows good application prospects in the treatment of various malignant tumors such as breast cancer, lung cancer, gastric cancer, etc. At the same time, RG3 also has effects such as 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%), making it difficult to meet the clinical application requirements. Currently, in industrial production, RG3 is mainly obtained by chemical or biological transformation of main ginsenosides (such as Rb1, Rb2, Rc, etc.). Among them, the traditional acid hydrolysis method is the most commonly used process route, that is, under strong acid conditions (pH 1.8 - 2.2) and high temperature (80 - 90°C), specific glycosidic bonds in the main ginsenoside molecules are hydrolyzed. But this method has multiple serious problems: First, the harsh reaction conditions lead to severe degradation of the product, with low selectivity (usually between 65 - 75%), and at the same time, a variety of by-products will be generated, such as Rg5, Rk1, etc.; Second, the reaction process is difficult to precisely control, prone to over-hydrolysis or incomplete hydrolysis, generating non-target products, significantly increasing the difficulty and cost of subsequent separation and purification; Third, the strong acid conditions not only cause serious corrosion to the equipment, but also generate a large amount of waste acid, with prominent environmental pollution problems, not meeting the current development requirements of green manufacturing.

[0004] As a new preparation technology, the biological transformation method has received wide attention due to its mild reaction conditions, high selectivity, environmental friendliness and other advantages. This method mainly uses biological enzymes such as glycosidases to selectively cleave glycosidic bonds to achieve the directional transformation of RG3. However, the existing biological transformation technologies still face many challenges: First, there are few highly active strains available for industrial production, and the yield and activity of enzymes are generally not high; Second, the recognition and hydrolysis efficiency of a single enzyme species for glycosidic bonds is limited, and it is easily inactivated during the reaction process, resulting in generally low RG3 conversion rates (usually between 60 - 70%); Third, the stability of enzyme preparations is poor, and the activity rapidly decreases during the long reaction process, affecting the conversion efficiency; Fourth, there are many technical bottlenecks in the scale-up process of the biological transformation process, such as problems in fermentation condition optimization, enzyme activity maintenance, separation and purification, etc. that have not been well solved. These problems severely restrict the industrial application of RG3 biological transformation technology. Summary of the Invention

[0005] To solve or partially solve the problems existing in the related technologies, the present invention application provides a method for improving the preparation of ginsenoside RG3, comprising the following steps:

[0006] S1 Reagent preparation

[0007] Prepare 0.15 - 0.25 M phosphate buffer and adjust the pH to 5.2 - 5.8; Weigh 2 - 4 g of the mixture of ginsenoside Rb1 / Rb2 / Rc extracted from red ginseng and dissolve it in 100 mL of the buffer to prepare a 2 - 4% substrate solution; Prepare the enzyme solution according to the ratio of 80 - 100 U / g of β-glucosidase, 40 - 60 U / g of α-L-arabinofuranosidase, and 20 - 30 U / g of the auxiliary enzyme system; Prepare another stabilizer mixed solution containing 4 - 6% glycerol and 0.05 - 0.15% Tween-80. At the same time, prepare the basic medium, and each liter of the medium contains: 15 - 25 g of glucose, 3 - 7 g of yeast extract, 8 - 12 g of peptone, 1.5 - 2.5 g of KH2PO4, 0.3 - 0.7 g of MgSO4, sterilize at 120 - 122 °C for 15 - 20 minutes and reserve for use;

[0008] S2 Strain culture

[0009] The strain activation starts from Bacillus licheniformis preserved at -80 °C. First, inoculate it on the slant medium, culture at 26 - 30 °C for 45 - 50 hours, then pick a single colony and inoculate it into the seed medium, and culture at 26 - 30 °C and 160 - 200 rpm for 22 - 26 hours. In the fermentation culture stage, transfer the seed liquid into the fermentation medium at an inoculation amount of 4 - 6%, control the fermentation temperature at 26 - 30 °C, the stirring speed at 160 - 200 rpm, maintain the dissolved oxygen level at 35 - 65%, and add 0.08 - 0.12% of ginsenoside Rb1 as an inducer after 22 - 26 hours of fermentation;

[0010] S3 Enzymatic conversion

[0011] In the enzymatic conversion stage, add the pre-prepared substrate solution and the stabilizer mixture to the fermentation broth, adjust the reaction temperature to 38 - 42 °C, and maintain stirring at 160 - 200 rpm. During the conversion process, sample every 1.5 - 2.5 hours to monitor the conversion progress, and the reaction duration is 45 - 50 hours. After the conversion is completed, enter the separation and purification stage: First, centrifuge the fermentation broth at 3800 - 4200 rpm for 12 - 18 minutes, filter the supernatant through a 0.45 μm filter membrane, and then perform membrane separation using an 8 - 12 kDa ultrafiltration membrane;

[0012] S4 Separation and purification

[0013] The separated filtrate was purified through a D101 macroporous resin column. First, pure water was used for elution to remove polar impurities, and then gradient elution was carried out with 25 - 35%, 45 - 55%, and 65 - 75% ethanol, and the target elution fraction was collected. The collected target component was concentrated under reduced pressure to an appropriate concentration, an appropriate amount of crystallization aid was added, and crystallization was carried out at 2 - 6°C for 22 - 26 hours. After crystallization was completed, the crystals were collected by suction filtration and vacuum dried at 38 - 42°C to obtain the finished product.

[0014] Furthermore, 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%.

[0015] Furthermore, in the enzyme solution ratio, the activity unit ratio of β - glucosidase, α - L - arabinosidase, and the auxiliary enzyme system is preferably 90:50:25 U / g.

[0016] Furthermore, the preferred amounts of each component in the basal medium are: glucose 20 g / L, yeast extract 5 g / L, peptone 10 g / L, KH2PO4 2 g / L, MgSO4 0.5 g / L.

[0017] Furthermore, the temperature in the strain culture stage is preferably 28°C, the stirring speed is preferably 180 rpm, and the fermentation time is preferably 24 hours.

[0018] Furthermore, the temperature in the enzymatic conversion stage is preferably 40°C, the stirring speed is preferably 180 rpm, and the reaction time is preferably 48 hours.

[0019] Furthermore, during the ethanol gradient elution process, it is preferably eluted successively with 30%, 50%, and 70% ethanol aqueous solutions.

[0020] Furthermore, the temperature in the crystallization process is preferably 4°C, and the crystallization time is preferably 24 hours.

[0021] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and cannot limit this application.

[0022] The beneficial technical effects of the present invention:

[0023] The method of the present invention realizes the stereoselective hydrolysis of the glycosidic bond at the C-20 position with a selectivity of over 95% by establishing a dual-enzyme co-catalysis system of β-glucosidase and α-L-arabinofuranosidase; by using a strain screened by direction, through optimizing the fermentation conditions and adding 0.1% ginsenoside Rb1 inducer, the expression level of the target enzyme gene 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 half-life of the enzyme activity is extended by 1.8 times, and a conversion rate of over 90% is achieved within 48 hours under the temperature control of 38 - 42°C; by adopting a multiple separation strategy of ultrafiltration - resin - crystallization, the purity of the final product is ensured to be ≥98%, providing a reliable process basis for large-scale production. Detailed implementation manners

[0024] The optional implementation manners of the present invention application will be described in more detail below. Although the optional implementation manners of the present invention application are described, it should be understood that the present invention application can be implemented in various forms and should not be limited by the implementation manners described herein. On the contrary, these implementation manners are provided to make the present invention application more thorough and complete, and to be able to convey the scope of the present invention application to those skilled in the art completely.

[0025] The terms used in the present invention application are only for the purpose of describing specific embodiments and are not intended to limit the present invention application. The singular forms "a", "the" and "said" used in the present invention application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term "and / or" used herein refers to and includes any and all possible combinations of one or more of the associated listed items.

[0026] The present invention application provides a method for improving the preparation of ginsenoside RG3, comprising the following steps:

[0027] S1 Reagent preparation

[0028] Prepare 0.15 - 0.25 M phosphate buffer and adjust the pH to 5.2 - 5.8 with hydrochloric acid and sodium hydroxide; weigh 2 - 4 g of the mixture of ginsenoside Rb1 / Rb2 / Rc extracted from red ginseng (the mass ratio of Rb1, Rb2 and Rc is 3:1:1) and dissolve it in 100 mL of buffer to make a 2 - 4% substrate solution; prepare the enzyme solution according to the ratio of β - glucosidase 80 - 100 U / g, α - L - arabinosidase 40 - 60 U / g, and auxiliary enzyme system (the auxiliary enzyme system is composed of cellulase, pectinase and xylanase, and the activity unit ratio of the three enzymes is 2:1:1) 20 - 30 U / g; prepare another stabilizer mixed solution containing 4 - 6% glycerol and 0.05 - 0.15% Tween - 80. At the same time, prepare the basal medium. Each liter of the medium contains: glucose 15 - 25 g, yeast extract 3 - 7 g, peptone 8 - 12 g, KH2PO4 1.5 - 2.5 g, MgSO4 0.3 - 0.7 g, sterilize at 120 - 122 °C for 15 - 20 minutes for standby;

[0029] Cultivation of Strain S2

[0030] Activation of the strain starts from Bacillus licheniformis preserved at - 80 °C (the Bacillus licheniformis used in this application is purchased from Zhengzhou Best Food Additive Co., Ltd.). First, inoculate it on the slant medium and culture it at 26 - 30 °C for 45 - 50 hours, then pick a single colony and inoculate it into the seed medium, and culture it at 26 - 30 °C and 160 - 200 rpm for 22 - 26 hours. In the fermentation culture stage, transfer the seed liquid into the fermentation medium at an inoculation amount of 4 - 6%, control the fermentation temperature at 26 - 30 °C, the stirring speed at 160 - 200 rpm, maintain the dissolved oxygen level at 35 - 65%, and add 0.08 - 0.12% ginsenoside Rb1 as an inducer after 22 - 26 hours of fermentation;

[0031] Enzymatic conversion in S3

[0032] In 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 - 200 rpm. During the conversion process, sample every 1.5 - 2.5 hours to monitor the conversion progress, and the reaction duration is 45 - 50 hours. After the conversion is completed, enter the separation and purification stage: first, centrifuge the fermentation broth at 3800 - 4200 rpm for 12 - 18 minutes, filter the supernatant through a 0.45 μm filter membrane, and then perform membrane separation using an 8 - 12 kDa ultrafiltration membrane;

[0033] Separation and purification in S4

[0034] The separated filtrate was purified through a D101 macroporous resin column. First, pure water was used for elution to remove polar impurities, and then gradient elution was carried out with 25 - 35%, 45 - 55%, and 65 - 75% ethanol, and the target elution fraction was collected. The collected target component was concentrated under reduced pressure to an appropriate concentration, and an appropriate amount of crystallization aid was added (anhydrous ethanol was used as the crystallization aid and was slowly added at a ratio of concentrated solution to anhydrous ethanol of 1:2 (V / V)). Crystallization was carried out at 2 - 6 °C for 22 - 26 hours. After crystallization was completed, the crystals were collected by suction filtration and vacuum dried at 38 - 42 °C to obtain the finished product.

[0035] This method has achieved systematic innovation at the molecular, cellular, and process levels, as follows:

[0036] I. Molecular-level mechanism of action

[0037] This method designed a specific dual-enzyme catalytic system. β-Glucosidase can specifically recognize and hydrolyze the β-D-glucoside bond at the C-20 position, while α-L-arabinofuranosidase selectively acts on the α-L-arabinofuranoside bond. The synergistic action of the two enzymes established a stereoselective hydrolysis network. Experimental data showed that the selective hydrolysis efficiency of this system for the target glycoside bond reached over 95%, significantly higher than 65 - 75% of the traditional chemical hydrolysis method.

[0038] The ionic strength (0.15 - 0.25 M) and pH value (5.2 - 5.8) of the buffer system optimized the formation of the enzyme-substrate complex by affecting the surface charge distribution of the enzyme molecules. Experiments confirmed that at pH 5.5, the enzyme activity reached the maximum value, and the conformational stability of the enzyme molecules increased by 30%. The added non-ionic surfactant Tween-80 (0.05 - 0.15%) improved the accessibility of the substrate by reducing the interfacial tension, making the substrate molecules more easily enter the enzyme active center.

[0039] II. Cellular metabolic regulation mechanism

[0040] During the fermentation process, a metabolic environment conducive to the expression of the enzyme system was established by precisely controlling the dissolved oxygen level (35 - 65%) and the stirring speed (160 - 200 rpm). 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.

[0041] 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 showed that the enzyme activity increased by 50 - 60% after induction. The optimized medium formula (15 - 25 g / L of glucose, 3 - 7 g / L of yeast extract, etc.) adjusted the C / N ratio to make the metabolic flow conducive to the accumulation of the target product.

[0042] III. Process control mechanism

[0043] 4 - 6% glycerol is added as an osmoprotectant to the reaction system to stabilize the conformation of enzyme molecules 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 a sufficient reaction rate but also avoids enzyme inactivation. The conversion rate can reach over 90% after 48 hours of reaction.

[0044] Multiple separation mechanisms based on molecular size and polarity differences are adopted for separation and purification. An ultrafiltration membrane with a molecular weight cut-off of 8 - 12 kDa can effectively remove macromolecular impurities; the D101 macroporous resin selectively adsorbs the target product through hydrophobic interaction and is eluted with 25 - 35%, 45 - 55%, and 65 - 75% ethanol for precise separation using polarity differences. The final crystallization process is carried out at 2 - 6 °C, and by controlling the temperature and concentration gradient, the orderly arrangement of product molecules is promoted to obtain a crystalline product with a purity ≥ 98%.

[0045] IV. Summary of Method Advantages

[0046] Selectivity: The dual-enzyme co-catalysis system achieves precise recognition and hydrolysis of specific glycosidic bonds.

[0047] Efficiency: Optimized fermentation conditions and induction expression strategies significantly improve the yield and activity of the enzyme.

[0048] Stability: Multiple protection mechanisms ensure the stability of enzyme molecules and the controllability of the reaction.

[0049] Purity: The multi-level separation strategy ensures high purity of the product.

[0050] In an embodiment of the present invention 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%.

[0051] In an embodiment of the present invention application, in the enzyme solution ratio, the activity unit ratio of β-glucosidase, α-L-arabinofuranosidase, and the auxiliary enzyme system is preferably 90:50:25 U / g.

[0052] In an embodiment of the present invention application, the preferred amounts of each component in the basal medium are: glucose 20 g / L, yeast extract 5 g / L, peptone 10 g / L, KH2PO4 2 g / L, MgSO4 0.5 g / L.

[0053] In an embodiment of the present invention application, the temperature in the strain culture stage is preferably 28 °C, the stirring speed is preferably 180 rpm, and the fermentation time is preferably 24 hours.

[0054] In one embodiment of the present invention application, the temperature in the enzymatic conversion stage is preferably 40°C, the stirring speed is preferably 180 rpm, and the reaction time is preferably 48 hours.

[0055] In one embodiment of the present invention application, during the ethanol gradient elution process, it is preferably eluted successively with 30%, 50%, and 70% ethanol aqueous solutions.

[0056] In one embodiment of the present invention application, the temperature in the crystallization process is preferably 4°C, and the crystallization time is preferably 24 hours.

[0057] For the sake of clarity, the following will be described in detail through the following examples.

[0058] Example 1: S1 Reagent preparation: Prepare 0.15 M phosphate buffer and adjust the pH to 5.2 with hydrochloric acid and sodium hydroxide; weigh 2 g of the mixture of ginsenoside Rb1 / Rb2 / Rc extracted from red ginseng (the mass ratio of Rb1, Rb2, and Rc is 3:1:1) and dissolve it in 100 mL of buffer to make a 2% substrate solution; prepare an 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 the auxiliary enzyme system (the auxiliary enzyme system is composed of cellulase, pectinase, and xylanase, and the activity unit ratio of the three enzymes is 2:1:1); prepare another stabilizer mixed solution containing 4% glycerol and 0.05% Tween-80. At the same time, prepare the basal medium, and each liter of the medium contains: 15 g of glucose, 3 g of yeast extract, 8 g of peptone, 1.5 g of KH2PO4, 0.3 g of MgSO4, and sterilize it at 120°C for 15 minutes for standby;

[0059] S2 Strain cultivation: The strain activation starts from Bacillus licheniformis stored at -80°C. First, inoculate it on the slant medium and culture it at 26°C for 45 hours, then pick a single colony and inoculate it into the seed medium, and culture it at 26°C and 160 rpm for 22 hours. In the fermentation culture stage, transfer the seed liquid into the fermentation medium at an inoculation amount of 4%, control the fermentation temperature at 26°C, the stirring speed at 160 rpm, maintain the dissolved oxygen level at 35%, and add 0.08% ginsenoside Rb1 as an inducer after 22 hours of fermentation;

[0060] S3 Enzymatic conversion: In the enzymatic conversion stage, add the pre-prepared substrate solution and stabilizer mixture to the fermentation broth, adjust the reaction temperature to 38°C, and maintain stirring at 160 rpm. During the conversion process, sample every 1.5 hours to monitor the conversion progress, and the reaction duration is 45 hours. After the conversion is completed, enter the separation and purification stage: First, centrifuge the fermentation broth at 3800 rpm for 12 minutes, filter the supernatant through a 0.45 μm filter membrane, and then perform membrane separation using an 8 kDa ultrafiltration membrane;

[0061] S4 Separation and purification: The obtained filtrate was purified through a D101 macroporous resin column. First, it was eluted with pure water to remove polar impurities, and then gradient elution was carried out with 25%, 45%, and 65% ethanol. The target elution section was collected (polar impurities were removed by eluting with 5 times the column volume of pure water, and then gradient elution was carried out successively with 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, and the 45% ethanol eluate was collected). The collected target component was concentrated under reduced pressure to an appropriate concentration (50 mg / mL), and an appropriate amount of crystallization aid was added (anhydrous ethanol was used as the crystallization aid and slowly added according to the ratio of concentrated solution to anhydrous ethanol of 1:2 (V / V)). Crystallization was carried out at 2°C for 22 hours. After crystallization was completed, the crystals were collected by suction filtration and dried in vacuo at 38°C to obtain the finished product, and the purity of the finished product was >98.8%.

[0062] Example 2: S1 Reagent preparation: Prepare 0.2 M phosphate buffer and adjust the pH to 5.5 with hydrochloric acid and sodium hydroxide; weigh 3 g of the mixture of ginsenoside Rb1 / Rb2 / Rc extracted from red ginseng (the mass ratio of Rb1, Rb2, and Rc is 3:1:1) and dissolve it in 100 mL of buffer to make a 3% substrate solution; prepare the enzyme solution according to the ratio of β-glucosidase 90 U / g, α-L-arabinosidase 50 U / g, and auxiliary enzyme system (the auxiliary enzyme system is composed of cellulase, pectinase, and xylanase, and the activity unit ratio of the three enzymes is 2:1:1) 25 U / g; prepare another stabilizer mixed solution containing 5% glycerol and 0.1% Tween-80. At the same time, prepare the basal medium, and each liter of the medium contains: 20 g of glucose, 5 g of yeast extract, 10 g of peptone, 2 g of KH2PO4, 0.5 g of MgSO4, and sterilize it at 121°C for 17.5 minutes for standby;

[0063] S2 Strain culture: The strain activation started from Bacillus licheniformis stored at -80°C. First, it was inoculated on the slant medium and cultured at 28°C for 47.5 hours, then a single colony was picked and inoculated into the seed medium and cultured at 28°C and 180 rpm for 24 hours. In the fermentation culture stage, the seed liquid was transferred to the fermentation medium at an inoculation amount of 5%, the fermentation temperature was controlled at 28°C, the stirring speed was 180 rpm, and the dissolved oxygen level was maintained at 50%. After 24 hours of fermentation, 0.1% ginsenoside Rb1 was added as an inducer;

[0064] S3 Enzymatic conversion: In the enzymatic conversion stage, the pre-prepared substrate solution and stabilizer mixture were added to the fermentation broth, the reaction temperature was adjusted to 40°C, and stirring was maintained at 180 rpm. During the conversion process, samples were taken every 2 hours to monitor the conversion progress, and the reaction duration was 47.5 hours. After the conversion was completed, it entered the separation and purification stage: First, the fermentation broth was centrifuged at 4000 rpm for 15 minutes, and the supernatant was filtered through a 0.45 μm filter membrane and then subjected to membrane separation using a 10 kDa ultrafiltration membrane;

[0065] S4 Separation and purification: The obtained filtrate was purified through a D101 macroporous resin column. First, it was eluted with pure water to remove polar impurities, and then gradient elution was carried out with 30%, 50%, and 70% ethanol. The target elution fraction was collected (polar impurities were removed by eluting with 5 column volumes of pure water, and then gradient elution was carried out successively with 3 column volumes of 30% ethanol, 5 column volumes of 50% ethanol, and 3 column volumes of 70% ethanol, and the 45% ethanol eluate was collected). The collected target component was concentrated under reduced pressure to an appropriate concentration (55 mg / mL), an appropriate amount of crystallization aid was added (anhydrous ethanol was used as the crystallization aid and added slowly according to the ratio of concentrated solution to anhydrous ethanol of 1:2 (V / V)), and crystallization was carried out at 4 °C for 24 hours. After crystallization was completed, the crystals were collected by suction filtration and dried in vacuo at 40 °C to obtain the finished product, and the purity of the finished product was >99.7%.

[0066] Example 3: S1 Reagent preparation: Prepare 0.25 M phosphate buffer and adjust the pH to 5.8 with hydrochloric acid and sodium hydroxide; weigh 4 g of the mixture of ginsenoside Rb1 / Rb2 / Rc extracted from red ginseng (the mass ratio of Rb1, Rb2, and Rc is 3:1:1) and dissolve it in 100 mL of buffer to make a 4% substrate solution; prepare the enzyme solution according to the ratio of β-glucosidase 100 U / g, α-L-arabinosidase 60 U / g, and auxiliary enzyme system (the auxiliary enzyme system is composed of cellulase, pectinase, and xylanase, and the activity unit ratio of the three enzymes is 2:1:1) 30 U / g; prepare another stabilizer mixed solution containing 6% glycerol and 0.15% Tween-80. At the same time, prepare the basal medium, and each liter of the medium contains: 25 g of glucose, 7 g of yeast extract, 12 g of peptone, 2.5 g of KH2PO4, 0.7 g of MgSO4, and sterilize it at 122 °C for 20 minutes for standby;

[0067] S2 Strain culture: The strain activation started from Bacillus licheniformis stored at -80 °C. First, it was inoculated on the slant medium and cultured at 30 °C for 50 hours, then a single colony was picked and inoculated into the seed medium, and cultured at 30 °C and 200 rpm for 26 hours. In the fermentation culture stage, the seed liquid was transferred into the fermentation medium at an inoculation amount of 6%, the fermentation temperature was controlled at 30 °C, the stirring speed was 200 rpm, and the dissolved oxygen level was maintained at 65%. After fermentation for 26 hours, 0.12% ginsenoside Rb1 was added as an inducer;

[0068] S3 Enzymatic conversion: In the enzymatic conversion stage, a pre-prepared substrate solution and stabilizer mixture were added to the fermentation broth, the reaction temperature was adjusted to 42 °C, and stirring was maintained at 200 rpm. Samples were taken every 2.5 hours during the conversion process to monitor the conversion progress, and the reaction duration was 50 hours. After the conversion was completed, the separation and purification stage began: First, the fermentation broth was centrifuged at 4200 rpm for 18 minutes, and the supernatant was filtered through a 0.45 μm filter membrane and then subjected to membrane separation using a 12 kDa ultrafiltration membrane;

[0069] S4 Separation and purification: The obtained filtrate was purified through a D101 macroporous resin column. First, pure water was used for elution to remove polar impurities, and then gradient elution was carried out with 35%, 55%, and 75% ethanol. The target elution fraction was collected (polar impurities were removed by eluting with 5 times the column volume of pure water, and then gradient elution was carried out successively with 3 times the column volume of 35% ethanol, 5 times the column volume of 55% ethanol, and 3 times the column volume of 75% ethanol, and the 45% ethanol eluate was collected). The collected target component was concentrated under reduced pressure to an appropriate concentration (60 mg / mL), and an appropriate amount of crystallization aid was added (anhydrous ethanol was used as the crystallization aid and slowly added according to the ratio of the concentrated solution to anhydrous ethanol of 1:2 (V / V)). Crystallization was carried out at 6 °C for 26 hours. After crystallization was completed, the crystals were collected by suction filtration and dried in vacuo at 42 °C to obtain the finished product, and the purity of the finished product > 99.1%.

[0070] Experimental example 1 Experimental scheme:

[0071] Experimental grouping (3 parallels in each group):

[0072] Group A: Chemical hydrolysis method (control group)

[0073] 2M hydrochloric acid solution

[0074] 80 °C water bath

[0075] React for 4 hours

[0076] Group B: Biotransformation method (method of Example 2)

[0077] Reaction system (100 mL):

[0078] Substrate: 3 g of ginsenoside Rb1 / Rb2 / Rc mixture (3%) extracted from red ginseng

[0079] Stabilizer: 5% glycerol + 0.1% Tween-80

[0080] Sampling time points: 0 h, 12 h, 24 h, 36 h, 48 h

[0081] Detection indexes:

[0082] Determination of RG3 content by HPLC

[0083] Calculate the conversion rate and selectivity

[0084] Analyze the composition of by-products by LC-MS

[0085] Experimental results:

[0086] Dynamic change of RG3 content

[0087] Table 1. Changes in RG3 content (mg / mL) under different conversion methods

[0088]

[0089] Detection results at the end point of 48h

[0090] Table 2. Comparison of conversion effects between two methods

[0091]

[0092] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications and improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

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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