CN115449536A - Method for converting Rb1 into CK by Aspergillus niger fermentation liquor - Google Patents

Method for converting Rb1 into CK by Aspergillus niger fermentation liquor Download PDF

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CN115449536A
CN115449536A CN202110644100.8A CN202110644100A CN115449536A CN 115449536 A CN115449536 A CN 115449536A CN 202110644100 A CN202110644100 A CN 202110644100A CN 115449536 A CN115449536 A CN 115449536A
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aspergillus niger
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谢海峰
马素敏
王鸿江
钟艾玲
谢期林
胡云岭
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Chengdu Biopurify Ltd
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Abstract

The invention relates to a method for converting Rb1 into CK by Aspergillus niger fermentation liquor, belonging to the technical field of traditional Chinese medicines. In the method for converting Rb1 into CK by using Aspergillus niger fermentation liquor, the Aspergillus niger is Aspergillus niger AS3.0739 with the preservation number of CGMCC 3.739. The method for converting ginsenoside Rb1 into ginsenoside CK by using the extracellular enzyme secreted by Aspergillus niger AS3.0739 for the first time has wide substrate source and the substrate concentration can reach 50g/L. The invention also optimizes the culture condition of Aspergillus niger AS3.0739, and the extracellular enzyme secreted by the invention can catalyze the CK conversion rate to reach 90 percent, which is far higher than that reported in the existing literature.

Description

Method for converting Rb1 into CK by Aspergillus niger fermentation liquor
Technical Field
The invention relates to a method for converting Rb1 into CK by Aspergillus niger fermentation liquor, belonging to the technical field of traditional Chinese medicines.
Background
Ginseng has a long medicinal history in China, and is well known as Shencao and Baicao king. According to the record of the earliest monograph of pharmacy in China, shen nong Ben Cao Jing, ginseng has the effects of nourishing five internal organs, calming the mind, stopping palpitation due to fright, eliminating pathogenic factors, improving eyesight, developing mind, improving intelligence, reducing weight and prolonging life after long-term use. The saponin is one of the main active ingredients of ginseng, and the ginsenoside Compound K is a main metabolite of protopanaxadiol type ginsenoside in a human body and an entity substance exerting the drug effect, and has various biological activities including anti-tumor, anti-inflammatory, anti-allergy and the like.
The ginsenoside components in Ginseng radix are various, but the main components are ginsenoside Rg1, rb2, rc, rd, re and notoginsenoside R1, etc., and the content of rare saponins such as ginsenoside CK, rg3 and Rh2 is very small. The preparation of the ginsenoside CK is divided into a chemical method and a biological method, the traditional thought is to hydrolyze C-3 and C-20 glycosyl groups by using an acid-base hydrolysis chemical method to obtain the CK, and the method has the defects of high cost, low yield, more byproducts and the like. The biological method has the advantages of high stereoselectivity, high yield, few byproducts, capability of obtaining rare ginsenoside and the like, and is the most potential method for producing the rare ginsenoside.
The prior patents related to the biological preparation of ginsenoside CK mainly comprise the following aspects: (1) Carrying out gene modification on wild beta-glucosidase to improve the activity of synthesizing ginsenoside CK; (CN 103805581) (2) preparation of ginsenoside CK by combination of enzyme method and biological method, and CN 105838770A introduction of Microbacterium oxydans (preservation number: CCTCC AB 205576) fermentation culture in combination with heteropolyacid H x YW 12 O 40 ·nH 2 Preparing ginsenoside CK by using an O catalyst; (3) CN 106047978 fermenting and culturing natural Ganoderma with fermentation bacteria to obtain ginsenoside CK, except thatThe concentration of the total saponins of the substrate is 2mg/mL, the conversion rate of the ginsenoside CK is less than 1%, the highest yield of the ginsenoside CK is 11.4mg/L, and the yield is low; (4) CN 105296587A introduces a beta-glucosidase from b.breve ATCC 15700 bifidobacterium, the concentration of ginsenoside Rb1 is 10g/L, the CK conversion rate is 62-68%, but the purification step of the beta-glucosidase is involved in the middle, which is complicated. (5) CN 109536561A introduces a method for preparing CK by catalyzing ginsenoside Rb1 with Ginseng radix endophyte, wherein the concentration of ginsenoside Rb1 substrate is 0.25mg/mL, and the culture is continued for 5-10 days by shaking to obtain rare ginsenoside CK.
Aspergillus niger degrades ginsenoside Rb1 to prepare rare saponin Compound K [ J ] biotechnological advances, 2016,6 (2): 98-104, discloses 9 strains of fungi separated from northeast agro-farming soil, and systematically researches the capability of the fungi in converting ginsenoside Rb1 to prepare CK. Wherein Aspergillus niger sp.J7 can efficiently convert ginsenoside Rb1 to generate CK, and the conversion pathway is Rb1 → Rd → F2 → CK. Optimizing the condition of preparing CK by converting Rb1 by Aspergillus niger J7, wherein the optimal substrate concentration is 1mg/mL, and 12h Rb1 can be completely converted into CK; however, the substrate concentration increased to 2mg/L,12h Rb1 was not completely converted. The conversion system is expanded to 200mL, rb1 can be converted into CK within 60h under the optimal condition, the conversion rate is 74.7%, and the literature reports that the concentration of a substrate in the process system is low, so that the process system is not beneficial to expanded production.
The mould is often used as a commercial enzyme source for decomposing starch sugar in food industry application, and enzyme preparations produced by a plurality of moulds can be used as processing aids in the food and health care product industry.
Aspergillus niger AS3.0739 is a common glucoamylase-producing strain.
Disclosure of Invention
The invention aims to provide a novel method for converting Rb1 into CK by Aspergillus niger fermentation liquor.
In order to solve the technical problem, in the method for converting Rb1 into CK by using the Aspergillus niger fermentation liquid, aspergillus niger AS3.0739 is adopted AS Aspergillus niger, and the preservation number is CGMCC 3.739.
In one embodiment, the preparation method of the aspergillus niger fermentation broth comprises the following steps of: 1.0-5.0 g/L of corn steep liquor, 2.0-10.0 g/L of yeast powder, 5.0-9.0 g/L of ammonium sulfate, 4.0-8.0 g/L of monopotassium phosphate, 1.0-3.0 g/L of magnesium sulfate, 0.5-2.0 g/L of calcium chloride dihydrate, 0-8.0 g/L of dipotassium phosphate and the balance of water; the water is preferably distilled water.
In one embodiment, the preparation method of the aspergillus niger fermentation liquid comprises the following steps: inoculating Aspergillus niger into the culture medium, and fermenting at 25-37 deg.c for 1-16 days.
In a specific embodiment, the preparation method of the aspergillus niger fermentation liquid further comprises standing the fermented solution, centrifuging, and taking the supernatant to obtain the aspergillus niger fermentation liquid.
In one embodiment, the method for converting Rb1 to CK by the aspergillus niger fermentation broth comprises: mixing ginsenoside Rb1, aspergillus niger fermentation liquor and organic solvent to obtain conversion solution, stirring, and reacting to obtain solution containing CK.
The ginsenoside Rb1 standard product or the ginsenoside crude product containing Rb1 can be used as the substrate ginsenoside Rb1 for reaction; for example, the ginsenoside crude products can be ginsenoside Rb1 crude products with different purities, and the ginsenoside Rb1 standard product can be more than 98% of the commercially available standard product; the ginsenoside Rb1 crude product is extract containing panaxadiol saponins such as ginsenoside Rb1, ginsenoside Rd and ginsenoside F2.
In one embodiment, the concentration of ginsenoside Rb1 in the conversion solution is 5 to 50g/L, preferably 10 to 20g/L.
In one embodiment, the organic solvent is DMSO, preferably the DMSO is added in an amount of 2 to 10wt%, more preferably 2 to 5wt% of the conversion solution.
In one embodiment, the temperature of the reaction is from 25 to 55 ℃, more preferably from 45 to 50 ℃; the reaction time is 24-240 h.
In one embodiment, the method further comprises maintaining the ph of the conversion solution at 4.0 to 7.0, preferably at 4.8;
the method for maintaining the pH value is to add buffer salt to maintain the pH value, preferably, the buffer salt is phosphate buffer solution or acetate buffer solution, and the concentration of the buffer salt is 0-0.2M; preferably, the concentration of the buffer salt is 0.05 to 0.2M, more preferably 0.1 to 0.2M. The phosphate is preferably an analytically pure phosphate.
In a specific embodiment, the method further comprises purifying the solution containing CK to obtain CK with a purity of 98% or more; the purification method comprises the steps of extracting a solution containing CK by using an organic solvent, purifying by using a Sephadex G-200 gel column and crystallizing.
Has the advantages that:
(1) The method for converting ginsenoside Rb1 into ginsenoside CK by using the extracellular enzyme secreted by Aspergillus niger AS3.0739 for the first time has the substrate concentration of 50g/L.
(2) Aspergillus niger AS3.0739 is a fermentation strain, experimental results are verified for many times and process optimization, and extracellular enzyme secreted by the Aspergillus niger AS3.0739 can catalyze CK with the relative percentage content reaching 90% by adopting the culture condition of the invention, which is far higher than that reported in the existing literature.
(3) The raw materials used by the invention are wide, and can be ginsenoside Rb1 98% standard substance, protopanaxadiol saponin mixture containing ginsenoside Rb1, rd and F2, and also can be crude extract containing ginsenoside Rb 1.
(4) The Aspergillus niger AS3.0739 fermentation liquor can be used AS a reaction catalyst without a complicated purification process, so that a large amount of purification cost and time are saved.
(5) The production process has the characteristics of mild reaction conditions, low organic solvent consumption, low industrial wastewater, high conversion rate, large-scale production and the like.
Drawings
FIG. 1 is a liquid chromatogram before and after conversion of a fermentation broth of Aspergillus niger AS3.0739 with ginsenoside Rb1 having a purity of 98% AS a substrate, A AS a substrate, and B AS a sample spectrum after enzyme conversion.
FIG. 2 is a liquid chromatogram before and after conversion of a fermentation broth of Aspergillus niger AS3.0739 with a crude product of ginsenoside Rb1 AS a substrate, A AS a substrate, and B AS a sample map after enzymatic conversion.
FIG. 3 is a standard sample liquid chromatogram.
FIG. 4-1 is a full view of the carbon spectrum of the obtained ginsenoside CK sample, wherein three large peaks near 151, 136 and 125 are solvent pyridine peaks, the peak values are higher, and the later signal peaks are normal; the peaks are too many and numbers easily overlap, and the numbers partially overlapping the peaks in fig. 4-1 are illustrated here as 0.90 and 16.95.
Fig. 4-2 is a partial enlarged view of the obtained ginsenoside CK sample.
FIG. 5-1 is a full view of the hydrogen spectrum of the obtained ginsenoside CK sample.
FIG. 5-2 is a partial enlarged view of the hydrogen spectrum of the obtained ginsenoside CK sample; CK has 60H's, each hydrogen will signal and so will overlap, and the number of peaks overlapping in FIG. 5-2 is illustrated as 1.573.
FIG. 6 is a mass spectrum of the prepared CK sample of ginsenoside; 681.3 in fig. 6 is the signal peak for CK, since some peaks are denser, with numbers partially overlapping with peaks, which in fig. 6 is illustrated as 659.2, 668.2, 682.3.
FIG. 7 is a comparison of enzyme activities of different media of example 1; the Rb1 hydrolyzing activity of enzyme liquid produced by the fermentation of the M3 culture medium is defined as 100%.
FIG. 8 is a diagram of the growth of Aspergillus niger in PDA solid medium; PDA solid medium: 200g of potato, 20g of glucose, 20g of agar and 1000mL of water, and the pH value is natural.
FIG. 9 shows the growth pattern of cells in a part of the medium of example 1; from left to right, PD medium (control), M1 medium, M2 medium, M3 medium, M4 medium; wherein the PD culture medium comprises 20g of potato, 2.0g of glucose and 100mL of water, and the pH value is natural.
FIG. 10 shows the CK percentage of ginsenoside in example 2.
FIG. 11 shows the CK percentage of ginsenoside in example 3.
FIG. 12 shows the CK percentage of ginsenoside in example 4.
FIG. 13 shows CK percent ginsenoside of example 5.
FIG. 14 shows the CK percentage of ginsenoside in example 6.
FIG. 15 shows the CK percentage of ginsenoside in example 7.
FIG. 16 shows the CK percentage of ginsenoside used in example 8.
Detailed Description
In order to solve the technical problem, in the method for converting Rb1 into CK by using the Aspergillus niger fermentation liquid, the Aspergillus niger AS3.0739 is adopted AS the Aspergillus niger, and the preservation number is CGMCC 3.739.
In one embodiment, the preparation method of the aspergillus niger fermentation broth comprises the following steps of: 1.0-5.0 g/L of corn steep liquor, 2.0-10.0 g/L of yeast powder, 5.0-9.0 g/L of ammonium sulfate, 4.0-8.0 g/L of monopotassium phosphate, 1.0-3.0 g/L of magnesium sulfate, 0.5-2.0 g/L of calcium chloride dihydrate, 0-8.0 g/L of dipotassium phosphate and the balance of water; the water is preferably distilled water.
In one embodiment, the preparation method of the aspergillus niger fermentation liquid comprises the following steps of: 3g/L of corn steep liquor, 5g/L of yeast powder, 7g/L of ammonium sulfate, 6g/L of monopotassium phosphate, 1g/L of magnesium sulfate, 1g/L of calcium chloride dihydrate and the balance of water.
In a specific embodiment, the preparation method of the aspergillus niger fermentation broth comprises the following steps: inoculating Aspergillus niger into the culture medium, and fermenting at 25-37 deg.c for 1-16 days.
In a specific embodiment, the preparation method of the aspergillus niger fermentation liquid further comprises the steps of standing the fermented solution, centrifuging, and taking the supernatant to obtain the aspergillus niger fermentation liquid.
In one embodiment, the method for converting Rb1 into CK by the aspergillus niger fermentation broth comprises: mixing ginsenoside Rb1, aspergillus niger fermentation liquor and organic solvent to obtain conversion solution, stirring, and reacting to obtain solution containing CK.
The ginsenoside Rb1 standard product or the ginsenoside crude product containing Rb1 can be used as the substrate ginsenoside Rb1 for reaction; for example, the ginsenoside crude products can be ginsenoside Rb1 crude products with different purities, and the ginsenoside Rb1 standard product can be more than 98% of the commercially available standard product; the ginsenoside Rb1 crude product is extract containing panaxadiol saponins such as ginsenoside Rb1, ginsenoside Rd and ginsenoside F2.
In a specific embodiment, the concentration of the ginsenoside Rb1 in the conversion solution is 5 to 50g/L, preferably 10 to 20g/L.
In one embodiment, the organic solvent is DMSO, preferably the DMSO is added in an amount of 2 to 10wt%, more preferably 2 to 5wt% of the conversion solution.
In one embodiment, the temperature of the reaction is from 25 to 55 ℃, more preferably from 45 to 50 ℃; the reaction time is 24-240 h.
In one embodiment, the method further comprises maintaining the ph of the conversion solution at 4.0 to 7.0, preferably at 4.8;
maintaining the pH value by adding buffer salt, preferably selecting the buffer salt as phosphate buffer solution or acetate buffer solution, wherein the concentration of the buffer salt is 0-0.2M; the concentration of the buffer salt is preferably 0.05 to 0.2M, more preferably 0.1 to 0.2M. The phosphate is preferably an analytically pure phosphate.
In one embodiment, the method further comprises purifying the solution containing CK to obtain CK with a purity of 98% or more; the purification method comprises the steps of extracting a solution containing CK by using an organic solvent, purifying by using a Sephadex G-200 gel column and crystallizing.
The following examples are provided to further illustrate the embodiments of the present invention and are not intended to limit the scope of the present invention.
In all examples, the percentage of CK in the ginsenoside material was approximately 0. The measure CK conversion is calculated as follows:
CK conversion = actual conversion to CK/theoretical conversion to CK 100%
Example 1
Inoculating separated and purified Aspergillus niger AS3.0739 (purchased from China general microbiological culture Collection center, number CGMCC 3.739) (see figure 8) into different culture media, culturing at 30 deg.C for 6 days (see figure 9), performing solid-liquid separation, and collecting supernatant for use.
The specific components of the culture medium are as follows:
m1 culture medium: 1.0g/L of cane sugar, 1.0g/L of soybean meal, 0.3g/L of sodium nitrate, 0.3g/L of monopotassium phosphate and 0.2g/L of dipotassium phosphate.
M2 culture medium: 2.0g/L of corn steep liquor, 6.0g/L of yeast powder, 6.0g/L of ammonium sulfate, 6.0g/L of monopotassium phosphate, 2.0g/L of magnesium sulfate and 1.0g/L of calcium chloride dihydrate.
M3 medium: 30.0g/L of sucrose, 3.0g/L of sodium nitrate, 0.5g/L of magnesium sulfate heptahydrate, 0.5g/L of potassium chloride, 0.01g/L of ferric chloride tetrahydrate and 1.0g/L of dipotassium phosphate.
M4 medium: 100.0g/L glucose, 5.0g/L peptone, 5.0g/L potassium nitrate, 30.0g/L calcium carbonate, 2.0g/L potassium dihydrogen phosphate, 0.7g/L magnesium sulfate heptahydrate and 0.5g/L potassium chloride.
Medium No. M5: 30.0g/L of corn flour, 10.0g/L of gluten powder, 5.0g/L of yeast powder, 7.0g/L of ammonium sulfate, 6.0g/L of monopotassium phosphate, 1.0g/L of magnesium sulfate heptahydrate, 1.0g/L of calcium chloride dihydrate and 0.1g/L of Twwen-80.
Medium No. M6: 15g/L of sucrose, 12g/L of beef extract, 1.0g/L of ammonium sulfate, 2g/L of monopotassium phosphate and 0.1g/L of magnesium sulfate heptahydrate.
Medium No. M7: 20g/L of konjac flour, 30g/L of gluten powder, 60g/L of corn steep liquor, 5g/L of monopotassium phosphate and 2.5g/L of magnesium sulfate.
M8 Medium (M2-V1): 3.5g/L of corn steep liquor, 6.0g/L of yeast powder, 7.0g/L of ammonium sulfate, 6.0g/L of monopotassium phosphate, 2.0g/L of magnesium sulfate, 1.0g/L of calcium chloride dihydrate and 12.5g/L of calcium carbonate.
M9 Medium (M2-V2): 3.0g/L of corn steep liquor, 6.0g/L of yeast powder, 7.0g/L of ammonium sulfate, 6.0g/L of monopotassium phosphate, 2.0g/L of magnesium sulfate, 1.0g/L of calcium chloride dihydrate and 6.0g/L of dipotassium phosphate.
M10 medium (M2-V3): 3.0g/L of corn steep liquor, 6.0g/L of yeast powder, 7.0g/L of ammonium sulfate, 6.0g/L of monopotassium phosphate, 2.0g/L of magnesium sulfate, 1.0g/L of calcium chloride dihydrate and 15g/L of cane sugar.
The collected supernatant was used as a reaction catalyst, and 98% Rb1,5wt% was added to assist in DMSO, 20mM citric acid-sodium citrate was maintained at pH =4.5, the final concentration of the substrate in the reaction solution was 10g/L, and the reaction was carried out at 50 ℃ for 48 hours. The reaction solution is detected by HPLC, and the conversion rate of the ginsenoside CK is taken as a measurement index, and specific data are shown in figure 7. The result shows that the M4 culture medium has the worst transformation effect, and the conversion rate of the ginsenoside CK is only 3.8%; ginsenoside, M2 and M9 culture medium are effective, and the conversion rate is 68.7% and 61.1% respectively. Therefore, M2 is the optimal medium.
Example 2
Preparing fermentation liquor: in M2 medium: 2.0g/L of corn steep liquor, 6.0g/L of yeast powder, 6.0g/L of ammonium sulfate, 6.0g/L of monopotassium phosphate, 2.0g/L of magnesium sulfate and 1.0g/L of calcium chloride dihydrate. The culture medium is prepared by fermenting one with tap water and the other with distilled water at 37 deg.C for 3 days to obtain supernatant.
Weighing 1kg of ginsenoside crude product (purity 50%), adding 2wt% DMSO for solubilization, adding 98L of fermented supernatant, and substrate concentration 5g/L. The reaction temperature is 50 ℃, the pH value is 4.8, the phosphate concentration is 0.2M, the reaction is carried out for 72 hours, and the relative percentage content of CK detected by liquid chromatography is used as a measurement index. As shown in FIG. 10, the conversion rates of CK in the enzyme-liquid catalytic reactions of the fermentation of tap water and distilled water were 52.72% and 77.31%, respectively. Extracting with organic solvent, purifying with Sephadex G-200 gel column, and crystallizing to obtain 98% CK containing ginsenoside 123.6G and 188.6G. Therefore, the enzyme preparation prepared from distilled water has obviously better effect on catalyzing the reaction CK than tap water.
Example 3
Preparing fermentation liquor: in M2 medium: 2.0g/L of corn steep liquor, 6.0g/L of yeast powder, 6.0g/L of ammonium sulfate, 6.0g/L of monopotassium phosphate, 2.0g/L of magnesium sulfate and 1.0g/L of calcium chloride dihydrate. The culture medium is prepared by fermenting with tap water at 37 deg.C for 3 days to obtain supernatant.
Adding phosphate buffer solution into the supernatant to adjust the pH value, enabling the reaction system to be respectively at pH4.8, pH6.0 and pH8.0, adding 2wt% of DMSO to assist dissolution, wherein the purity of the ginsenoside Rb1 as a raw material is 98%, the concentration of a substrate is 10g/L, the concentration of phosphate is 0.2M, the reaction temperature is 50 ℃, sampling and measuring are carried out at regular time, and the conversion rate of CK detected by liquid chromatography is used as a measurement index. The data are shown in FIG. 11, at pH4.8, pH6.0 and pH8.0, the 72h CK conversion is 97.67%, 88.23% and 1.13%, respectively. The conversion rate of the ginsenoside CK is the highest under the acidic condition of pH 4.8.
Example 4
Preparing fermentation liquor: in M2 medium: 2.0g/L of corn steep liquor, 6.0g/L of yeast powder, 6.0g/L of ammonium sulfate, 6.0g/L of monopotassium phosphate, 2.0g/L of magnesium sulfate and 1.0g/L of calcium chloride dihydrate. The culture medium is prepared by fermenting with tap water at 37 deg.C for 3 days to obtain supernatant.
Adding crude product of ginsenoside Rb1 with purity of 50% into the supernatant, adding 2wt% DMSO for solubilization, with substrate concentration of 10g/L, phosphate concentration of 0.2M and pH of 4.8, reacting for 48h at room temperature of 45 deg.C, 50 deg.C and 55 deg.C, and detecting CK conversion rate by liquid chromatography as measurement index. Sampling at regular time for determination, wherein the experimental result is shown in figure 12, the reaction lasts for 96h, the reaction temperature is 50 ℃, and the conversion rate of the ginsenoside CK is 88.5%; the highest CK conversion rate is only 22.2 percent at the temperature of 55 ℃; CK conversion 76.9% at 45 ℃. Reaction at 25 ℃ for 240h, the CK conversion rate reaches 63.4 percent.
Example 5
Preparing fermentation liquor: in M2 medium: 2.0g/L of corn steep liquor, 6.0g/L of yeast powder, 6.0g/L of ammonium sulfate, 6.0g/L of monopotassium phosphate, 2.0g/L of magnesium sulfate and 1.0g/L of calcium chloride dihydrate. The culture medium is prepared by fermenting with tap water at 37 deg.C for 3 days to obtain supernatant.
Adding crude product of ginsenoside Rb1 with purity of 50% into the above supernatant, 2wt% adding DMSO for solubilization, phosphate concentration of 0.2M, pH4.8, reaction temperature of 50 deg.C, and reacting for 96h. The concentration of a substrate ginsenoside Rb1 is selected from three gradients of 10g/L, 20g/L and 50g/L, and the CK conversion rate detected by liquid chromatography is used as a measurement index. Specific data As shown in FIG. 13, CK conversions of 10g/L, 20g/L and 50g/L were 91.45%, 81.29% and 42.66% at 96h.
Example 6
Preparing fermentation liquor: in M2 medium: 2.0g/L of corn steep liquor, 6.0g/L of yeast powder, 6.0g/L of ammonium sulfate, 6.0g/L of monopotassium phosphate, 2.0g/L of magnesium sulfate and 1.0g/L of calcium chloride dihydrate. The culture medium is prepared by fermenting with tap water at 37 deg.C for 3 days to obtain supernatant.
Adding crude product of ginsenoside Rb1 with the purity of 50% into the supernatant, carrying out DMSO cosolvent, wherein the adding amount of DMSO is 2wt%, 5wt% and 10wt%, the substrate concentration is 10g/L, the phosphate concentration is 0.2M, the pH value is 4.8, the reaction temperature is 50 ℃, reacting for 96h, and detecting the relative percentage content of CK by liquid chromatography as a measurement index. Specific data are shown in FIG. 14, 96h, DMSO addition levels are 2wt%, 5wt%, and 10wt%, and CK conversions are 91.45%, 88.81%, and 51.67%, respectively.
Example 7
Preparing fermentation liquor: in M2 medium: 2.0g/L of corn steep liquor, 6.0g/L of yeast powder, 6.0g/L of ammonium sulfate, 6.0g/L of monopotassium phosphate, 2.0g/L of magnesium sulfate and 1.0g/L of calcium chloride dihydrate. The culture medium is prepared by fermenting with tap water at 37 deg.C for 3 days to obtain supernatant.
Adding crude product of ginsenoside Rb1 with purity of 50% into the supernatant, adding DMSO at 2wt% to assist dissolution, with substrate concentration of 10g/L, pH of 4.8, reaction temperature of 50 deg.C, and measuring CK relative percentage content by liquid chromatography. And (3) investigating the influence of different grades of phosphate on the percentage content of CK, wherein the phosphate is selected from analytically pure phosphate, industrial phosphate and phosphate not added to adjust the pH value, the phosphate concentration is 0.2M, the specific result is shown in figure 15, and the CK conversion rates of the analytically pure phosphate, the industrial phosphate and the phosphate not added are respectively 92.39%, 56.32% and 86.63% after 102 h. Industrial phosphate has more impurities, seriously influences the enzyme reaction effect, and is selected to be analytically pure; in order to save cost in industrial production, phosphate can not be added in the enzyme reaction stage.
Example 8
Preparing fermentation liquor: in M2 medium: 2.0g/L of corn steep liquor, 6.0g/L of yeast powder, 6.0g/L of ammonium sulfate, 6.0g/L of monopotassium phosphate, 2.0g/L of magnesium sulfate and 1.0g/L of calcium chloride dihydrate. The culture medium is prepared by fermenting with tap water at 37 deg.C for 3 days to obtain supernatant.
Adding crude product of ginsenoside Rb1 with purity of 50% into the above supernatant, 2wt% DMSO for solubilization, substrate concentration of 10g/L, reaction temperature of 50 deg.C, phosphate concentration of 5 gradients tested, and reaction time of 48h, and the specific data is shown in FIG. 16. Phosphate concentrations 0, 0.02M, 0.05M, 0.1M and 0.2M, CK conversions 57.96%, 58.82%, 63.17%, 70.67% and 80.79%, respectively.

Claims (10)

1. The method for converting Rb1 into CK by Aspergillus niger fermentation liquor is characterized in that Aspergillus niger AS3.0739 is adopted AS Aspergillus niger, and the preservation number is CGMCC 3.739.
2. The method for converting Rb1 to CK using Aspergillus niger fermentation broth according to claim 1, wherein the preparation method of Aspergillus niger fermentation broth comprises using the following media: 1.0-5.0 g/L of corn steep liquor, 2.0-10.0 g/L of yeast powder, 5.0-9.0 g/L of ammonium sulfate, 4.0-8.0 g/L of monopotassium phosphate, 1.0-3.0 g/L of magnesium sulfate, 0.5-2.0 g/L of calcium chloride dihydrate, 0-8.0 g/L of dipotassium phosphate and the balance of water; the water is preferably distilled water.
3. The method for converting Rb1 to CK using Aspergillus niger fermentation broth according to claim 2, wherein the preparation method of Aspergillus niger fermentation broth comprises: inoculating Aspergillus niger into the culture medium, and fermenting at 25-37 deg.c for 1-16 days.
4. The method for converting Rb1 to CK using the Aspergillus niger fermentation liquid of claim 3, wherein the method for preparing the Aspergillus niger fermentation liquid further comprises the step of separating the solid from the liquid of the fermentation liquid, and collecting the supernatant as the reaction solution.
5. The method for converting Rb1 to CK using Aspergillus niger fermentation broth according to any of claims 1 to 4, wherein the method for converting Rb1 to CK using Aspergillus niger fermentation broth comprises: mixing ginsenoside Rb1, aspergillus niger fermentation liquor and organic solvent to obtain conversion solution, stirring, and reacting to obtain solution containing CK.
6. The method for converting Rb1 into CK by the Aspergillus niger fermentation liquid according to claim 5, wherein the concentration of the ginsenoside Rb1 in the conversion solution is 5 to 50g/L, preferably 10 to 20g/L.
7. The method for converting Rb1 to CK using Aspergillus niger fermentation broth according to claim 5 or 6, wherein the organic solvent is DMSO, preferably the DMSO is added in an amount ranging from 2 to 10wt%, more preferably from 2 to 5wt% of the conversion solution.
8. The method for converting Rb1 to CK using an Aspergillus niger fermentation broth according to any of claims 5 to 7, wherein the temperature of the reaction is between 25 ℃ and 55 ℃, more preferably between 45 ℃ and 50 ℃; the reaction time is 24-240 h.
9. The method of converting Rb1 to CK by an aspergillus niger fermentation broth according to any of claims 5 to 8, further comprising maintaining the ph of the conversion solution at 4.0 to 7.0, preferably at 4.8;
the method for maintaining the pH value is to add buffer salt to maintain the pH value, preferably, the buffer salt is phosphate buffer solution or acetate buffer solution, and the concentration of the buffer salt is 0-0.2M; the concentration of the buffer salt is preferably 0.05 to 0.2M, more preferably 0.1 to 0.2M.
10. The method for converting Rb1 to CK using Aspergillus niger fermentation broth according to any of claims 5 to 9, further comprising purifying the solution containing CK to obtain CK with a purity of 98% or more; the purification method comprises the steps of extracting a solution containing CK by using an organic solvent, purifying by using a Sephadex G-200 gel column and crystallizing.
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