A composite enzyme for efficiently converting high-purity rare ginsenoside CK and its preparation method and application

Abstract

The present invention relates to a composite enzyme for efficiently converting high-purity rare ginsenoside CK and its preparation method and application, belonging to the field of microbial technology. The composite enzyme of the present invention includes β-glucosidase, fructosyltransferase, lactase, cellulase and β-glucanase. The present invention obtains a composite enzyme by selecting β-glucosidase, fructosyltransferase, lactase, cellulase and β-glucanase, and can prepare ginsenoside CK in an enzyme engineering process, which has the effect of synergistically increasing the conversion rate of ginsenoside Rb1 into ginsenoside CK.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a composite enzyme for efficiently converting high-purity rare ginsenoside CK, and a preparation method and application thereof. Background Art

[0002] Rare ginsenoside Compound k (CK) is a non-natural ginsenoside, which is mainly converted from other diol ginsenosides. It has good biological activities, including anti-tumor, anti-skin aging, anti-allergy and anti-inflammatory, and has a wide range of uses and high economic benefits.

[0003] At present, the methods for preparing ginsenoside CK mainly include microbial fermentation and enzymatic hydrolysis. Patent CN106047978A prepares ginsenoside CK by microbial fermentation. The patent uses Ganoderma lucidum as the fermentation bacteria for fermentation culture. After at least 3 days of fermentation, natural saponins are added to the fermentation liquid, and the rare ginsenoside CK is obtained by continuous fermentation. The yield of rare ginsenoside CK is the highest after 6 days of fermentation, and the average can reach 11.415 mg / mL. Although this method can obtain a high yield of ginsenoside CK in the laboratory, in industrial production, there is no matching and feasible production equipment, and it is impossible to achieve mass production of ginsenoside CK.

[0004] Patent CN105296587B is to prepare ginsenoside CK by enzymatic hydrolysis, and the β-glucosidase produced by bifidobacterium reacts with ginsenoside Rb1 with a concentration of 10mg / mL to generate ginsenoside CK, and the conversion efficiency of ginsenoside CK is 62-68%. However, the substrate concentration of the patent is 1% (10mg / mL), which does not meet the requirements of industrial production. In industrial production, if the substrate concentration is less than 10%, the ginsenoside CK produced after conversion will be too low in concentration, and heating and stirring in the reactor, as well as subsequent purification, extraction, rotary evaporation and other processing steps will cause ginsenoside CK to cause a large amount of losses, and a large amount of electric energy will be consumed, so that the production cost is greatly increased. At the same time, most of the β-glucosidases have low expression and poor stability in microorganisms, and can only be enzymolyzed when the substrate concentration is low. Once the substrate concentration increases, the concentration and activity of the enzyme are not enough to support the conversion of high-concentration substrates, and ginsenoside CK cannot be efficiently converted.

[0005] Patent CN 108138212 B uses an enzymatic hydrolysis method to prepare ginsenoside CK, wherein pectinase produced by Aspergillus aculeatus and Trichoderma reesei reacts with ginsenosides to produce ginsenoside CK, the enzymatic hydrolysate is heated to inactivate the enzyme, ethyl acetate is added for extraction, and ginsenoside CK is separated by silica gel column chromatography. This method uses a variety of organic solvents and a variety of production processes, which puts a burden on the environment and has high production costs. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a composite enzyme for efficiently converting high-purity rare ginsenoside CK and its preparation method and application. The present invention also provides a method for efficiently converting high-purity rare ginsenoside CK, which can improve the conversion rate of ginsenoside Rb1 into ginsenoside CK, is suitable for industrial production, and the prepared ginsenoside CK has the advantages of low conductivity and good stability.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] In a first aspect, the present invention provides a method for preparing β-glucosidase, the preparation method comprising the following steps:

[0009] A1. Codon optimization of β-glucosidase from soil Sinomonas sp. was performed, and an expression vector with a coding sequence was prepared by gene editing technology. The amino acid sequence of the codon-optimized β-glucosidase is shown in SEQ ID NO.1.

[0010] A2, transforming the expression vector described in step A1 into Escherichia coli, and obtaining positive clones through screening and identification;

[0011] A3, preparing the positive clones obtained in step A2 as a secondary seed solution, and using the secondary seed solution to perform fed-batch fermentation to obtain a fermentation solution;

[0012] A4. Centrifuge the fermentation liquid obtained in step A3, collect the bacterial precipitate, and crush, centrifuge, concentrate and freeze-dry the obtained bacterial precipitate to obtain β-glucosidase.

[0013] The β-glucosidase used in the composite enzyme of the present invention is obtained by the inventor's own production. The enzymatic conversion pathway of ginsenosides is: Rb1→Rd→F2→CK, and different enzymes have different rate-limiting sites. For example, the rate-limiting site of the commercial composite enzyme is F2, which causes a large amount of raw materials to gather in the F2→CK stage, thereby reducing the overall conversion rate of CK; and the self-produced β-glucosidase of the present invention is a special CK conversion enzyme prepared by computer software-assisted design and directional optimization based on the shortcomings of commercial enzymes, and its rate-limiting site is in the Rd→F2 stage. The complementary use of the β-glucosidase with other enzymes can effectively avoid the influence of all rate-limiting sites and realize the efficient conversion of the rare ginsenoside CK. At present, the self-produced β-glucosidase of the present invention can reach 12000U / g and above through the optimization of the fermentation process, which is the key to achieving effective supplementation of traditional commercial enzymes.

[0014] As a preferred embodiment of the preparation method of the present invention, in step A3, the fed-batch fermentation comprises the following operations:

[0015] B1. Inoculate the secondary seed liquid into the fermentation medium, adjust the pH to 6.6-7.0, and set the temperature to 35-37° C.; the volume ratio of the secondary seed liquid to the fermentation medium is secondary seed liquid: fermentation medium = (0.8-1):30;

[0016] B2. When the dissolved oxygen drops to 60-70%, adjust the temperature to 23-27°C and start feeding. The feeding rate is 190-200mL / h in the first hour, 220-230mL / h in the second hour, and 250-260mL / h in the third hour and maintain it unchanged; control the dissolved oxygen to slowly drop to 12-18% by controlling the tank pressure, rotation speed, and air volume; sample the fermentation liquid every 4 hours after feeding to detect the OD600 value, and terminate the fermentation when the OD600 value no longer rises.

[0017] As a preferred embodiment of the preparation method of the present invention, in step B1, the formula of the fermentation medium is: yeast extract powder 10g / L, citric acid 1g / L, potassium dihydrogen phosphate 6g / L, ammonium sulfate 5g / L, magnesium sulfate heptahydrate 1g / L, dipotassium hydrogen phosphate 16g / L, peptone 8g / L, complex vitamins 0.1g / L, glucose 7g / L, and kanamycin is added to the final concentration of 20μg / mL according to the volume of the fermentation liquid before inoculation.

[0018] As a preferred embodiment of the preparation method of the present invention, in step B2, the feed is a 20 wt % sterilized lactose solution.

[0019] In a second aspect, the present invention provides a β-glucosidase, which is mainly prepared by the above preparation method.

[0020] In a third aspect, the present invention provides a composite enzyme for efficiently converting high-purity rare ginsenoside CK, comprising β-glucosidase, fructosyltransferase, lactase, cellulase and β-glucanase, wherein the weight ratio of the β-glucosidase, fructosyltransferase, lactase, cellulase and β-glucanase is β-glucosidase: fructosyltransferase: lactase: cellulase: β-glucanase = (0.05-0.2): (0.005-0.2): (0.01-0.1): (0.01-0.1): (0.02-0.1);

[0021] The β-glucosidase is the above-mentioned β-glucosidase.

[0022] The present invention obtains a composite enzyme by selecting β-glucosidase, fructosyltransferase, lactase, cellulase and β-glucanase, which can prepare ginsenoside CK in an enzyme engineering process, has a synergistic effect of improving the conversion rate of ginsenoside Rb1 into ginsenoside CK, and the content of ginsenoside CK can reach up to 69.3%.

[0023] One of the pain points of the fermentation process is that the growth state of microorganisms is difficult to control, resulting in large fluctuations in the fermentation level between different batches, leading to an unstable production of rare ginsenoside CK. At the same time, the microbial fermentation technology requires the addition of carbon sources and nitrogen sources, which increases the impurity content during ginsenoside extraction and causes product instability. The present invention effectively converts ginsenosides into rare ginsenoside CK through composite enzyme technology, and the parameters such as enzyme activity and concentration can be accurately controlled, so that the properties of ginsenoside CK between batches are highly repeated, thereby improving the stability of the production process.

[0024] As a preferred embodiment of the composite enzyme of the present invention, the weight ratio of β-glucosidase, fructosyltransferase, lactase, cellulase and β-glucanase is β-glucosidase:fructosyltransferase:lactase:cellulase:β-glucanase=0.1:0.01:0.04:0.05:0.05.

[0025] Under the preferred weight ratio, the conversion efficiency of ginsenoside CK by the composite enzyme of the present invention is higher.

[0026] As a preferred embodiment of the composite enzyme of the present invention, the enzyme activity of the β-glucosidase is 12000-13000U / g, the enzyme activity of the fructosyltransferase is 2500-3000U / g, the enzyme activity of the lactase is 5500-6000U / g, the enzyme activity of the cellulase is 9000-10000U / g, and the enzyme activity of the β-glucanase is 6000-6500U / g.

[0027] As a preferred embodiment of the composite enzyme of the present invention, the enzyme activity of the β-glucosidase is 12000U / g, the enzyme activity of the fructosyltransferase is 3000U / g, the enzyme activity of the lactase is 6000U / g, the enzyme activity of the cellulase is 10000U / g, and the enzyme activity of the β-glucanase is 6500U / g.

[0028] In a fourth aspect, the present invention provides a method for preparing the above-mentioned composite enzyme, wherein β-glucosidase, fructosyltransferase, lactase, cellulase and β-glucanase are mixed to obtain the composite enzyme.

[0029] As a preferred embodiment of the preparation method of the present invention, the complex enzyme is mixed with sodium phosphate buffer to prepare a complex enzyme solution, and the weight ratio of the complex enzyme to the sodium phosphate buffer is complex enzyme: sodium phosphate buffer = 1:15.

[0030] The preparation method of the composite enzyme of the present invention is simple and quick, and the composite enzyme can be prepared by simply mixing the components according to a specific weight ratio.

[0031] In a fifth aspect, the present invention provides the use of the above-mentioned complex enzyme in the preparation of ginsenoside CK.

[0032] In a sixth aspect, the present invention provides a method for efficiently converting high-purity rare ginsenoside CK, comprising the following steps:

[0033] (1) mixing an extract containing ginsenoside Rb1 with 0.5-1 mM phosphate buffer, adding the above-mentioned complex enzyme, adjusting the pH to 6.0-6.5, and performing enzymolysis at 45-55° C. and 200-800 rpm for 70-74 h to obtain an enzymolysis solution, wherein the weight ratio of the extract containing ginsenoside Rb1 to the phosphate buffer is ginsenoside Rb1:phosphate buffer=1:(10-100);

[0034] (2) centrifuging the enzymatic hydrolyzate obtained in step (1), collecting precipitate A, mixing the precipitate A with pure water, homogenizing and centrifuging, and collecting precipitate B when the conductivity of the supernatant is less than 100 μs / cm;

[0035] (3) Mixing the precipitate B obtained in step (2) with an ethanol solution having a volume percentage of 40-95% as a solvent to prepare a reconstituted solution, and subjecting the reconstituted solution to pressure filtration, microfiltration and concentration treatment to obtain high-purity rare ginsenoside CK.

[0036] The conversion method of ginsenoside CK of the present invention has the advantages of simple production process and green environmental protection, avoids complex processes such as column chromatography, and uses reagents such as water and ethanol, which can be recycled and reused. At the same time, the price of ginsenoside CK powder with a content of 50% on the market is 36,000-75,000 yuan / kg. The present invention does not need to undergo additional purification steps such as column chromatography to produce rare ginsenoside CK powder with a content of more than 50%, and the cost per kilogram is within 12,000 yuan, which has higher economic benefits.

[0037] In general industrial production, a substrate concentration lower than 10% will result in too low a content of ginsenoside CK. However, in the conversion method of the present invention, when the substrate concentration is 1%, 526g of ginsenoside CK (content is 66.3%) can be obtained for every 1000g of ginsenoside Rb1 extract. This is because the present invention adopts a specific composite enzyme to react with ginsenoside Rb1. The high-activity β-glucosidase in the composite enzyme can remove the rate limitation in the ginsenoside CK pathway, and synergize with other enzymes to overcome the problem of low ginsenoside CK yield caused by low substrate concentration, and effectively improve the conversion rate of ginsenoside CK.

[0038] The enzymatic hydrolysis and conversion pathway of ginsenosides is: Rb1→Rd→F2→CK. When Rb1 begins to be further converted, the generated saponins are all water-insoluble saponins. A paste precipitate will be formed during the enzymatic hydrolysis process, which is not conducive to the contact between the enzyme and Rd and F2 inside the paste precipitate, thereby hindering the conversion efficiency of CK. The present invention optimizes the rotation speed of the enzymatic hydrolysis process, and disperses the precipitate by impacting the stirring paddle, thereby increasing the contact frequency between the enzyme molecules and the saponins inside the precipitate, thereby greatly improving the bioconversion efficiency of CK.

[0039] The method of the present invention adds a homogenization step. By introducing a homogenization centrifugation process, a large amount of enzymatically converted paste is added with pure water and dispersed with a homogenizer, and salt ions and some water-soluble substances are dispersed in the supernatant. The homogenate is centrifuged, the supernatant is discarded and the precipitate is recovered, and the product can be further purified. The prepared ginsenoside CK has low conductivity, light color, high clarity, high stability, and few impurities. The powder is not easy to harden when exposed to the air, and can be used in cosmetics, and is suitable for various cosmetic application scenarios such as water, milk, ointment, and cream.

[0040] In the enzymatic hydrolysis process of ginsenoside CK, enzyme residue is one of the pain points of enzyme engineering. The commonly used treatment method is to inactivate the enzyme by high-temperature heating. However, in this treatment, the inactivated enzyme still exists in the ginsenoside CK in the form of protein impurities, reducing its purity; at the same time, the heating treatment will have the risk of destroying the stability, efficacy, color, etc. of the raw materials to a certain extent. In order to solve the above pain points, the present invention removes the water-soluble enzyme through a water washing process (i.e., adding pure water for homogenization and using ethanol aqueous solution for re-dissolution), leaving the water-insoluble CK, and realizing non-heating and protein-free enzyme removal.

[0041] As a preferred embodiment of the application of the present invention, in step (1), the pH is adjusted to 6.0, and enzymolysis is performed at 50° C. and 400-800 rpm for 72 hours to obtain an enzymolysis solution.

[0042] As a preferred embodiment of the application of the present invention, in step (1), the weight ratio of the extract containing ginsenoside Rb1 to the phosphate buffer is extract containing ginsenoside Rb1:phosphate buffer=1:(10-90).

[0043] As a preferred embodiment of the application of the present invention, in step (1), the weight ratio of the extract containing ginsenoside Rb1 to the phosphate buffer is extract containing ginsenoside Rb1:phosphate buffer=1:(20-50).

[0044] As a preferred embodiment of the application of the present invention, in step (1), the weight ratio of the extract containing ginsenoside Rb1 to the phosphate buffer is 1:50. Under the preferred weight ratio, the ginsenoside CK content prepared by the present invention is 69.3%.

[0045] As a preferred embodiment of the application of the present invention, in step (1), the weight ratio of the extract containing ginsenoside Rb1 to the complex enzyme is extract containing ginsenoside Rb1:complex enzyme=1:(0.095-0.7).

[0046] As a preferred embodiment of the application of the present invention, in step (1), the weight ratio of the extract containing ginsenoside Rb1 to the complex enzyme is extract containing ginsenoside Rb1:complex enzyme=1:0.25.

[0047] As a preferred embodiment of the application of the present invention, in step (1), the complex enzyme is configured into a complex enzyme solution with a phosphate buffer having a pH of 6.5-7.5 and 0.5-1 mM before being added, and the weight ratio of the complex enzyme to the phosphate buffer is complex enzyme: phosphate buffer = 1: (10-15).

[0048] As a preferred embodiment of the application of the present invention, in step (1), the complex enzyme is configured into a complex enzyme solution with a phosphate buffer of pH=7.0 and 1mM before being added, and the weight ratio of the complex enzyme to the phosphate buffer is complex enzyme: phosphate buffer = 1:10.

[0049] As a preferred embodiment of the application of the present invention, in step (2), the precipitate B is collected when the conductivity of the supernatant is 1-90 μs / cm.

[0050] As a preferred embodiment of the application of the present invention, in step (2), the weight ratio of the precipitate A to pure water is precipitate A:pure water=1:(10-50).

[0051] As a preferred embodiment of the application of the present invention, in step (2), the weight ratio of the precipitate A to pure water is precipitate A:pure water=1:10.

[0052] As a preferred embodiment of the application of the present invention, in step (3), the weight ratio of the ethanol solution to the precipitate B is 1:(10-15), and the volume percentage of the ethanol solution is 50-60%.

[0053] As a preferred embodiment of the application of the present invention, in step (3), the weight ratio of the ethanol solution to the precipitate B is 1:10, and the volume percentage of the ethanol solution is 50%.

[0054] Compared with the prior art, the present invention has the following beneficial effects:

[0055] 1. The present invention independently constructs a strain expressing β-glucosidase through computer software simulation calculation and codon optimization process, optimizes the production process to produce high-activity β-glucosidase, and mixes and matches with four enzymes, namely fructosyltransferase, lactase, cellulase and β-glucanase, to synergistically improve the efficiency of converting ginsenoside Rb1 into ginsenoside CK, which is suitable for industrial mass production of ginsenoside CK.

[0056] 2. The present invention obtains ginsenoside CK by enzymatic hydrolysis, centrifugation, homogenization, redissolution, filter pressing, microfiltration, concentration and drying of an extract containing ginsenoside Rb1, thereby simplifying the preparation process of ginsenoside CK, reducing production costs, and making the production process simple and environmentally friendly. At the same time, the conversion rate of ginsenoside CK is improved. The obtained ginsenoside CK has low conductivity, light color, high clarity, high stability, and few impurities, and can be used in cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is a plasmid spectrum of the β-glucosidase expression vector in Example 1 of the present invention;

[0058] Figure 2 The liquid chromatogram of the ginsenoside CK standard substance in Effect Example 1 of the present invention;

[0059] Figure 3 This is a liquid chromatogram of ginsenoside CK prepared by the composite enzyme obtained in Example 1 of Effect Example 1 of the present invention according to the method of Example 4;

[0060] Figure 4 These are the stability test results of different ginsenosides CK in Effect Example 3 of the present invention, wherein A is the observation result on the 0th day of the stability test, and B is the observation result on the 90th day of the stability test. DETAILED DESCRIPTION

[0061] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0062] Unless otherwise specified, other materials, reagents, etc. used in the following examples, comparative examples and effect examples can be obtained from commercial sources.

[0063] The source of the extract containing ginsenoside Rb1 described in the present invention is not limited, it only needs to be clear that it contains ginsenoside Rb1. The extract containing ginsenoside Rb1 used in the following embodiments, comparative examples and effect examples was purchased from Xi'an Xiaocao Plant Technology Co., Ltd., and the content of ginsenoside Rb1 was 70.52% after testing.

[0064] The vector pET28a(+) plasmid was purchased from Hunan Fenghui Biotechnology Co., Ltd.

[0065] Plasmid extraction kit, DNA purification kit, and gel recovery kit were purchased from Tiangen Biochemical Technology Co., Ltd.

[0066] LB medium formula (for BL21 growth): 5g / L yeast powder, 10g / L peptone, 10g / L NaCl, sterilized at 121°C for 20min; if prepared as solid medium, add 15-20g / L agar powder.

[0067] LBR medium (for shake flask fermentation): 5 g / L yeast powder, 10 g / L peptone, 10 g / L NaCl, sterilized at 121°C for 20 min.

[0068] SOC medium (for BL21 transformation): 20 g / L tryptone, 5 g / L yeast extract, 0.5 g / L sodium chloride, 10 mL 250 mM potassium chloride solution, 10 mL 20 mM glucose solution, 10 mL 10 mM magnesium chloride, sterilized at 121°C for 20 min; if prepared as solid medium, add 15-20 g / L agar powder.

[0069] In the following examples and comparative examples, the culture, storage and competent transformation methods of BL21 are as follows:

[0070] 1. Cultivation: Select the strain and streak or spread it on a solid LB plate with corresponding resistance, then invert and culture it in a constant temperature incubator at 37°C for 12-16 hours, perform colony PCR identification or seal it with sealing film and store it inverted in a refrigerator at 4°C;

[0071] A single clone of freshly revived E. coli was picked from the LB solid culture medium plate, inoculated into a certain volume of LB liquid culture medium, and corresponding resistance was added as needed, and cultured at 37°C and 220 rpm for 12-16 hours.

[0072] 2. Storage: Take 500 μL of fresh overnight culture seed solution, add it to a cryovial containing 500 μL of 50% sterile glycerol, and store it in a -80°C ultra-low temperature refrigerator.

[0073] 3. Transformation: Add the transformation system to DH5α and BL21 competent cells melted on ice, ice bath for 30 minutes, heat shock at 42℃ for 90 seconds, ice bath again for 2 minutes, then add 900μL SOC medium preheated at 37℃, and culture at 37℃, 220rpm for 45min; take 100μL of bacterial solution and add it to LB solid medium containing corresponding antibiotics, culture at 37℃ for 12-16h, pick a certain number of clones from the plate for colony PCR identification, and streak them onto the corresponding plates, and culture at 37℃ for 12-16h for use.

[0074] Homologous recombination technology IIOne Step Cloning Kit (single fragment) instructions, ligation conditions were 37°C for 30 min, IIOne Step Cloning Kit was provided by Nanjing Novozymes Biotech Co., Ltd., with the catalog number C112. The plasmid or expression gene cluster assembled by homologous recombination technology was transferred into BL21, and the corresponding primers were used for cloning PCR amplification and agarose gel electrophoresis detection. After positive transformants were identified, they were stored or activated according to the above method for later use.

[0075] The clone detection PCR method is to pick a single transformant into the PCR reaction solution for reaction. The PCR premix used is 2×Rapid Taq Master Mix purchased from Nanjing Novozymes Biotech Co., Ltd., with the catalog number P222-01. The PCR amplification system is shown in Table 1, and the amplification conditions are shown in Table 2. Agarose gel electrophoresis was performed according to the operation in "Molecular Biology Experiment".

[0076] Table 1 PCR amplification system for clone detection

[0077] Reagents Dosage 2× Rapid Taq Master Mix 25μL 10 μM upstream primer 2μL 10 μM downstream primer 2μL <![CDATA[ddH2O]]> Make up to 50 μL

[0078] Table 2 Cloning PCR amplification conditions

[0079]

[0080] The operation of 3,5-dinitrosalicylic acid colorimetric method (DNS method) for detecting enzyme activity is as follows:

[0081] 1.1 Weigh 10g of 3,5-dinitrosalicylic acid and add it to 500mL of water. Add 16g of sodium hydroxide several times and stir to dissolve (temperature <45°C). Add 300g of potassium sodium tartrate several times and stir until completely dissolved. After cooling, dilute to 1000mL with water. Store the brown bottle in a dark place at room temperature and use it after one week (if there is precipitation, filter it before use).

[0082] 1.2 Absorb 2.4 ml of glacial acetic acid, add appropriate amount of water, add 4.92 g of sodium acetate to dissolve, make up to 1000 mL, adjust the pH value to 4.80±0.01 and prepare 0.05 mol / L, pH=4.80 acetic acid-sodium acetate buffer, which is valid within 2 months when stored at room temperature.

[0083] 1.3 Dry anhydrous glucose at 80℃ to constant weight, weigh 0.1000g and dissolve it in a beaker with appropriate amount of water, transfer it to a volumetric flask and make up to 100mL to obtain 0.1wt% (1mg / mL) glucose standard solution.

[0084] 1.4 Weigh 1.00 g of salicin and dissolve it in a beaker. Add 70 mL of the corresponding buffer to dissolve it. Transfer it to a volumetric flask and dilute it to 100 mL with buffer to obtain a 1.0 wt% salicin solution, which can be placed in a refrigerator for later use.

[0085] 1.5 Drawing of glucose standard curve: Pipette 0, 0.2, 0.4, 0.6, 0.8, 1.0 mL of 1 mg / mL glucose standard solution into a test tube, add water to 2 mL, add 3 mL of DNS reagent, mix and boil in boiling water for 10 min, cool and dilute to 15 mL, and measure absorbance (A) at 540 nm wavelength on a spectrophotometer. Draw a standard curve with absorbance as the ordinate and glucose content as the abscissa. The mean of three repeated tests is fitted to the linear equation y=ax+b by the least squares method to find the relationship between absorbance and glucose content.

[0086] 1.6 Treatment of the enzyme sample to be tested: weigh an appropriate amount into a beaker (accurate to 0.0001g), moisten it with a small amount of water and make it into a paste, then dissolve it with water, and dilute the supernatant appropriately after centrifugation.

[0087] 1.7 Enzyme activity determination, take a 15mL stoppered graduated test tube and operate according to the reaction sequence in Table 3 below. During the reaction, starting from the addition of substrate (shake well before aspiration), the time interval for adding reagents to each test tube must be absolutely consistent. Hydrolyze at 50℃ for 30min. The reaction steps and the amounts of reagents and solutions are shown in Table 3.

[0088] Table 3 DNS method operation steps and reagents and solution dosage

[0089]

[0090] Enzyme activity unit calculation formula:

[0091] Enzyme activity U / g = (SD1000) / (0.2×30)

[0092] Where: S is the amount of glucose corresponding to the measured absorbance on the standard curve, mg;

[0093] D is the dilution multiple of enzyme solution or enzyme powder;

[0094] 1000 is the conversion factor between mg and μg;

[0095] 0.2 is the amount of enzyme solution taken for determination, mL;

[0096] 30 is the reaction time, min.

[0097] The formula of the fermentation medium is: yeast extract powder 10g / L, citric acid 1g / L, potassium dihydrogen phosphate 6g / L, ammonium sulfate 5g / L, magnesium sulfate heptahydrate 1g / L, potassium dihydrogen phosphate 16g / L, peptone 8g / L, complex vitamins 0.1g / L, glucose 7g / L, kanamycin was added to the final concentration of 20μg / mL according to the volume of the fermentation liquid before inoculation;

[0098] The compound vitamins were purchased from Zhengzhou Ruipu Bioengineering Co., Ltd. with the product code of 210521B-02-16V.

[0099] The feed in the fed-batch fermentation was a 20 wt% sterile lactose solution.

[0100] The fructosyltransferase and lactase in the following examples, comparative examples and effect examples were purchased from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd., and the cellulase and β-glucanase were purchased from Angel Enzyme (Yichang) Co., Ltd.

[0101] The β-glucosidase A in the following comparative example 8 was purchased from Angel Enzyme (Yichang) Co., Ltd.

[0102] The content of commercially available ginsenoside CK in the following Effect Example 3 was 50.19% as determined by HPLC.

[0103] The C18 column was purchased from Agilent, with the specifications of 250 mm×4.6 mm×5 μm and the part number being 990967-902.

[0104] In the following examples, comparative examples and effect examples, the content of ginsenoside CK refers to the purity of ginsenoside CK.

[0105] Example 1

[0106] An embodiment of the β-glucosidase and the preparation method thereof of the present invention, the preparation method of the composite enzyme described in this embodiment comprises the following steps:

[0107] 1.1 Entrust GenScript Biotech Co., Ltd. to perform codon optimization on β-glucosidase UGT-SH (GenBank No. KHL04625.1) from Sinomonas humi, and connect it to the vector pET28a(+) through homologous recombination technology to obtain an expression vector pET28a(+)-UGT-SH carrying UGT-SH, wherein the amino acid sequence of the codon-optimized β-glucosidase UGT-SH is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2;

[0108] 1.2 The expression vector pET28a(+)-UGT-SH obtained in step 1.1 was transformed into Escherichia coli BL21, and positive clones were obtained by screening and identification;

[0109] 1.3 Activate the positive clones obtained in step 1.2 on the LB plate, pick a single colony and add it to 10 mL LB liquid culture medium, culture at 37°C and 220 rpm for 12-16 hours to obtain the primary seed solution;

[0110] 1.4 Inoculate 10 mL of the first-level seed solution obtained in step 1.3 into 1 L of LB liquid medium containing kanamycin, and culture at 37°C and 220 rpm for 12-16 h to obtain the second-level seed solution;

[0111] 1.5 Inoculate 1L of the secondary seed solution obtained in step 1.4 into a 50L fermenter containing 30L of fermentation medium. Use ammonia water to control the pH of the fermentation liquid to 6.8±0.2, set the temperature to 37°C, and when the dissolved oxygen drops to 60-70%, adjust the temperature to 25°C and start feeding. The feeding rate is 200mL / h in the first hour, 230mL / h in the second hour, and 260mL / h in the third hour and remains unchanged. Control the tank pressure (maximum 0.08Mpa), rotation speed (maximum 360rpm), and air volume (maximum 55L / min) to control the dissolved oxygen to slowly drop to about 15%. Take samples every 4h after feeding to detect OD600, and end the fermentation when the bacterial cell volume no longer increases.

[0112] 1.6 The fermentation liquid was centrifuged at 16000 rpm, the supernatant was removed, the bacteria were collected, 1 mM sodium phosphate buffer (pH = 7.0) was added at a mass ratio of bacteria: sodium phosphate buffer = 1:20, and the bacteria were crushed with a high-pressure homogenizer after stirring. The homogenization pressure was 1000 bar, and the feed liquid temperature was controlled within 40°C to obtain a homogenized liquid;

[0113] 1.7 The homogenate obtained in step 1.6 was centrifuged at 16000rpm, the supernatant (about 50L) was collected, and ultrafiltration was concentrated using a filter element with a pore size of 10000 molecular weight to about one twentieth of the original volume (about 2.5L), and the concentrate was collected, trehalose with a final concentration of 3wt% was added, and lyophilized after complete dissolution and stirring to obtain about 300g of β-glucosidase lyophilized powder. The enzyme activity was 12000U / g according to the DNS method, and the powder was stored in a -20℃ refrigerator;

[0114] The primer sequences mentioned above are shown in Table 4.

[0115] Table 4 Primers and sequences used in the preparation of β-glucosidase

[0116]

[0117] Example 2

[0118] An embodiment of the composite enzyme and the preparation method thereof of the present invention. The components and amounts of the composite enzyme described in this embodiment are shown in Table 5. The preparation method of the composite enzyme described in this embodiment comprises the following steps:

[0119] The β-glucosidase, fructosyltransferase, lactase, cellulase and β-glucanase prepared above were mixed according to the ratio in Table 5, and 1 mM sodium phosphate buffer (pH=7.0) was added according to the weight ratio of complex enzyme: sodium phosphate buffer=1:15, and the complex enzyme solution was obtained after complete dissolution;

[0120] According to the DNS method, the enzyme activity of fructosyltransferase was 3000U / g, the enzyme activity of lactase was 6000U / g, the enzyme activity of cellulase was 10000U / g, and the enzyme activity of β-glucanase was 6500U / g.

[0121] Table 5 Components and dosages of different complex enzymes (in parts by weight)

[0122]

[0123]

[0124] Embodiment 3-4

[0125] Two embodiments of the composite enzyme and the preparation method thereof of the present invention, the components and dosages of the composite enzyme described in Embodiment 3-4 are shown in Table 5, and the preparation method of the composite enzyme described in Embodiment 3-4 is the same as that of Embodiment 2.

[0126] Example 5

[0127] An embodiment of a method for efficiently converting high-purity rare ginsenoside CK of the present invention comprises the following steps:

[0128] S1. The extract containing ginsenoside Rb1 and 1 mM sodium phosphate buffer (pH=7.0) were mixed in a reactor at a speed of 200 rpm according to a weight ratio of ginsenoside Rb1: sodium phosphate buffer = 1:50, and the composite enzyme solution obtained in Example 1 was added according to a weight ratio of ginsenoside Rb1: composite enzyme = 1:0.25, and the pH was adjusted to 6.5±0.1 with 10 wt % sodium hydroxide solution, and the high and low temperature oil tank was opened, the temperature was set to 50° C., the stirring speed of the reactor was 800 rpm, and the enzymolysis was performed for 72 h to obtain an enzymolysis solution;

[0129] S2, using a scraper to collect the precipitate on the wall of the reaction kettle and add it to the enzymolysis solution obtained in step S1, centrifuging the enzymolysis solution at 16000 rpm for 10 min, discarding the supernatant, collecting the precipitate, and washing the precipitate with a small amount of pure water to obtain precipitate A;

[0130] S3, adding the precipitate A obtained in step S2 and pure water into a homogenizer at a weight ratio of precipitate A:pure water = 1:10, and homogenizing and mixing. The obtained homogenate is centrifuged at 16000 rpm for 10 min, and the conductivity of the obtained supernatant is measured. If the conductivity is lower than 100 μs / cm, the precipitate is collected to obtain precipitate B. If the conductivity is higher than 100 μs / cm, pure water is added to the precipitate to continue homogenizing and centrifuging until the conductivity is lower than 100 μs / cm;

[0131] S4, placing the precipitate B obtained in step S3 and the 50 v / v% ethanol solution in a reaction kettle at a weight ratio of precipitate B: ethanol solution = 1:10, and stirring at 50° C. and 200 rpm until the precipitate is completely dissolved to obtain a reconstituted solution;

[0132] S5. After cleaning and installing the filter press plate frame, weigh 2 wt% diatomaceous earth into the reactor, stir evenly, open the feed port of the reactor, start the peristaltic pump, and introduce the complex solution in the reactor into the plate frame to obtain a clear solution;

[0133] S6, wash the 0.22μm PTFE folded filter element with pure water, wait until the pH of the liquid discharged from the liquid outlet is about 7.0, install the stainless steel filter, turn on the peristaltic pump, open the exhaust port to relieve pressure, open the liquid outlet of the stainless steel filter to drain the internal residual liquid, add the clear liquid obtained in step S5, self-circulate for 5min, and then discharge. After filtration, introduce 500mL of 95v / v% ethanol solution for filtration, and collect and combine the filtrate;

[0134] S7, the reactor is first cleaned with an appropriate amount of 95v / v% ethanol solution, and the tank wall and the tank bottom are checked for visible foreign matter. If there is any, it is cleaned with ethanol and finally rinsed with an appropriate amount of pure water. The filtrate obtained in step S6 is transferred to the reactor, and the reactor is started. The high and low temperature circulation box is set to 150±5℃, and the cooling water tank is set to -10±5℃ for concentration. During the concentration process, the vacuum degree is controlled to be less than -0.04Mpa until the filtrate is concentrated into a paste;

[0135] S8, vacuum drying the paste obtained in step S7 at 90° C. until the paste has a moisture content of less than 5% and becomes a block;

[0136] S9, crushing the block obtained in step S8 in a grinder, passing through an 80-mesh sieve, and the sieved material is ginsenoside CK.

[0137] Example 6

[0138] An embodiment of a method for efficiently converting high-purity rare ginsenoside CK of the present invention is similar to that of Example 5, except that:

[0139] In step S1, the weight ratio of the extract containing ginsenoside Rb1 and the complex enzyme is adjusted to ginsenoside Rb1:complex enzyme=1:0.095;

[0140] In step S1, the enzymatic hydrolysis conditions were adjusted to 45°C, 200 rpm, and the enzymatic hydrolysis time was adjusted to 70 h;

[0141] In step S4, the 50 v / v% ethanol solution is adjusted to a 40 v / v% ethanol solution;

[0142] In step S5, 2 wt% diatomaceous earth is adjusted to 1 wt% diatomaceous earth;

[0143] The remaining steps and their parameters remain unchanged.

[0144] Example 7

[0145] An embodiment of a method for efficiently converting high-purity rare ginsenoside CK of the present invention is similar to that of Example 5, except that:

[0146] In step S1, the weight ratio of the extract containing ginsenoside Rb1 and the sodium phosphate buffer is adjusted to 1:100 of the extract containing ginsenoside Rb1:sodium phosphate buffer;

[0147] In step S1, the weight ratio of the extract containing ginsenoside Rb1 and the complex enzyme is adjusted to ginsenoside Rb1:complex enzyme=1:0.7;

[0148] In step S1, the enzymatic hydrolysis conditions were adjusted to 55°C, 400 rpm, and the enzymatic hydrolysis time was adjusted to 74 h;

[0149] In step S3, the weight ratio of precipitate A and pure water is adjusted to precipitate A:pure water=1:50;

[0150] In step S4, the 50 v / v% ethanol solution is adjusted to a 95 v / v% ethanol solution;

[0151] In step S5, 2 wt% diatomaceous earth is adjusted to 10 wt% diatomaceous earth;

[0152] The remaining steps and their parameters remain unchanged.

[0153] Example 8

[0154] An embodiment of a method for efficiently converting high-purity rare ginsenoside CK of the present invention is similar to that of Example 5, except that:

[0155] In step S1, the weight ratio of the extract containing ginsenoside Rb1 and the sodium phosphate buffer is adjusted to 1:20 of the extract containing ginsenoside Rb1:sodium phosphate buffer;

[0156] The remaining steps and their parameters remain unchanged.

[0157] Example 9

[0158] An embodiment of a method for efficiently converting high-purity rare ginsenoside CK of the present invention is similar to that of Example 5, except that:

[0159] In step S1, the weight ratio of the extract containing ginsenoside Rb1 and the sodium phosphate buffer is adjusted to 1:10 of the extract containing ginsenoside Rb1:sodium phosphate buffer;

[0160] The remaining steps and their parameters remain unchanged.

[0161] Comparative Examples 1-6

[0162] Two comparative examples of the composite enzyme and the preparation method thereof of the present invention, the components and dosages of the composite enzymes described in comparative examples 1-6 are shown in Table 5, and the preparation method of the composite enzymes described in comparative examples 1-6 is the same as that of Example 2.

[0163] Comparative Example 7

[0164] A comparative example of the composite enzyme of the present invention and the preparation method thereof, the components and the dosage of the composite enzyme in this comparative example are similar to those in Example 2, except that the β-glucosidase used is replaced by β-glucosidase A, and the other components and the dosage remain unchanged; the preparation method of the composite enzyme in this comparative example is the same as that in Example 2.

[0165] Comparative Example 8

[0166] A comparative example of a method for efficiently converting high-purity rare ginsenoside CK of the present invention, the method described in this comparative example is similar to that of Example 5, except that:

[0167] In step S1, the weight ratio of the extract containing ginsenoside Rb1 and the sodium phosphate buffer is adjusted to 1:150 of the extract containing ginsenoside Rb1:sodium phosphate buffer;

[0168] In step S1, the weight ratio of the extract containing ginsenoside Rb1 and the complex enzyme is adjusted to ginsenoside Rb1:complex enzyme=1:0.001;

[0169] In step S1, the enzymatic hydrolysis conditions were adjusted to 60°C, 800 rpm, and the enzymatic hydrolysis time was adjusted to 85 h;

[0170] In step S3, the weight ratio of precipitate A and pure water is adjusted to precipitate A:pure water=1:60;

[0171] In step S5, 2 wt% diatomaceous earth is adjusted to 20 wt% diatomaceous earth;

[0172] The remaining steps and their parameters remain unchanged.

[0173] Effect Example 1

[0174] According to the method of Example 5, the composite enzyme preparations obtained in Examples 2-4 and Comparative Examples 1-7 were used to prepare ginsenoside CK, and the content and conversion rate of ginsenoside CK were detected and calculated by liquid chromatography.

[0175] The liquid chromatography operation is as follows:

[0176] Weigh 25 mg (accurate to 0.0001 g) of ginsenoside CK standard substance, place it in a 25 mL volumetric flask, add appropriate amount of methanol to dissolve, continue to add methanol to the scale, shake well to obtain a standard solution (stock solution) with a concentration of 1000 mg / L, seal it and store it at 4°C for one month. The purity of ginsenoside CK standard is ≥98%.

[0177] Take 0.50mL, 1.0mL, 2.5mL, and 5mL of the stock solution of ginsenoside CK standard respectively and place them in a 10mL volumetric flask, add methanol to make up to the scale, and obtain a series of ginsenoside CK standard solutions with corresponding concentrations of 50mg / L, 100mg / L, 250mg / L, 500mg / L, and 1000mg / L.

[0178] Weigh 0.2-0.5g (accurate to 0.0001g) of solid powder of ginsenoside CK with ginsenoside CK content ≥10% of the sample to be tested; weigh 2-10g (accurate to 0.0001g) of liquid sample with content <10%, and appropriately increase the sample weight for samples with lower ginsenoside CK content), place the sample in a 25mL volumetric flask, add methanol to the scale, ultrasonically vibrate for 30 minutes, shake well, filter with a 0.22μm organic filter membrane, and use it as the sample to be tested. When the content in the sample exceeds the range of the standard curve, the sample should be appropriately diluted and the appropriate standard curve range should be selected for detection.

[0179] The chromatographic column is a C18 column, the detection wavelength is 311nm, the flow rate is 1.0mL / min, the column temperature is 35℃, the injection volume is 10μL, the mobile phase A is first-grade water in accordance with GB / T 6682, and the mobile phase B is chromatographic grade acetonitrile. Gradient elution is performed. The elution program is shown in Table 6. The standard series solutions are injected in sequence, and the chromatogram is recorded. The standard curve is drawn with the concentration of the standard series solutions as the horizontal axis and the peak area as the vertical axis. The samples to be tested are injected in sequence, and the chromatogram is recorded. The retention time and ultraviolet spectrum are used for qualitative analysis, and the peak area is measured. The mass concentration of each component in the solution to be tested is obtained according to the standard curve, and the content and conversion rate of each component in the sample are calculated. The calculation formula is shown as follows:

[0180]

[0181] In formula 1, ω is the mass fraction of each component in the sample, %; ρ is the mass concentration of each component to be measured obtained from the standard curve, mg / L; V is the sample volume, mL; m is the sample volume, g; n is the dilution multiple;

[0182]

[0183] In Formula 2, n is the amount of ginsenoside CK or ginsenoside Rb1, which is obtained by multiplying the content of ginsenoside by the weight of ginsenoside and then dividing by the relative molecular mass of ginsenoside (1109.29 for ginsenoside Rb1 and 622.87 for ginsenoside CK).

[0184] The liquid chromatogram of ginsenoside CK standard is shown in Figure 2 The liquid chromatogram of ginsenoside CK prepared by the composite enzyme of Example 2 is shown in Figure 3 The rest of the data is shown in Table 7.

[0185] Table 6 Elution procedure for liquid chromatography detection of ginsenoside CK

[0186] Time / min Volume (mobile phase A) / % Volume (mobile phase B) / % 0 80 20 4 50 50 12 20 80 16 0 100 16.5 60 40 17 80 20 20 80 20

[0187] Table 7 Effects of different complex enzymes on the transformation of ginsenoside CK

[0188]

[0189]

[0190] like Figure 2-3 As shown in Table 7, the retention time of the ginsenoside CK standard is 13.78 min. The composite enzyme of the present invention can efficiently utilize ginsenoside Rb1 to convert it into ginsenoside CK, and the content of ginsenoside CK in the prepared product reaches more than 60.54%.

[0191] The composite enzyme obtained in Example 2 was used to convert ginsenoside CK into ginsenoside CK in three batches of extracts containing ginsenoside Rb1 according to the method of Example 5. The content of ginsenoside CK was detected and calculated according to the above method. The results are shown in Table 8.

[0192] Table 8 Conversion effect of ginsenoside CK in three batches of repeated verification of the optimal process

[0193]

[0194] As shown in Table 8, different batches confirmed that the composite enzyme and the method for efficiently converting high-purity ginsenoside CK of the present invention can stably convert ginsenoside CK to a content of 64.52% or more.

[0195] Effect Example 2

[0196] The composite enzyme preparation obtained in Example 2 was used to prepare ginsenoside CK according to the methods of Examples 5-9 and Comparative Example 8, and the content and conversion rate of ginsenoside CK were detected and calculated by liquid chromatography according to the method of Effect Example 1. The results are shown in Table 9.

[0197] Table 9 Effect of different processes on the conversion efficiency of ginsenoside CK

[0198]

[0199] As shown in Table 9, the method for efficiently converting high-purity ginsenoside CK of the present invention can effectively increase the conversion of ginsenoside Rb1 into ginsenoside CK, so that the content and conversion rate of ginsenoside CK are higher.

[0200] Effect Example 3

[0201] According to the method of Example 5, the composite enzyme obtained in Example 2 was used to prepare ginsenoside CK, and the stability of ginsenoside CK (named ginsenoside CK ①) and commercially available ginsenoside CK with a content of 50% (named ginsenoside CK ②) were tested.

[0202] Ginsenoside CK ① and ginsenoside CK ② were added to butanediol to prepare a 10 g / 100 mL solution, which was placed at 48°C for 3 months (90 days) and its color and state were observed. Figure 4 .

[0203] like Figure 4 As shown, after ginsenoside CK② was dissolved and placed at 48°C for 3 months, obvious precipitation occurred, and the liquid was yellow, while after ginsenoside CK① was dissolved and placed for 3 months, no precipitate was precipitated, and the color was relatively clear and transparent, indicating that the ginsenoside CK prepared by the composite enzyme obtained by the present invention and the method for converting ginsenosides has high stability, lighter color and is more suitable for application in the fields of cosmetics, pharmaceuticals, etc.

[0204] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A complex enzyme for efficiently converting high-purity rare ginsenoside CK, characterized in that: The invention comprises β-glucosidase, fructosyltransferase, lactase, cellulase and β-glucanase, wherein the weight ratio of β-glucosidase, fructosyltransferase, lactase, cellulase and β-glucanase is β-glucosidase:fructosyltransferase:lactase:cellulase:β-glucanase=(0.05-0.2):(0.005-0.2):(0.01-0.1):(0.01-0.1):(0.02-0.1); The preparation method of the β-glucosidase comprises the following steps: A1. Codon optimization of β-glucosidase from soil Sinomonas was performed, and an expression vector carrying a β-glucosidase coding sequence was prepared by gene editing technology, wherein the amino acid sequence of the codon-optimized β-glucosidase is shown in SEQ ID NO.1; A2, transforming the expression vector described in step A1 into Escherichia coli, and obtaining positive clones through screening and identification; A3, preparing the positive clones obtained in step A2 as a secondary seed solution, and using the secondary seed solution to perform fed-batch fermentation to obtain a fermentation solution; A4, centrifuging the fermentation liquid obtained in step A3, collecting the bacterial precipitate, crushing, centrifuging, concentrating and freeze-drying the obtained bacterial precipitate to obtain β-glucosidase; In step A3, the fed-batch fermentation comprises the following operations: B1. Inoculate the secondary seed liquid into the fermentation medium, adjust the pH to 6.6-7.0, and set the temperature to 35-37° C.; the volume ratio of the secondary seed liquid to the fermentation medium is secondary seed liquid: fermentation medium = (0.8-1):30; B2. When the dissolved oxygen drops to 60-70%, adjust the temperature to 23-27°C and start feeding. The feeding rate is 190-200mL / h in the first hour, 220-230mL / h in the second hour, and 250-260mL / h in the third hour and maintained unchanged. The dissolved oxygen is slowly reduced to 12-18% by controlling the tank pressure, rotation speed and air volume. After feeding, sample the fermentation liquid every 4 hours to detect the OD600 value. When the OD600 value no longer rises, the fermentation is terminated.

2. The complex enzyme according to claim 1, characterized in that The weight ratio of the β-glucosidase, fructosyltransferase, lactase, cellulase and β-glucanase is β-glucosidase:fructosyltransferase:lactase:cellulase:β-glucanase=0.1:0.01:0.04:0.05:0.

05.

3. The complex enzyme according to claim 1 or 2, characterized in that The enzyme activity of the β-glucosidase is 12000-13000 U / g, the enzyme activity of the fructosyltransferase is 2500-3000 U / g, the enzyme activity of the lactase is 5500-6000 U / g, the enzyme activity of the cellulase is 9000-10000 U / g, and the enzyme activity of the β-glucanase is 6000-6500 U / g.

4. Use of the complex enzyme as described in any one of claims 1 to 3 in the preparation of ginsenoside CK.

5. A method for efficiently converting high-purity rare ginsenoside CK, characterized in that: The following steps are involved: (1) mixing an extract containing ginsenoside Rb1 with 0.5-1 mM phosphate buffer, adding the complex enzyme described in any one of claims 1-3, adjusting the pH to 6.0-6.5, and performing enzymolysis at 45-55° C. and 200-800 rpm for 70-74 h to obtain an enzymolysis solution, wherein the weight ratio of the extract containing ginsenoside Rb1 to the phosphate buffer is 1:(10-100); (2) centrifuging the enzymatic hydrolyzate obtained in step (1), collecting precipitate A, mixing the precipitate A with pure water, homogenizing and centrifuging, and collecting precipitate B when the conductivity of the supernatant is less than 100 μs / cm; (3) Mixing the precipitate B obtained in step (2) with an ethanol solution having a volume percentage of 40-95% as a solvent to prepare a reconstituted solution, and subjecting the reconstituted solution to pressure filtration, microfiltration and concentration treatment to obtain high-purity rare ginsenoside CK.

6. The method according to claim 5, characterized in that Including at least one of the following (I)-(III): (I) In step (1), the weight ratio of the extract containing ginsenoside Rb1 to the phosphate buffer is: extract containing ginsenoside Rb1: phosphate buffer = 1: (10-90); (II) In step (1), the weight ratio of the extract containing ginsenoside Rb1 to the complex enzyme is: extract containing ginsenoside Rb1: complex enzyme = 1: (0.095-0.7); (III) In step (1), the complex enzyme is prepared into a complex enzyme solution with a phosphate buffer having a pH of 6.5-7.5 and a value of 0.5-1 mM before being added, and the weight ratio of the complex enzyme to the phosphate buffer is complex enzyme: phosphate buffer = 1: (10-15).

7. The method according to claim 5, characterized in that Including at least one of the following (IV)-(V): (IV) In step (2), the weight ratio of the precipitate A to pure water is precipitate A:pure water=1:(10-50); (V) In step (3), the weight ratio of the ethanol solution to the precipitate B is 1:(10-15), and the volume percentage of the ethanol solution is 50-60%.

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