CN117660579A - Dual-drug-effect cordyceps militaris solid-state fermentation ginseng product and preparation method thereof - Google Patents
Dual-drug-effect cordyceps militaris solid-state fermentation ginseng product and preparation method thereof Download PDFInfo
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Abstract
The invention provides a double-drug-effect cordyceps militaris solid-state fermentation ginseng product and a preparation method thereof, wherein the preparation method of the cordyceps militaris solid-state fermentation ginseng product comprises the following steps: s1, preparing cordyceps militaris liquid strains: inoculating fungus blocks into a liquid culture medium after PDA plate activation culture of Cordyceps militaris strain test tube seeds, and obtaining Cordyceps militaris liquid strain after shake cultivation, wherein the liquid culture medium contains glucose, potato, tomato and Arabidopsis thaliana; s2, bidirectional solid state fermentation: slicing dried ginseng, adding ginseng stems and leaves which are treated by auxiliary materials and smashed into water, soaking, stirring and sterilizing to obtain a solid fermentation substrate, inoculating the solid fermentation substrate into the cordyceps militaris liquid strain obtained in the part S1, culturing to obtain fermented ginseng slices, drying to constant weight, and crushing to obtain a fermented ginseng slice sample. The cordyceps militaris solid-state fermentation ginseng product provided by the invention has dual drug effects provided by cordycepin and ginseng rare adenosine, can be widely applied to the technical field of cordyceps militaris solid-state fermentation ginseng, and has good commercial application value.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to the technical field of cordyceps militaris solid-state fermentation ginseng, and particularly relates to a dual-efficacy cordyceps militaris solid-state fermentation ginseng product and a preparation method thereof.
Background
Cordyceps militaris is a famous medicinal fungus, and cordycepin is one of main medicinal components of the cordycepin and exists in fruiting bodies, culture media after grass emergence and liquid fermentation products. Ginseng is a traditional rare Chinese medicine in China, ginsenoside is one of main active ingredients which play pharmacological roles, the content of the single ginsenoside which is separated at present reaches more than 50, the content and the pharmacological roles of each saponin in ginseng are greatly different, and the ginseng has the pharmacological properties of resisting cancer, diabetes, depression, aging and the like.
The bidirectional solid state fermentation is that medicinal bacteria are subjected to solid state fermentation by using a substrate containing medicinal components, the product is called mycoplasm, and the medicinal components in the substrate are changed along with the fermentation, so that the bacterial growth generates abundant secondary metabolites, and the active components in the mycoplasm are more and more complex. At present, the content change research of the medicinal components before and after the bidirectional solid state fermentation mainly uses the components contained in a fermentation substrate, and the research on the medicinal components of the strain per se is less. Ginsenoside Rg 3 And F is equal to 2 Called rare saponins, rg 3 Has effect in inhibiting cancer cell proliferation, F 2 Is an intermediate product of converting glycol group saponin into Compound-K, and can induce apoptosis and protective autophagy of breast cancer stem cells. The rare saponins in the ginseng have complicated structures, and chemical synthesis is not feasible, but the content of the rare saponins in the ginseng is small, and even some parts do not contain the rare saponins.
The modern Chinese medicine fermentation is a new technology, which combines the modern technologies such as fermentation process and traditional Chinese medicine fermentation, wherein one or more beneficial bacteria are mainly added into the Chinese medicine in the fermentation process, and enzyme generated in the growth and metabolism process of microorganisms is utilized to react with complex active ingredients in the Chinese medicine, so that the content of the active ingredients can be improved, and the completion of the reaction can be accelerated by utilizing the catalysis of the enzyme. Has been widely used in the field of extracting ginsenoside.
For example, patent CN113621673B discloses a method for fermenting ginseng by a composite strain, and a fermentation product and use thereof, which comprises the following steps: (1) Adding water into ginseng powder to prepare fermentation homogenate, adding vitamin C, and adjusting the pH of the fermentation homogenate to 3.0-6.0; (2) Adding to the fermentation homogenateEnzymatic hydrolysate is subjected to enzymolysis for 120-180 min at 50-60 ℃ and pH is regulated to 5.5-7.0, so as to obtain enzymatic hydrolysate; (3) Preserving the temperature of the enzymolysis liquid at 60-90 ℃ for 35-55 min to obtain a sterilizing liquid; (4) Cooling the sterilization liquid to 10-30 ℃, adding the zymophyte powder solution into the sterilization liquid, starting timing by inoculating the zymophyte powder solution, and fermenting for 24-72 hours at the fermentation temperature of 20-30 ℃ to obtain fermentation liquid. The method can improve the ginsenoside Rg in fermentation broth 3 Is contained in the composition. However, the patent focuses on improving the rare ginsenoside Rg in fermentation liquor 3 Rather than the cordycepin and rare ginsenoside Rg in the cordyceps militaris bi-directional solid state fermentation ginseng product 3 And F 2 Study of dual pharmacodynamic ingredients.
Therefore, there is a need in the market for a solid ginseng fermentation product with dual efficacy provided by cordycepin and rare adenosine of ginseng and a simple, low cost preparation method.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention selects the liquid culture medium with specific components to prepare the cordyceps militaris liquid strain, then slices the ginseng into water, then adds the ginseng stems and leaves treated by specific auxiliary materials to obtain the solid fermentation matrix, and utilizes the two-way solid fermentation technology to connect the solid fermentation matrix into the liquid strain to prepare the cordyceps militaris solid fermentation ginseng product by culture, thereby solving the problems that the content of the ginseng rare saponins in the existing ginseng is low and the solid fermentation product cannot have dual drug effects of cordycepin and the ginseng rare saponins.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the invention provides a preparation method of a double-drug-effect cordyceps militaris solid-state fermentation ginseng product, which comprises the following steps of:
s1, preparing cordyceps militaris liquid strains: after the test tube strain of the cordyceps militaris strains is subjected to PDA flat plate activation culture, fungus blocks are taken and inoculated into a liquid culture medium, and the cordyceps militaris liquid strain is obtained after shaking culture,
wherein the liquid medium contains glucose, potato, tomato and Arabidopsis;
s2, bidirectional solid state fermentation: and (3) putting the dried ginseng slices into water, adding the ginseng stems and leaves which are treated by auxiliary materials and smashed, soaking, stirring and sterilizing to obtain a solid fermentation substrate, inoculating the solid fermentation substrate into the cordyceps militaris liquid strain obtained in the part S1, culturing to obtain fermented ginseng slices, drying to constant weight, and crushing to obtain a fermented ginseng slice sample, namely the cordyceps militaris solid fermentation ginseng product.
Preferably, the preparation method of the double-drug-effect cordyceps militaris solid-state fermentation ginseng product comprises the following steps of:
s1, preparing cordyceps militaris liquid strains: culturing Cordyceps militaris strain test tube strain in PDA plate at 23deg.C for 8d, perforating with a puncher (diameter of 4 mm), inoculating the bacterial block into liquid culture medium, shake culturing at 22deg.C under 160r/min for 7d to obtain Cordyceps militaris liquid strain,
wherein the liquid medium contains glucose, potato, tomato and Arabidopsis;
s2, bidirectional solid state fermentation: slicing dried ginseng, adding the sliced ginseng into water, adding ginseng stems and leaves which are treated by auxiliary materials and smashed, soaking for 5-6 hours, stirring for 5 times, sterilizing at 121 ℃ for 20 minutes to obtain a solid fermentation substrate, inoculating the solid fermentation substrate into the cordyceps militaris liquid strain obtained in the part S1, culturing in dark at 23 ℃ for 70 days to obtain fermented ginseng slices, drying at 40 ℃ to constant weight, and crushing to obtain a fermented ginseng slice sample, namely the cordyceps militaris solid fermentation ginseng product.
In some embodiments of the invention, the volume ratio of the number of bacterial pieces to the liquid medium in step S1 is 1: (41-45).
Preferably, the volume ratio of the number of the bacterial pieces to the liquid culture medium in the step S1 is 1:43.3.
the accumulation of the active ingredient cordycepin in the cordyceps militaris requires a certain space and oxygen, and the applicant ensures that the growth of the cordyceps militaris fungus blocks in the liquid culture medium has enough space and oxygen by controlling the size of the cordyceps militaris fungus blocks and the ratio of the fungus block number to the volume of the liquid culture medium, and simultaneously ensures that the nutrition synthesis in the liquid culture medium can be fully absorbed by the cordyceps militaris, so that the active ingredient in the cordyceps militaris is more, and the subsequently prepared cordyceps militaris solid fermentation product has better double drug effects.
In some embodiments of the invention, the mass ratio of glucose, potato, tomato, and arabidopsis in the liquid medium in step S1 is 1: (9-11): (1-2): (1.5-2.5).
Preferably, the mass ratio of glucose, potato, tomato and arabidopsis in the liquid medium in step S1 is 1:10:1.5:2.
glucose and potatoes are taken as a common culture medium, a necessary carbon source and an energy source are provided for the growth of cordyceps militaris, the growth and reproduction of cordyceps militaris colonies are promoted, but the content of extractable active ingredients cordycepin in cordyceps militaris liquid strains cultured by a liquid culture medium containing only glucose and potatoes is low, the applicant selects to add two natural green plants of tomatoes and arabidopsis thaliana into the liquid culture medium, the tomatoes contain regulatory genes SlZF3, the content of ascorbic acid (AsA) can be remarkably improved, the AsA can be used for enhancing the oxidation resistance and the salt tolerance of cordyceps militaris by removing active oxygen, the arabidopsis thaliana contains regulatory genes ZAT18, the liquid absorption capacity and the cold resistance of cordyceps militaris can be remarkably improved, besides, the tomatoes can also promote the carbon source and the energy source in the glucose and the potatoes to be absorbed by the cordyceps militaris, the applicant further controls the proportion of tomatoes, arabidopsis thaliana and the glucose to the potatoes to the maximum extent, so that the best nutritional ingredients can be absorbed, the oxidation substances, high salt substances and adverse substances and the cordyceps militaris substances in the cordyceps militaris can be prevented from growing in natural environment from being produced, and the cordyceps militaris can be effectively accumulated in the cordyceps militaris strain, and the side products can be produced by the cordyceps militaris can be effectively accumulated by the side products.
In some embodiments of the present invention, the ginseng slice in step S2 has a thickness of 0.3-0.7cm; the volume ratio of the mass of the ginseng slice to the water is 1: (1.5-2.5).
Preferably, the thickness of the ginseng slice in step S2 is 0.5cm; the volume ratio of the mass of the ginseng slice to the water is 1:2. the applicant ensures that the ginseng slices uniformly absorb water by controlling the thickness of the ginseng slices and the addition amount of ginseng in water so as to ensure the full reaction of the fermented ginseng sample.
In some embodiments of the invention, the preparation of the adjuvant in step S2 comprises the steps of:
(1) Mixing sorbitan oleate with 1, 2-propylene glycol, and stirring to obtain a mixed solution 1;
(2) Mixing caprylic/capric glyceride and sodium pyruvate, stirring, and adding into the mixed solution 1 to obtain a mixed solution 2;
(3) And (3) dropwise adding acetic acid into the mixed solution 2, regulating the pH, adding n-butanol, and stirring until the system is uniform and transparent to obtain the auxiliary material.
In some embodiments of the invention, the mass ratio of sorbitan oleate to 1, 2-propanediol is 1: (1-4); the mass ratio of the sorbitan oleate, the caprylic/capric glyceride and the sodium pyruvate is 1: (6-8): (3-4); the mass ratio of the sorbitan oleate to the ginseng stem and leaf is (1.5-2.5): 1.
preferably, the mass ratio of the sorbitan oleate to the 1, 2-propanediol is 1:2.5; the mass ratio of the sorbitan oleate, the caprylic/capric glyceride and the sodium pyruvate is 1:7:3.5; the mass ratio of the sorbitan oleate to the ginseng stems and leaves is 2:1.
in some embodiments of the invention, the pH in step (3) is from 6.5 to 7.5.
Preferably, the pH in step (3) is 7.0.
In some embodiments of the invention, the mass ratio of ginseng slice to ginseng stem and leaf in step S2 is 1: (0.5-0.7).
Preferably, in the step S2, the mass ratio of the ginseng slice to the ginseng stem and leaf is 1:0.6.
ginseng is a rare traditional Chinese medicine in China, contains various saponins which exert different pharmacological actions, but the ginseng is expensive and not easy to obtain, the types and the content of the saponins at each part of the ginseng are very different, compared with the whole raw ginseng, the ginseng stem and leaf is low in cost and relatively easy to obtain and contains rich saponin components, the applicant adds the ginseng stem and leaf into uniformly water-absorbed ginseng slices to prepare a solid fermentation matrix, prepares auxiliary materials of components such as sorbic acid sorbitol, 1, 2-propanediol, caprylic acid glyceride and sodium pyruvate and the like to treat the ginseng stem and leaf, and on one hand, the addition of the sorbic acid sorbitol, 1, 2-propanediol and caprylic acid glyceride is beneficial to construct an emulsion system with hydrophilic-lipophilic balance, and can be used as a good carrier for dispersing the ginseng stem and leaf and the ginseng slice in water, so that the ginseng stem and leaf prepared solid fermentation matrix has higher content of the ginsenosides; on the other hand, sodium pyruvate is used as a precursor substance of ginsenoside, so that the cell proliferation capability of ginseng can be improved, thereby ensuring the necessary capability in the ginsenoside synthesis path and being beneficial to the growth of callus in ginseng and the synthesis of the ginsenoside.
Further, in order to increase the content of rare saponins in ginseng stems and leaves, the applicant adds a small amount of acetic acid dropwise to adjust the pH to about 7.0 in the preparation process of auxiliary materials, which is favorable for properly increasing the acid hydrolysis product of the ginsenosides, not only can increase the medicinal value of the ginsenosides in the ginseng stems and leaves, but also can be used as a raw material for preparing the rare saponins, and is favorable for increasing the content of the ginsenosides in the solid fermentation product and reducing the fermentation cost. Furthermore, the applicant controls the mass ratio of ginseng slices to ginseng stems and leaves, and ensures the maximization of the yield of ginsenoside on the basis of reducing the cost to a certain extent.
In some embodiments of the invention, the ratio of the mass of the ginseng slice to the volume of the cordyceps militaris liquid strain is 1: (0.5-1.5).
Preferably, the volume ratio of the ginseng slice mass to the cordyceps militaris liquid strain is 1:1. the applicant limits the inoculation amount of the solid fermentation matrix into the liquid strain by controlling the volume ratio of the ginseng slice to the liquid strain of the cordyceps militaris, so as to ensure the full synthesis of the solid fermentation ginseng product of the cordyceps militaris, thereby ensuring that the bi-directional solid fermentation product has the highest cordycepin content and ginsenoside content, and ensuring that the rare ginsenoside Rg 3 And F 2 The content is improved.
The invention also provides the double-drug-effect cordyceps militaris solid-state fermentation ginseng product, wherein the cordycepin content is not less than 4mg/g, and the total ginsenoside content is not less than 3.3%.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention provides a preparation method of a cordyceps militaris solid-state fermentation ginseng product, which is characterized in that tomato and arabidopsis thaliana are introduced into a traditional liquid culture medium, and ginseng stems and leaves treated by auxiliary materials are introduced into a solid-state fermentation matrix, so that the cordyceps militaris solid-state fermentation ginseng product with dual effects of cordycepin and ginseng rare saponin is obtained.
(2) Glucose, potatoes, tomatoes and arabidopsis thaliana are prepared into a novel liquid culture medium, and the content of cordycepin which is an effective component in cordyceps militaris is further improved by enhancing the inhibition effect on cordycepin synthesis in cordyceps militaris.
(3) According to the invention, the ginseng stems and leaves and the raw ginseng slices are added for matching use, and the self-made auxiliary materials are processed on the ginseng stems and leaves, so that the dispersibility of ginseng in water and the cell proliferation capability of ginseng are promoted, and the contents of ginsenoside and ginseng rare saponin are improved.
(4) The solid-state fermentation ginseng product of the dual-efficacy cordyceps militaris has the cordycepin content of not less than 4mg/g, the total ginsenoside content of not less than 3.3 percent, and rare ginsenoside Rg 3 And F 2 The content is greatly improved.
Detailed Description
The invention will be described below in connection with specific embodiments. The following examples are illustrative of the present invention and are not intended to limit the present invention. Other combinations and various modifications within the spirit of the invention may be made without departing from the spirit or scope of the invention.
In the following examples, except for the liquid medium and the auxiliary materials, the cordyceps militaris strains, related compounds and nutrients used are all available from the market, wherein the cordyceps militaris strains are purchased from the edible fungus research center of Shanghai national academy of agricultural sciences; the ginseng is Jilin Fusong 4-year ginseng, and is purchased from Jilin Fusong ten thousand good ginseng markets; ginseng stems and leaves are purchased from natural bioengineering limited company in Fusong county, china; ginsenoside [ Re, rb ] 1 、Rb 2 、Rb 3 、Rc、Rd、F 2 、20(S)-Rg 3 ]Standards were purchased from Sichuan Vickers biotechnology Co., ltd; cordycepin was purchased from Guangzhou Wide forest technologies Inc.
Preparation example 1
The synthesis method of the liquid culture medium A comprises the following steps: 130mL of liquid medium A consisting of 20g/L glucose, 200g/L potato, 30g/L tomato and 40g/L Arabidopsis were prepared at natural pH.
Preparation example 2
The liquid culture medium B, the synthesis method is the same as the liquid culture medium A, and the difference is that the concentration of the components in the liquid culture medium is respectively as follows: 20g/L glucose, 200g/L potato, 16g/L tomato, and 20g/L Arabidopsis.
Preparation example 3
The synthesis method of the auxiliary material A comprises the following steps:
(1) 6g of sorbitan oleate and 15g of 1, 2-propanediol are mixed and stirred for 10min to obtain a mixed solution 1;
(2) 42g of caprylic/capric glyceride and 21g of sodium pyruvate are mixed, stirred for 10min and then added into the mixed solution 1 to obtain a mixed solution 2;
(3) And (3) dropwise adding acetic acid into the mixed solution 2, regulating the pH to 7.0, adding 20ml of n-butanol, and stirring for 20min until the system is uniform and transparent, thus obtaining the auxiliary material.
Preparation example 4
The auxiliary material B is synthesized by the same method as the auxiliary material A, and the difference is that the mass of the 1, 2-propanediol, the caprylic/capric glyceride and the sodium pyruvate in the auxiliary material are respectively 4.8g, 33g and 15g.
Example 1
A preparation method of a double-drug-effect cordyceps militaris solid-state fermentation ginseng product comprises the following steps:
s1, preparing cordyceps militaris liquid strains: after the cordyceps militaris strain test tube strain is subjected to PDA flat plate activation culture for 8d at the temperature of 23 ℃, a puncher is used for punching (the diameter is 4 mm), 3 fungus blocks are inoculated into 130mL of liquid culture medium A, and shake cultivation is carried out for 7d at the temperature of 22 ℃ and the speed of 160r/min to obtain cordyceps militaris liquid strain;
s2, bidirectional solid state fermentation: cutting dry ginseng slices into 0.5cm thick by a small guillotine, putting 5g of dry ginseng slices into 10mL of water, adding the auxiliary material A into 3g of ginseng stems and leaves, mashing, soaking for 5.5h, stirring for 5 times, sterilizing at 121 ℃ for 20min to obtain a solid fermentation matrix, inoculating the solid fermentation matrix into 5mL of cordyceps militaris liquid strain obtained by S1, culturing in dark at 23 ℃ for 70d to obtain fermented ginseng slices, drying to constant weight at 40 ℃, and crushing to obtain a fermented ginseng slice sample, namely the cordyceps militaris solid fermentation ginseng product.
Example 2
A preparation method of a double-drug-effect cordyceps militaris solid-state fermentation ginseng product comprises the following steps:
s1, preparing cordyceps militaris liquid strains: after the cordyceps militaris strain test tube strain is subjected to PDA flat plate activation culture for 8d at the temperature of 23 ℃, a puncher is used for punching (the diameter is 4 mm), 3 fungus blocks are inoculated into 123mL of liquid culture medium A, and shake cultivation is carried out for 7d at the temperature of 22 ℃ and the speed of 160r/min to obtain cordyceps militaris liquid strain;
s2, bidirectional solid state fermentation: cutting dry ginseng slices into 0.3cm thick by a small guillotine, taking 5g of dry ginseng slices, putting into 7.5mL of water, adding the auxiliary material A into 2.5g of ginseng stems and leaves, mashing, soaking for 5.5h, stirring for 5 times, sterilizing at 121 ℃ for 20min to obtain a solid fermentation matrix, inoculating the solid fermentation matrix into 1.25mL of cordyceps militaris liquid strain obtained in S1, culturing in dark at 23 ℃ for 70d to obtain fermented ginseng slices, drying at 40 ℃ to constant weight, and crushing to obtain a fermented ginseng slice sample, namely the cordyceps militaris solid fermentation ginseng product.
Example 3
A preparation method of a double-drug-effect cordyceps militaris solid-state fermentation ginseng product comprises the following steps:
s1, preparing cordyceps militaris liquid strains: after the cordyceps militaris strain test tube strain is subjected to PDA flat plate activation culture for 8d at the temperature of 23 ℃, a puncher is used for punching (the diameter is 4 mm), 3 fungus blocks are inoculated into 135mL of liquid culture medium A, and shake cultivation is carried out for 7d at the temperature of 22 ℃ and the speed of 160r/min to obtain cordyceps militaris liquid strain;
s2, bidirectional solid state fermentation: cutting dry ginseng slices into 0.7cm thick by a small guillotine, taking 5g of dry ginseng slices, putting the dried ginseng slices into 12.5mL of water, adding the auxiliary material A into 3.5g of ginseng stems and leaves, mashing, soaking for 5.5h, stirring for 5 times, sterilizing at 121 ℃ for 20min to obtain a solid fermentation matrix, inoculating the solid fermentation matrix into 5.25mL of cordyceps militaris liquid strain obtained in S1, culturing in dark at 23 ℃ for 70d to obtain fermented ginseng slices, drying at 40 ℃ to constant weight, and crushing to obtain a fermented ginseng slice sample, namely the cordyceps militaris solid fermentation ginseng product.
Example 4
The embodiment provides a preparation method of a double-drug-effect cordyceps militaris solid-state fermentation ginseng product, which is the same as the embodiment 1, and is different in that a liquid culture medium B is used for replacing a liquid culture medium A in an equivalent way.
Example 5
The embodiment provides a preparation method of a double-drug-effect cordyceps militaris solid-state fermentation ginseng product, which is the same as the embodiment 1, and is different in that auxiliary material A is replaced by auxiliary material B in an equivalent manner.
Comparative example 1
The comparative example provides a preparation method of a double-effect cordyceps militaris solid-state fermentation ginseng product, and the specific implementation mode is the same as that of example 1, except that tomatoes and arabidopsis thaliana are not added in a liquid culture medium.
Comparative example 2
The comparative example provides a preparation method of a double-drug-effect cordyceps militaris solid-state fermentation ginseng product, and the specific implementation mode is the same as example 1, except that no auxiliary materials are added.
Comparative example 3
In this comparative example, 5g of dry ginseng was additionally pulverized to obtain a ginseng sample.
Comparative example 4
The comparative example further provides a preparation method of the cordyceps militaris fruiting body, comprising the following steps:
a. preparing cordyceps militaris liquid strains: the specific preparation method is the same as in example 1;
b. inoculating 7mL of the liquid strain obtained in the step a into a 550mL culture bottle filled with 100g of culture medium, culturing to obtain fruiting bodies, drying the fruiting bodies at 40 ℃, and crushing to obtain fruiting body samples, wherein the culture medium comprises 85% of rice, 15% of silkworm chrysalis meal and 65% of water content.
Comparative example 5
The comparative example further provides a method for preparing a substrate after grass emergence, comprising the following steps:
a. preparing cordyceps militaris liquid strains: the specific preparation method is the same as in example 1;
b. inoculating 7mL of the liquid strain obtained in the step a into a 550mL culture bottle filled with 100g of culture medium, culturing to obtain fruiting bodies, collecting the substrate after harvesting the fruiting bodies, namely a substrate after grass emergence, drying the substrate after grass emergence at 40 ℃, and crushing to obtain a substrate sample after grass emergence, wherein the formula of the culture medium is 85% of rice, 15% of silkworm chrysalis powder and 65% of water content.
Performance testing
The solid fermentation products of Cordyceps militaris described in examples 1 to 5 and comparative examples 1 to 2, and the ginseng samples, the fruiting bodies of Cordyceps militaris and the substrates after fruiting, the total saponins content and the types and contents of ginsenoside monomers were tested as described in comparative examples 3 to 5, and the test results are shown in tables 1 and 2, wherein table 1 is the test result of cordycepin content and total saponins content, and table 2 is the content of various ginsenoside monomers.
(1) Cordycepin content determination
And measuring the cordycepin content by adopting an ultrahigh pressure liquid phase method. Chromatographic conditions: acquity UPLC BEH C18 chromatography column (2.1 mm. Times.50 mm,1.7 μm), mobile phase acetonitrile-water (7:93), flow rate 0.2mL/min, detection wavelength 260nm, column temperature 35 ℃, and sample injection amount 2. Mu.L.
The cordycepin standard is dissolved by ultrapure water to prepare a series of mixed labels. And drawing a standard curve by taking the peak area as an ordinate and the sample injection quality as an abscissa. Continuously measuring the precision of 6 times by taking the same mixed standard solution; taking the same sample solution to be tested and respectively sampling for 0,4,8,12 and 16 hours for testing the stability; taking the same sample, and preparing 6 samples of test solution for testing repeatability; taking 6 parts of cordyceps militaris sample with known content for sample adding recovery rate experiment;
100mg of sample is added with ultrapure water (25 mL of fruiting body sample, 5mL of substrate sample after grass emergence and 50mL of fermented ginseng slice sample), ultrasonic (500W, 40 kHz) is carried out for 30min, centrifugation is repeated for 10min for 2 times, the supernatant is taken to measure peak area, and the cordycepin content is calculated according to a standard curve.
(2) Determination of total saponins and content
1g of the sample is weighed, 10mL of methanol is added for ultrasonic treatment (500W, 40 kHz) for 30min, the process is repeated for 2 times, water saturated n-butanol is added into the extract for extraction for 3 times, the n-butanol is volatilized, methanol is redissolved, the volume is fixed to 10mL, the total saponin content is measured by a vanillin-sulfuric acid method, and the experiment is repeated for 3 times to obtain an average value.
(3) Ginsenoside monomer content determination
Chromatographic conditions: acquity UPLC BEH C18 column (2.1 mm. Times.50 mm,1.7 μm), mobile phase acetonitrile-water, gradient elution: 0-5min,17% -19% acetonitrile; 5-7min,19% -21% acetonitrile; 7-11min,21% -26% acetonitrile; 11-13min, 26-27% acetonitrile, 13-15min, 27-32% acetonitrile, 15-17min, 32-43% acetonitrile, 17-19min, 43-60% acetonitrile, the flow rate is 0.5mL/min, the detection wavelength is 203nm, the column temperature is 35 ℃, and the sample injection amount is 2 mu L. Ginsenoside Rb 1 ，Rb 2 ，Rb 3 ，Rc，Rd，F 2 ，20(S)-Rg 3 Adding proper amount of methanol to dissolve and prepare a series of mixed marks. And drawing a standard curve by taking the peak area as an ordinate and the sample injection quality as an abscissa, performing precision, stability, repeatability and sample injection recovery experiments, measuring the peak area according to the chromatographic conditions, and calculating the ginsenoside monomer content according to the standard curve.
TABLE 1
| Group of | Cordycepin content (mg/g) | Total saponin content (%) |
| Example 1 | 4.14 | 3.37％ |
| Example 2 | 4.13 | 3.36％ |
| Example 3 | 4.12 | 3.35％ |
| Example 4 | 3.85 | 2.94％ |
| Example 5 | 3.76 | 2.88％ |
| Comparative example 1 | 2.98 | 2.47％ |
| Comparative example 2 | 2.87 | 2.38％ |
| Comparative example 3 | / | 2.95％ |
| Comparative example 4 | 0.28 | / |
| Comparative example 5 | 0.23 | / |
TABLE 2
As can be seen from the data in Table 1, the solid-state fermentation ginseng product of Cordyceps militaris in examples 1-3 of the invention has the characteristics of high total cordycepin content and high total saponin content. Wherein, example 4 changes the addition amount of tomato and arabidopsis in the liquid culture medium, so that the stress resistance of the liquid culture medium is reduced, thereby reducing the content of cordycepin and total saponins; example 5 changes the proportion of the main components in the auxiliary materials, and the result shows that the main components of the auxiliary materials are controlled within a proper range, so that the improvement of the cordycepin content and the total saponin content is facilitated; comparative examples 1 and 2 were prepared by synthesizing a cordyceps militaris solid-state fermentation ginseng product in the presence of a liquid medium without tomatoes and arabidopsis thaliana added and without auxiliary materials, respectively, and the test shows that both cordycepin and total saponin content of the prepared cordyceps militaris solid-state fermentation ginseng product show lower results; comparative examples 4 to 5 are cordyceps militaris fruiting bodies and substrates after grass emergence respectively, and test results show that the cordycepin content of the ginseng product subjected to solid state fermentation of cordyceps militaris is far higher than that of the cordyceps militaris fruiting bodies and the substrates after grass emergence.
As can be seen from the data in Table 2, the content of various ginsenosides was changed during the solid fermentation of the ginseng slice of Cordyceps militaris, wherein the sample of the ginseng slice of comparative example 3 does not contain rare saponin F 2 、Rg 3 While the fermented ginseng pieces of examples 1 to 5 and comparative examples 1 to 2 contained F 2 、Rg 3 The cordyceps militaris is obtained through solid state fermentation and conversion.
The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the present invention and to implement it, but not limit the scope of the present invention, and all equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of the present invention.
Claims (10)
1. The preparation method of the double-drug-effect cordyceps militaris solid-state fermentation ginseng product is characterized by comprising the following steps of:
s1, preparing cordyceps militaris liquid strains: after the test tube strain of the cordyceps militaris strains is subjected to PDA flat plate activation culture, fungus blocks are taken and inoculated into a liquid culture medium, and the cordyceps militaris liquid strain is obtained after shaking culture,
wherein the liquid medium contains glucose, potato, tomato and Arabidopsis;
s2, bidirectional solid state fermentation: and (3) putting the dried ginseng slices into water, adding the ginseng stems and leaves which are treated by auxiliary materials and smashed, soaking, stirring and sterilizing to obtain a solid fermentation substrate, inoculating the solid fermentation substrate into the cordyceps militaris liquid strain obtained in the part S1, culturing to obtain fermented ginseng slices, drying to constant weight, and crushing to obtain a fermented ginseng slice sample, namely the cordyceps militaris solid fermentation ginseng product.
2. The method for preparing a double-effect cordyceps militaris solid-state fermentation ginseng product according to claim 1, wherein the volume ratio of the number of bacteria to the liquid culture medium in the step S1 is 1: (41-45).
3. The method for preparing the solid state fermentation ginseng product of double-effect cordyceps militaris according to claim 1, wherein the mass ratio of glucose, potato, tomato and arabidopsis thaliana in the liquid medium in the step S1 is 1: (9-11): (1-2): (1.5-2.5).
4. The method for preparing a double-effect cordyceps militaris solid-state fermentation ginseng product according to claim 1, wherein the thickness of the ginseng slice in the step S2 is 0.3-0.7cm; the volume ratio of the mass of the ginseng slice to the water is 1: (1.5-2.5).
5. The method for preparing the solid state fermentation ginseng product of double drug effect cordyceps militaris according to claim 1, wherein the preparation of the auxiliary materials in the step S2 comprises the following steps:
(1) Mixing sorbitan oleate with 1, 2-propylene glycol, and stirring to obtain a mixed solution 1;
(2) Mixing caprylic/capric glyceride and sodium pyruvate, stirring, and adding into the mixed solution 1 to obtain a mixed solution 2;
(3) And (3) dropwise adding acetic acid into the mixed solution 2, regulating the pH, adding n-butanol, and stirring until the system is uniform and transparent to obtain the auxiliary material.
6. The method for preparing the solid state fermentation ginseng product of double-drug-effect cordyceps militaris according to claim 5, wherein the mass ratio of sorbitan oleate to 1, 2-propanediol is 1: (1-4); the mass ratio of the sorbitan oleate, the caprylic/capric glyceride and the sodium pyruvate is 1: (6-8): (3-4).
7. The method for producing a double-effect cordyceps militaris solid state fermentation ginseng product according to claim 5, wherein the PH in the step (3) is 6.5 to 7.5.
8. The method for preparing a double-effect cordyceps militaris solid-state fermentation ginseng product according to claim 1, wherein the mass ratio of ginseng slices to ginseng stems and leaves in the step S2 is 1: (0.5-0.7).
9. The method for preparing the solid state fermentation ginseng product of double-drug-effect cordyceps militaris, which is characterized in that the volume ratio of the mass of ginseng slices to the liquid strain of cordyceps militaris is 1: (0.5-1.5).
10. A solid-state fermented ginseng product comprising a double-effect cordyceps militaris obtained by the preparation method of any one of claims 1 to 9, wherein the cordycepin content is not less than 4mg/g, and the total ginsenoside content is not less than 3.3%.
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