Beta-(1,3)-(1,6)-glucan and method for its preparation, cosmetic

Abstract

The application relates to the field of cosmetics, and relates to beta-(1,3)-(1,6)-glucan and a preparation method and cosmetics thereof. Fermentation seed liquid is inoculated into a culture medium for fermentation; the culture medium comprises, in percentage by mass, 1-5% of glucose, 0.5-5% of tryptone, 0.1-1% of yeast extract powder, 0.01-0.1% of magnesium sulfate, 0.05-0.5% of potassium dihydrogen phosphate and the balance of water; in the culture medium, the concentration of vitamin B1 is 5-20 mg / L; and the concentration of folic acid is 1-8 mg / L. The fermentation strategy of using the culture medium containing vitamin B1 and folic acid in the above content range for fermentation can be used to prepare beta-(1,3)-(1,6)-glucan produced by schizophyllum commune, can significantly increase the weight of schizophyllum commune mycelium in the same time, effectively improves the content of beta-(1,3)-(1,6)-glucan, and has the advantages of shortening the production cycle and reducing the production cost.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cosmetics, in particular, a kind of β-(1,3)-(1,6)-dextran and its preparation method, cosmetics. BACKGROUND

[0002] At present, Schizophyllum polysaccharide preparation production cycle is long, and mycelium grows slowly, which affects its application and opposite.

[0003] And the specific structure of the Schizophyllum polysaccharide prepared at present is unknown, and the composition contained is complex. SUMMARY

[0004] The purpose of the embodiment of the present application is to provide a kind of β-(1,3)-(1,6)-dextran and its preparation method, cosmetics.

[0005] In the first aspect, the present application provides a kind of β-(1,3)-(1,6)-dextran preparation method, comprising:

[0006] The fermentation seed liquid is inoculated into the culture medium for fermentation;

[0007] The culture medium is the mixed solution of the first component and the second component;

[0008] The first component includes, by mass percentage: 1% to 5% glucose, 0.5% to 5% tryptone, 0.1% to 1% yeast extract powder, 0.01% to 0.1% magnesium sulfate, 0.05% to 0.5% potassium dihydrogen phosphate, and the balance is water;

[0009] The second component includes: vitamin B1 and folic acid;

[0010] In the culture medium, the concentration of vitamin B1 is 5mg / L to 20mg / L;The concentration of folic acid is 1mg / L to 8mg / L.

[0011] In the above technical solution, the culture medium containing the above content range of vitamin B1 and folic acid is used for fermentation, and the product β-(1,3)-(1,6)-dextran of Schizophyllum is prepared, which can significantly increase the weight of Schizophyllum mycelium in the same time, and effectively improve the content of β-(1,3)-(1,6)-dextran, with the advantages of shortening production cycle and reducing production cost.

[0012] In other embodiments of the present application, by mass percentage, the first component includes: 2% to 5% glucose, 1% to 5% tryptone, 0.25% to 0.5% yeast extract powder, 0.05% to 0.1% magnesium sulfate, 0.1% to 0.5% potassium dihydrogen phosphate, and the balance is water;

[0013] The second component includes: vitamin B1 and folic acid;

[0014] The concentration of vitamin B1 in the culture medium is 7.5 mg / L-15 mg / L; and the concentration of folic acid is 2.5 mg / L-5 mg / L.

[0015] In other embodiments of the present application, the fermentation seed liquid is inoculated into the culture medium for fermentation, including a first fermentation stage and a second fermentation stage.

[0016] The second fermentation stage includes:

[0017] The promoting factor is added to the culture medium for the second fermentation stage.

[0018] In other embodiments of the present application, the promoting factor includes: litchi wood chips.

[0019] In other embodiments of the present application, during the second fermentation stage, the weight of the added promoting factor: the volume of the culture medium is (0.5-1.5):(98.5-99.5).

[0020] In other embodiments of the present application, the second fermentation stage includes:

[0021] The fermentation is carried out at 25-30℃, 150-200 r / min for 24-72 hours.

[0022] In other embodiments of the present application, during the first fermentation stage, the volume of the added fermentation seed liquid: the volume of the culture medium is (5-10):(90-95).

[0023] In other embodiments of the present application, the first fermentation stage includes:

[0024] The fermentation seed liquid is inoculated into the culture medium, and the fermentation is carried out at 25-30℃, 150-200 r / min for 24-100 hours.

[0025] In a second aspect, the present application provides a β-(1,3)-(1,6)-glucan prepared by the preparation method of the β-(1,3)-(1,6)-glucan provided in any one of the preceding aspects.

[0026] In a third aspect, the present application provides a cosmetic product including the β-(1,3)-(1,6)-glucan provided in any one of the preceding aspects. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly introduced as follows. It should be understood that the following drawings only show some of the embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those of ordinary skill in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0028] Figure 1 A Fourier transform infrared spectrum test chart of the dried sample;

[0029] Figure 2 A nuclear magnetic resonance hydrogen spectrum test chart of the sample;

[0030] Figure 3 A BJ cell scratch test result;

[0031] Figure 4 A change trend chart of the stratum corneum moisture content of the volunteers before and after using the sample;

[0032] Figure 5 A change chart of the facial skin red area color of the volunteers before and after using the sample;

[0033] Figure 6 A change chart of the facial color score and lactic acid stinging score of the volunteers before and after using the sample. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be clearly and completely described as follows. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments.

[0035] Therefore, the following detailed description of the embodiments of the present application is not intended to limit the scope of the claimed present application, but only represents selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor are within the scope of protection of the present application.

[0036] The present application provides a preparation method of β-(1,3)-(1,6)-glucan, comprising:

[0037] The fermentation seed liquid is inoculated into the culture medium for fermentation;

[0038] The culture medium is a mixed solution of the first component and the second component;

[0039] The first component includes, in percentage by mass: 1-5% glucose, 0.5-5% tryptone, 0.1-1% yeast extract, 0.01-0.1% magnesium sulfate, 0.05-0.5% potassium dihydrogen phosphate, and the rest is water;

[0040] The second component includes vitamin B1 and folic acid.

[0041] In the culture medium, the concentration of vitamin B1 is 5-20 mg / L, and the concentration of folic acid is 1-8 mg / L.

[0042] In the above technical solution, the fermentation strategy is to ferment the culture medium containing vitamin B1 and folic acid in the above content range to prepare the Schizophyllum product β-(1,3)-(1,6)-glucan. The weight of the Schizophyllum mycelium can be significantly increased in the same time, and the content of β-(1,3)-(1,6)-glucan can be effectively improved, which has the advantages of shortening the production cycle and reducing the production cost.

[0043] Further, in some embodiments of the present application, the method for preparing β-(1,3)-(1,6)-glucan includes the following steps:

[0044] Step S1, preparing a fermentation seed liquid.

[0045] Further, in some embodiments of the present application, the fermentation seed liquid is prepared by culturing the Schizophyllum at 25-30°C for 5-7 days and then expanding the culture.

[0046] The above technical solution can activate the Schizophyllum, so that it can be used for fermentation culture of β-(1,3)-(1,6)-glucan, which is beneficial to increase the weight of the Schizophyllum mycelium and effectively improve the content of β-(1,3)-(1,6)-glucan.

[0047] Further, in some embodiments of the present application, the Schizophyllum mentioned above can be GIM 5.43 provided by Guangdong Microbial Institute.

[0048] Further optionally, in some embodiments of the present application, the fermentation seed liquid is prepared by transferring the Schizophyllum to a slant medium for activation, culturing at 25-30°C for 5-7 days until the mycelium covers the slant, and then transferring the slant strain to a liquid culture medium for expansion culture for 1-3 days to obtain the fermentation seed liquid.

[0049] Illustratively, in some embodiments of the present application, the fermentation seed liquid is prepared by transferring the Schizophyllum to a slant medium for activation, culturing at 28°C for 5-7 days until the mycelium covers the slant, and then transferring the slant strain to a liquid culture medium for expansion culture for 2 days to obtain the fermentation seed liquid.

[0050] Step S2, preparing the culture medium.

[0051] Further, in some embodiments of the present application, the culture medium is a mixture of the first component and the second component;

[0052] The first component comprises, in percentage by mass, 1% to 5% glucose, 0.5% to 5% tryptone, 0.1% to 1% yeast extract, 0.01% to 0.1% magnesium sulfate, 0.05% to 0.5% potassium dihydrogen phosphate, and the balance being water.

[0053] The second component comprises vitamin B1 and folic acid.

[0054] In the culture medium, the concentration of vitamin B1 is 5 mg / L to 20 mg / L; and the concentration of folic acid is 1 mg / L to 8 mg / L.

[0055] Exemplarily, in some embodiments of the present application, the first component comprises, in percentage by mass:

[0056] 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, or a range between any two of the foregoing, of glucose;

[0057] 0.5%, 0.8%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, or a range between any two of the foregoing, of tryptone;

[0058] 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, or a range between any two of the foregoing, of yeast extract;

[0059] 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.08%, 0.1%, or a range between any two of the foregoing, of magnesium sulfate;

[0060] 0.05%, 0.08%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, or a range between any two of the foregoing, of potassium dihydrogen phosphate; and the balance being water.

[0061] Further, exemplarily, in some embodiments of the present application, the concentration of vitamin B1 in the culture medium is 5 mg / L, 6 mg / L, 8 mg / L, 10 mg / L, 12 mg / L, 15 mg / L, 16 mg / L, 18 mg / L, 19 mg / L, 20 mg / L or a range between any two of the aforementioned values; and the concentration of folic acid is 1 mg / L, 1.5 mg / L, 2 mg / L, 2.5 mg / L, 3 mg / L, 3.5 mg / L, 4 mg / L, 4.5 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L or a range between any two of the aforementioned values.

[0062] Further, in some embodiments of the present application, the first component comprises, in percentage by mass: 2% to 5% glucose, 1% to 5% tryptone, 0.25% to 0.5% yeast extract powder, 0.05% to 0.1% magnesium sulfate, 0.1% to 0.5% potassium dihydrogen phosphate, and the balance being water.

[0063] The second component comprises vitamin B1 and folic acid.

[0064] In the culture medium, the concentration of vitamin B1 is 7.5 mg / L to 15 mg / L; and the concentration of folic acid is 2.5 mg / L to 5 mg / L.

[0065] Step S3, fermentation of β-(1,3)-(1,6)-glucan (Schizophyllan β-glucan, SPG).

[0066] Further, in some embodiments of the present application, the fermentation of β-(1,3)-(1,6)-glucan (Schizophyllan β-glucan, SPG) comprises inoculating the fermentation seed liquid obtained in step S1 into the culture medium obtained in step S2 for fermentation.

[0067] Further, in some embodiments of the present application, inoculating the fermentation seed liquid into the culture medium for fermentation comprises a first fermentation stage and a second fermentation stage.

[0068] The second fermentation stage comprises:

[0069] The second fermentation stage is performed by adding a promoting factor to the culture medium.

[0070] In the above technical solution, the two-stage fermentation is used as the fermentation strategy, and the β-(1,3)-(1,6)-glucan product of Schizophyllum is prepared in stages, which can significantly increase the weight of Schizophyllum mycelium in the same time, effectively improve the content of β-(1,3)-(1,6)-glucan, and has the advantages of shortening the production cycle and reducing the production cost.

[0071] Further, in some embodiments of the present application, the promoting factor comprises: litchi wood chips.

[0072] In the above technical solution, by adding litchi wood chips into the culture medium in the second fermentation stage, the litchi wood chips serve as a factor for promoting the synthesis of SPG (special wood); the weight of the Schizophyllum hyphae can be effectively increased, and the content of β-(1, 3)-(1, 6)-glucan can be effectively improved.

[0073] Further, in some embodiments of the present application, when the second fermentation stage is performed, the weight of the promoting factor added: the volume of the culture medium is (0.5-1.5):(98.5-99.5).

[0074] Exemplarily, in some embodiments of the present application, when the second fermentation stage is performed, the weight of the promoting factor added: the volume of the culture medium is 0.5:99.5; 0.6:99.4; 0.8:99.2; 1:99; 1.2:98.8; 1.3:98.7; 1.4:98.6; 1.5:98.5 or a ratio between any two of the above values.

[0075] (0.5-1.5):(98.5-99.5). Or the promoting factor in a range between any two of the above values is added into the culture medium to perform the second fermentation stage.

[0076] In the above technical solution, by adding 0.5%-1.5% of the promoting factor into the culture medium to perform the second fermentation stage in the second fermentation stage, the weight of the Schizophyllum hyphae can be effectively increased, and the content of β-(1, 3)-(1, 6)-glucan can be effectively improved.

[0077] Further, in some embodiments of the present application, the second fermentation stage comprises:

[0078] The fermentation is performed at 25-30°C and 150-200 r / min for 24-72 hours.

[0079] Exemplarily, in some embodiments of the present application, the second fermentation stage comprises:

[0080] fermentation is carried out at 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, or a range between any two of the aforementioned values, at 150 r / min, 160 r / min, 170 r / min, 180 r / min, 190 r / min, 200 r / min, or a range between any two of the aforementioned values, for 24 hours, 25 hours, 28 hours, 30 hours, 32 hours, 36 hours, 38 hours, 40 hours, 42 hours, 46 hours, 50 hours, 60 hours, 72 hours, or a range between any two of the aforementioned values.

[0081] Further, in some embodiments of the present application, the volume of the fermentation seed liquid added: the volume of the culture medium is (5-10):(90-95) during the first fermentation stage.

[0082] Illustratively, in some embodiments of the present application, the volume of the fermentation seed liquid added: the volume of the culture medium is 5:95; 6:94; 7:93; 8:92; 10:90, or a ratio between any two of the aforementioned values during the first fermentation stage.

[0083] Further, in some embodiments of the present application, the first fermentation stage comprises:

[0084] The fermentation seed liquid is inoculated into the culture medium, and fermentation is carried out at 25-30℃, 150-200 r / min for 24-100 hours.

[0085] Illustratively, in some embodiments of the present application, the first fermentation stage comprises:

[0086] The fermentation seed liquid is inoculated into the culture medium, and fermentation is carried out at 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, or a range between any two of the aforementioned values, at 150 r / min, 160 r / min, 170 r / min, 180 r / min, 190 r / min, 200 r / min, or a range between any two of the aforementioned values, for 24 hours, 26 hours, 30 hours, 32 hours, 35 hours, 38 hours, 40 hours, 45 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, or a range between any two of the aforementioned values.

[0087] Step S4, purification.

[0088] Further, in some embodiments of the present application, the purification comprises:

[0089] The fermentation product prepared in step S3 is subjected to removal of the bacterial cells, to obtain a fermentation product, and the polysaccharide is obtained from the fermentation product, i.e., β-(1,3)-(1,6)-glucan.

[0090] Exemplarily, in some embodiments of the present application, the purifying comprises: centrifuging the fermentation product to remove the bacteria bodies, to obtain the fermentation product. Then, 2 times volume of absolute ethyl alcohol is added to the fermentation product for alcohol precipitation, filtration, collection of the precipitated crude polysaccharide, and multiple washing with absolute ethyl alcohol, and drying to obtain the β-(1,3)-(1,6)-glucan (Schizophyllan β-glucan, SPG).

[0091] Further, some embodiments of the present application provide a β-(1,3)-(1,6)-glucan, which is prepared by the method for preparing the β-(1,3)-(1,6)-glucan provided in any of the preceding embodiments.

[0092] The Schizophyllan polysaccharide prepared in the present application is determined by Fourier transform infrared spectrometer and nuclear magnetic resonance spectrometer to be β-(1,3)-(1,6)-glucan. The SPG with the structure can remove excess free radicals, and achieve the effects of moisturizing and soothing.

[0093] Further, some embodiments of the present application provide a cosmetic product comprising the β-(1,3)-(1,6)-glucan provided in any of the preceding embodiments.

[0094] The cosmetic product provided in the present application can remove excess free radicals by containing the β-(1,3)-(1,6)-glucan provided in any of the preceding embodiments, and achieve the effects of moisturizing and soothing.

[0095] The features and performances of the present application are further described in detail below in combination with examples:

[0096] Example 1

[0097] A method for preparing a β-(1,3)-(1,6)-glucan is provided, which is prepared according to the following steps:

[0098] (1) Schizophyllum sp. activation: Schizophyllum is transferred to a slant medium for activation, and is cultured at 28°C and 160 r / min for 5-7 days until the mycelium covers the slant; then the slant strain is transferred to a liquid culture medium for expansion culture for 2 days to obtain a fermentation seed liquid.

[0099] (2) Preparation of culture medium: the culture medium is a mixture of the first component and the second component.

[0100] The first component includes, by mass percentage, 2% glucose, 1% tryptone, 0.5% yeast extract powder, 0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate, and the balance of water.

[0101] The second component is vitamin B1 and folic acid. The concentration of vitamin B1 in the culture medium is 15 mg / L; the concentration of folic acid is 5 mg / L. The pH of the culture medium is natural (about 6.0).

[0102] (3) Fermentation of β-(1,3)-(1,6)-glucan (Schizophyllan β-glucan, SPG): A two-stage fermentation strategy is adopted. First fermentation stage: Schizophyllum sp. is inoculated and mixed with the culture medium at a volume ratio of 1:9, and fermentation is carried out at 28°C and 160 r / min for 50 hours. Second fermentation stage: litchi sawdust is mixed with the culture medium at a weight:volume ratio of 1.5:98.5, and fermentation is continued at 28°C and 160 r / min for 50 hours.

[0103] (4) SPG purification: The fermentation product is centrifuged to remove the bacterial bodies, and the fermentation product is obtained. Then 2 times the volume of anhydrous ethanol is added for alcohol precipitation, filtration is performed, the precipitated crude polysaccharide is collected, and anhydrous ethanol is used for multiple washes, and after drying, SPG is obtained.

[0104] Example 2

[0105] A method for preparing β-(1,3)-(1,6)-glucan is provided, which is prepared according to the following steps:

[0106] (1) Activation of Schizophyllum sp.: Schizophyllum is transferred to a slant medium for activation, and is cultured at 28°C and 160 r / min for 5-7 days until the mycelium covers the slant; then the slant strain is transferred to a liquid culture medium for expansion culture for 2 days to obtain a fermentation seed liquid.

[0107] (2) Preparation of fermentation medium: The culture medium is a mixture of the first component and the second component.

[0108] The first component includes, by mass percentage: 2% glucose, 0.5% tryptone, 0.25% yeast extract powder, 0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate, and the balance is water.

[0109] The second component is vitamin B1 and folic acid. The concentration of vitamin B1 in the culture medium is 15 mg / L; the concentration of folic acid is 5 mg / L. The pH of the culture medium is natural (about 6.0).

[0110] (3) Fermentation of β-(1,3)-(1,6)-glucan (Schizophyllan β-glucan, SPG): Two-stage fermentation strategy was adopted. First fermentation stage: Schizophyllum sp. was inoculated and mixed with culture medium at a volume ratio of 1:9, and then fermented at 28°C and 160 r / min for 50 hours. Second fermentation stage: Litchi wood chips were mixed with culture medium at a weight:volume ratio of 1.5:98.5, and then continued to be fermented at 28°C and 160 r / min for 50 hours.

[0111] (4) SPG purification: The fermentation product was centrifuged to remove the bacterial bodies, and then 2 times the volume of absolute ethanol was added for alcohol precipitation. The precipitated crude polysaccharide was collected by filtration and washed with absolute ethanol for multiple times. After drying, SPG was obtained.

[0112] Example 3

[0113] A method for preparing β-(1,3)-(1,6)-glucan is provided, which is prepared according to the following steps:

[0114] (1) Activation of Schizophyllum sp.: Schizophyllum sp. was transferred to a slant medium for activation, and then cultured at 28°C and 160 r / min for 5-7 days until the mycelium covered the slant. Then, the slant medium was transferred to a liquid culture medium for expansion culture for 2 days to obtain a fermentation seed liquid.

[0115] (2) Preparation of culture medium: The culture medium was a mixture of a first component and a second component.

[0116] The first component included 2% glucose, 1% tryptone, 0.5% yeast extract powder, 0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate, and the rest was water, by mass percentage. The second component was vitamin B1 and folic acid. In the culture medium, the concentration of vitamin B1 was 7.5 mg / L, and the concentration of folic acid was 2.5 mg / L. The pH of the culture medium was natural (about 6.0).

[0117] (3) Fermentation of SPG: Two-stage fermentation strategy was adopted. In the first stage, Schizophyllum sp. was inoculated and mixed with culture medium at a volume ratio of 1:9, and then fermented at 28°C and 160 r / min for 50 hours. In the second stage, Litchi wood chips were mixed with culture medium at a weight:volume ratio of 1.5:98.5, and then continued to be fermented at 28°C and 160 r / min for 50 hours.

[0118] (4) SPG purification: The fermentation product was centrifuged to remove the bacterial bodies, and then 2 times the volume of absolute ethanol was added for alcohol precipitation. The precipitated crude polysaccharide was collected by filtration and washed with absolute ethanol for multiple times. After drying, SPG was obtained.

[0119] Example 4

[0120] A method for preparing β-(1,3)-(1,6)-glucan is provided, which is prepared according to the following steps:

[0121] (1) Schizophyllum sp. activation: Schizophyllum is inoculated on a slant medium for activation, and is cultured at 28°C and 160 r / min for 5-7 days until the mycelium covers the slant; then the slant strain is inoculated into a liquid medium for expansion culture for 2 days to obtain a fermentation seed liquid.

[0122] (2) Preparation of fermentation medium: the medium is a mixture of a first component and a second component.

[0123] The first component includes, by mass percentage, 2% glucose, 1% tryptone, 0.5% yeast extract powder, 0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate, and the balance water.

[0124] The second component is vitamin B1 and folic acid. In the medium, the concentration of vitamin B1 is 15 mg / L; the concentration of folic acid is 5 mg / L. The pH of the medium is natural (about 6.0).

[0125] (3) Fermentation of β-(1,3)-(1,6)-glucan (Schizophyllan β-glucan, SPG): a two-stage fermentation strategy is adopted. First fermentation stage: Schizophyllum sp. is inoculated and mixed with the medium at a volume ratio of 1:9, and is fermented at 28°C and 160 r / min for 50 hours. Second fermentation stage: litchi wood chips are mixed with the medium at a weight:volume ratio of 0.5:99.5 as special wood for promoting SPG synthesis, and are continuously fermented at 28°C and 160 r / min for 50 hours.

[0126] (4) SPG purification: the fermentation product is subjected to centrifugal treatment to remove the bacterial bodies, to obtain a fermentation product. Then 2 times the volume of anhydrous ethanol is added for alcohol precipitation, filtration is performed, the precipitated crude polysaccharide is collected, and anhydrous ethanol is used for multiple washing, and after drying, SPG is obtained.

[0127] Example 5

[0128] A method for preparing β-(1,3)-(1,6)-glucan is provided, which is prepared according to the following steps:

[0129] (1) Schizophyllum sp. activation: Schizophyllum is inoculated on a slant medium for activation, and is cultured at 28°C and 160 r / min for 5-7 days until the mycelium covers the slant; then the slant strain is inoculated into a liquid medium for expansion culture for 2 days to obtain a fermentation seed liquid;

[0130] (2) Preparation of fermentation medium: the medium is a mixture of the first component and the second component.

[0131] The first component includes, in percentage by mass: 2% glucose, 1% tryptone, 0.5% yeast extract powder, 0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate, and the balance water.

[0132] The second component is vitamin B1 and folic acid. In the medium, the concentration of vitamin B1 is 15 mg / L; the concentration of folic acid is 5 mg / L. The pH of the medium is natural (about 6.0).

[0133] (3) Fermentation of β-(1,3)-(1,6)-glucan (Schizophyllan β-glucan, SPG): the Schizophyllum sp. is inoculated and mixed with the medium at a volume ratio of 1:9, and is fermented at 28°C and 160 r / min for 100 hours.

[0134] (4) Purification of SPG: the fermentation product is subjected to centrifugal treatment to remove the bacterial bodies, to obtain a fermentation product. Subsequently, 2 times the volume of anhydrous ethanol is added to the fermentation product for alcohol precipitation, filtration is performed, the precipitated crude polysaccharide is collected, and anhydrous ethanol is used for multiple washing, and after drying, the SPG is obtained.

[0135] Example 6

[0136] A method for preparing β-(1,3)-(1,6)-glucan is provided, which is prepared according to the following steps:

[0137] (1) Activation of Schizophyllum sp.: the Schizophyllum is transferred to a slant medium for activation, and is cultured at 28°C and 160 r / min for 5-7 days until the mycelium covers the slant; then the slant strain is transferred to a liquid medium for expansion culture for 2 days to obtain a fermentation seed liquid;

[0138] (2) Preparation of fermentation medium: the medium is a mixture of the first component and the second component.

[0139] The first component includes, in percentage by mass: 2% glucose, 1% tryptone, 0.5% yeast extract powder, 0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate, and the balance water.

[0140] The second component is vitamin B1 and folic acid. In the medium, the concentration of vitamin B1 is 15 mg / L; the concentration of folic acid is 5 mg / L. The pH of the medium is natural (about 6.0).

[0141] (3) Fermentation of β-(1,3)-(1,6)-glucan (Schizophyllan β-glucan, SPG): Two-stage fermentation strategy was adopted. First fermentation stage: Schizophyllum sp. was inoculated and mixed with culture medium at a volume ratio of 1:9, and then fermentation was carried out at 28°C, 160 r / min for 24 hours. Second fermentation stage: Litchi wood chips were mixed with culture medium at a weight:volume ratio of 0.5:99.5, and then fermentation was continued at 28°C, 160 r / min for 24 hours.

[0142] (4) Purification of SPG: The fermentation product was subjected to centrifugal treatment to remove the bacterial bodies, and then 2 times the volume of anhydrous ethanol was added for alcohol precipitation. The precipitated crude polysaccharide was collected by filtration, washed with anhydrous ethanol for multiple times, and then dried to obtain SPG.

[0143] Example 7

[0144] A method for preparing β-(1,3)-(1,6)-glucan is provided, which is prepared according to the following steps:

[0145] (1) Activation of Schizophyllum sp.: Schizophyllum sp. was transferred to a slant medium for activation, and then cultured at 28°C, 160 r / min for 5-7 days until the mycelium covered the slant. Then the slant strain was transferred to a liquid culture medium for expansion culture for 2 days to obtain a fermentation seed liquid;

[0146] (2) Preparation of fermentation medium: The culture medium is a mixture of the first component and the second component.

[0147] The first component includes, by mass percentage, 2% glucose, 1% tryptone, 0.5% yeast extract powder, 0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate, and the balance of water.

[0148] The second component is vitamin B1 and folic acid. In the culture medium, the concentration of vitamin B1 is 15 mg / L; the concentration of folic acid is 5 mg / L. The pH of the culture medium is natural (about 6.0).

[0149] (3) Fermentation of β-(1,3)-(1,6)-glucan (Schizophyllan β-glucan, SPG): Two-stage fermentation strategy was adopted. First fermentation stage: Schizophyllum sp. was inoculated and mixed with culture medium at a volume ratio of 1:9, and then fermentation was carried out at 28°C, 160 r / min for 24 hours. Second fermentation stage: Litchi wood chips were mixed with culture medium at a weight:volume ratio of 0.5:99.5, and then fermentation was continued at 28°C, 160 r / min for 24 hours.

[0150] (4) SPG purification: centrifugal treatment is performed on the fermentation product to remove the bacterial bodies, and a fermentation product is obtained. Subsequently, 2 times the volume of anhydrous ethanol is added to the fermentation product for alcohol precipitation, filtration is performed, the precipitated crude polysaccharide is collected, and multiple washes are performed using anhydrous ethanol. After drying, SPG is obtained.

[0151] Comparative Example 1

[0152] A preparation method of a Schizophyllum fermentation product is provided, and is prepared according to the following steps:

[0153] (1) Schizophyllum strain activation: the Schizophyllum is transferred to a slant medium for activation, and is cultured at 28°C and 160 r / min for 5-7 days until the mycelium covers the slant. Subsequently, the slant strain is transferred to a liquid medium for expansion culture for 2 days, and a fermentation seed liquid is obtained.

[0154] (2) Preparation of fermentation medium: the medium is a mixture of the first component and the second component.

[0155] The first component includes, in terms of mass percentage, 2% glucose, 1% tryptone, 0.5% yeast extract powder, 0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate, and the balance of water. The pH of the medium is natural (about 6.0). The second component is vitamin C and vitamin A. In the medium, the concentration of vitamin C is 15 mg / L; the concentration of vitamin A is 5 mg / L. The pH of the medium is natural (about 6.0).

[0156] (3) Schizophyllum polysaccharide: a two-stage fermentation strategy is adopted. First fermentation stage: the Schizophyllum strain is inoculated and mixed with the medium at a volume ratio of 1:9, and is fermented at 28°C and 160 r / min for 50 hours. Second fermentation stage: the litchi wood chips are mixed with the medium at a weight:volume ratio of 0.5:99.5, and are continuously fermented at 28°C and 160 r / min for 50 hours.

[0157] (4) SPG purification: centrifugal treatment is performed on the fermentation product to remove the bacterial bodies, and a fermentation product is obtained. Subsequently, 2 times the volume of anhydrous ethanol is added to the fermentation product for alcohol precipitation, filtration is performed, the precipitated crude polysaccharide is collected, and multiple washes are performed using anhydrous ethanol. After drying, SPG is obtained.

[0158] Comparative Example 2

[0159] A preparation method of a Schizophyllum fermentation product is provided, and is prepared according to the following steps:

[0160] (1) Schizophyllum strain activation: the Schizophyllum is transferred to a slant medium for activation, and is cultured at 28°C and 160 r / min for 5-7 days until the mycelium covers the slant. Subsequently, the slant strain is transferred to a liquid medium for expansion culture for 2 days, and a fermentation seed liquid is obtained.

[0161] (2) Preparation of fermentation medium: the medium is a mixture of the first component and the second component.

[0162] The first component includes, in mass percentage: 2% glucose, 1% tryptone, 0.5% yeast extract, 0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate, and the balance water. The second component is vitamin D and vitamin K1. In the medium, the concentration of vitamin D is 15 mg / L; the concentration of vitamin K1 is 5 mg / L. The pH of the medium is natural (about 6.0).

[0163] (3) Fermentation of SPG: a two-stage fermentation strategy is adopted. First fermentation stage: the Schizophyllum sp. is inoculated and mixed with the medium at a volume ratio of 1:9, and fermentation is carried out at 28°C and 160 r / min for 50 hours. Second fermentation stage: the litchi sawdust is mixed with the medium at a weight:volume ratio of 0.5:99.5, and the fermentation is continued at 28°C and 160 r / min for 50 hours.

[0164] (4) Purification of SPG: the fermentation product is subjected to centrifugal treatment to remove the bacterial bodies, and the fermentation product is obtained. Subsequently, 2 times the volume of anhydrous ethanol is added for alcohol precipitation, filtration is performed, the precipitated crude polysaccharide is collected, and anhydrous ethanol is used for multiple washing, and after drying, the SPG is obtained.

[0165] Comparative Example 3

[0166] A preparation method of a Schizophyllum fermentation product is provided, which is prepared according to the following steps:

[0167] (1) Activation of Schizophyllum sp.: the Schizophyllum is transferred to a slant medium for activation, and is cultured at 28°C and 160 r / min for 5-7 days until the mycelium covers the slant; then the slant strain is transferred to a liquid medium for expansion culture for 2 days to obtain a fermentation seed liquid.

[0168] (2) Preparation of fermentation medium: the medium includes, in mass percentage: 0.3% yeast extract, 0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate, 3% glucose, and the balance water, and the pH of the medium is natural (about 6.0).

[0169] (3) Fermentation of Schizophyllum fermentation product: the Schizophyllum sp. is inoculated and mixed with the medium at a volume ratio of 5:95, and is cultured at 28°C and 160 r / min for 100 h, and the fermentation product is subjected to centrifugal treatment to remove the bacterial bodies, and the fermentation product is obtained.

[0170] (4) SPG purification: the fermentation product was centrifuged to remove the bacteria, and the fermentation product was obtained. Then 2 times the volume of absolute ethanol was added to the fermentation product for alcohol precipitation, filtration, collection of the precipitated crude polysaccharide, and multiple washes with absolute ethanol. After drying, SPG was obtained.

[0171] Comparative Example 4

[0172] A preparation method of Schizophyllum commune fermentation product is provided, which is prepared according to the following steps:

[0173] (1) Schizophyllum commune strain activation: the Schizophyllum commune was transferred to a slant medium for activation, and cultured at 28°C and 160 r / min for 5-7 days until the mycelium covered the slant. Then the slant strain was transferred to a liquid medium for expansion culture for 2 days to obtain a fermentation seed liquid.

[0174] (2) Preparation of culture medium: the culture medium includes 2% glucose, 1% tryptone, 0.5% yeast extract powder, 0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate, and the balance is water. The pH of the culture medium is natural (about 6.0).

[0175] (3) Fermentation of SPG: a two-stage fermentation strategy was adopted. First fermentation stage: the Schizophyllum commune strain was inoculated and mixed with the culture medium at a volume ratio of 1:9, and fermented at 28°C and 160 r / min for 50 hours. Second fermentation stage: the lychee sawdust was mixed with the culture medium at a weight:volume ratio of 1.5:98.5, and continued to be fermented at 28°C and 160 r / min for 50 hours.

[0176] (4) SPG purification: the fermentation product was centrifuged to remove the bacteria, and the fermentation product was obtained. Then 2 times the volume of absolute ethanol was added to the fermentation product for alcohol precipitation, filtration, collection of the precipitated crude polysaccharide, and multiple washes with absolute ethanol. After drying, SPG was obtained.

[0177] Table 1: Medium composition of each example and comparative example

[0178]

[0179]

[0180] Table 2: First fermentation stage of each example and comparative example

[0181]

[0182] Table 3: Second fermentation stage of each example and comparative example

[0183]

[0184]

[0185] Experimental Example 1

[0186] The structure of the SPG samples prepared in each example and comparative example was identified.

[0187] (1) Infrared identification method of SPG structure:

[0188] Each of the above-mentioned dried SPG samples (about 2 mg) was added to 200 mg of KBr powder, ground gently in an infrared-lamp-equipped agate mortar, and then pressed into a KBr tablet. Subsequently, Fourier transform infrared spectroscopy (model: Nicolet IS10) was performed, with a wave number of 4000-400 cm -1 , a resolution of 0.8 cm -1 , and a scanning number of 32. The Fourier transform infrared spectrum of the dried SPG sample was obtained, as shown in Figure 1 .

[0189] Table 4: Assignment table of Fourier transform infrared data of each sample after drying

[0190] Home Vibration frequency (cm -1 )]]> O-H stretching vibration 3257.50 C-H stretching vibration 2883.72 C=O stretching vibration 1654.09 C-H bending vibration 1368.73 Saccharide C-O-C and C-O-H stretching vibration 1023.39 β glycosidic bond 886.79

[0191] From the above test results, it can be seen that 3257.50 cm -1 is the O-H stretching vibration peak, 2883.72 cm -1 is the C-H stretching vibration peak, 1654.09 cm -1 is the C=O stretching vibration absorption peak, 1368.73 cm -1 is the weak absorption peak of C-H bending vibration absorption peak, 1023.39 cm -1 is the sugar C-O-C and C-O-H stretching vibration peak, and the absorption peak at 886.79 cm-1 is the characteristic absorption peak of the β glycosidic bond.

[0192] (2) NMR identification method of SPG structure:

[0193] Each of the above-mentioned dried SPG samples (about 10 mg) was added to 0.55 mL of tritiated water for dissolution, and after complete dissolution, freeze-drying was performed. After freeze-drying, 0.55 mL of tritiated water was added again for dissolution, and freeze-drying was performed. The above steps were repeated 3 times. Detection was performed using a 400 MHz nuclear magnetic resonance spectrometer (model: Bruker Avance III), and the nuclear magnetic resonance spectrum was recorded; as shown in Figure 2 .

[0194] According to the -1H-NMR test results of each sample, the proton information in the CH-O group of the saccharide (3.0-4.0 ppm is the characteristic shrinkage peak thereof) is known, which indicates that the structure of the above SPG sample is β-(1, 3)-(1, 6)-glucan.

[0195] The present application realizes the characterization of the characteristic structure of Schizophyllum polysaccharide (SPG) for the first time, confirms that the structure of the above SPG sample is β-(1, 3)-(1, 6)-glucan, and makes it more scientific.

[0196] Experimental Example 2

[0197] The Schizophyllum biomass and SPG content in the fermentation products of each example and comparative example were determined.

[0198] The determination method of Schizophyllum biomass: the fermentation product of each example and comparative example was centrifuged at 15000xg and 4°C for 10 min, and the precipitate was collected; it was dried at 60°C to constant weight and weighed, and the biomass was obtained (if special wood was added, the final biomass of the microbial body was obtained by subtracting the amount of special wood added).

[0199] SPG content determination: the fermentation product of each example and comparative example was centrifuged at 15000xg and 4°C for 10 min, and the supernatant was collected; 2 times the volume of anhydrous ethanol was added for alcohol precipitation, the obtained solid was filtered, washed with anhydrous ethanol for 3 times, dried at 60°C to constant weight, and weighed, and the SPG content was obtained.

[0200] The test results are shown in the following table.

[0201] Table 5 Schizophyllum biomass and SPG content of each example and comparative example

[0202]

[0203] As can be seen from the data in the above table, compared with Examples 1-3, it can be seen that under the premise of the same fermentation time and the same amount of added promoting factor, the Schizophyllum biomass and SPG content of Comparative Example 4 are significantly lower than those of Examples 1-3.

[0204] Comparative Examples 1-2 and Example 4 can be compared, and under the premise of the same fermentation time and the same amount of added promoting factor, the Schizophyllum biomass and SPG content of Comparative Examples 1-2 are significantly lower than those of Example 4.

[0205] Comparative Example 3 is a conventional fermentation method of Schizophyllum commune. Compared with Example 1, it can be seen that the biomass and SPG of Schizophyllum commune are significantly lower than those of Example 1 at the same fermentation time. It can be seen that the conventional medium cannot increase the biomass of Schizophyllum commune and cannot promote the production of SPG by Schizophyllum commune.

[0206] From the above data, it can be seen that by adding vitamin B1 and folic acid in the medium, the growth of Schizophyllum commune can be significantly promoted, the biomass of Schizophyllum commune can be increased, and the production of SPG by Schizophyllum commune can be induced, and the content of SPG can be increased.

[0207] Further, compared with Example 1, under the premise of the same fermentation time, by using a two-stage fermentation strategy and adding lychee sawdust as a factor for promoting the synthesis of SPG in the second fermentation stage, the production of SPG by Schizophyllum commune can be significantly induced, and the content of SPG can be increased.

[0208] Experimental Example 3

[0209] The antioxidant capacity of the fermentation products of each example and comparative example is tested.

[0210] (1) One of the antioxidant capacities

[0211] DPPH·, also known as 1,1-diphenyl-2-trinitrobenzene hydrazine, is a very stable nitrogen-centered free radical. Its stability mainly comes from the spatial barrier of the three benzene rings of resonance stabilization, so that the unpaired electrons on the nitrogen atom in the middle cannot play their due role of electron pairing. Its anhydrous ethanol solution is purple, and has a maximum absorption at a wavelength of 517 nm. The absorbance is linearly related to the concentration. When a free radical scavenger is added, it can bind or replace DPPH·, reducing the number of free radicals, reducing the absorbance, and making the solution color lighter. Therefore, the ability to scavenge free radicals can be evaluated.

[0212] The specific experimental steps are as follows:

[0213] 2.0 mL of each example, comparative example sample solution (or blank group with anhydrous ethanol) and 2.0 mL of DPPH· solution were taken into a stoppered test tube and mixed; avoid light reaction for 30 min, and measure the absorbance at 517 nm wavelength.

[0214]

[0215] In the formula:

[0216] A0: The absorbance at 517 nm wavelength measured by adding 2.0 mL of anhydrous ethanol and adding 2.0 mL of DPPH· solution;

[0217] A1: absorbance value at 517 nm wavelength determined by adding 2.0 mL of sample and 2.0 mL of DPPH· solution;

[0218] A2: absorbance value at 517 nm wavelength determined by adding 2.0 mL of sample and 2.0 mL of anhydrous ethanol.

[0219] The test results are shown in the following table:

[0220] Table 6 DPPH free radical scavenging rate of different examples and comparative examples (10%, v / v)

[0221] Sample DPPH radical scavenging rate (%) Example 1 74.52 Example 2 50.36 Example 3 41.69 Example 4 44.48 Example 5 42.50 Example 6 41.03 Example 7 62.37 Comparative Example 1 36.77 Comparative Example 2 32.17 Comparative Example 3 39.10 Comparative Example 4 32.64

[0222] As can be seen from the above table data, each example has a certain scavenging ability on DPPH free radicals, and the scavenging ability of example 1 is the strongest. The scavenging ability of each example on DPPH free radicals is higher than the scavenging ability of each comparative example on DPPH free radicals.

[0223] (2) The second anti-oxidation ability

[0224] The scavenging ability of each example and comparative example sample on ABTS+· was investigated. ABTS is oxidized to green ABTS+· under the action of a suitable oxidant. When an antioxidant substance is added to the reaction, the generation of ABTS+· will be inhibited. The absorbance at 734 nm can be measured to determine and calculate the total antioxidant capacity of the sample.

[0225] The specific experimental steps are as follows:

[0226] (1) Take 7 mmol / L of ABTS solution, dilute 60 times with 10 mmol / L of PBS (pH = 7.4) to obtain ABTS working solution;

[0227] (2) Take 190 μL of ABTS working solution (or PBS), add 10 μL of each example or comparative example sample solution (blank group uses PBS instead of sample solution), shake for 10 s, and measure the absorbance A at 734 nm after standing for 6 min at 30°C.

[0228]

[0229] In the formula:

[0230] A0: absorbance value at 734 nm determined by adding 10 μL of PBS and 190 μL of ABTS;

[0231] A1: absorbance value at 734 nm determined by adding 10 μL of sample and 190 μL of ABTS;

[0232] A2: 10 μL of sample was added, and 90 μL of PBS was not added. The absorbance value at 734 nm was measured.

[0233] The test results are shown in the following table:

[0234] Table 7 ABTS+·radical scavenging capacity of different examples and comparative examples (10%, v / v)

[0235]

[0236]

[0237] From the data in the above table, it can be seen that the fermentation products of each example have good ABTS+·radical scavenging capacity and can be used as antioxidants in skin care products. The ABTS+·radical scavenging capacity of the fermentation products of each example is higher than that of the fermentation products of each comparative example.

[0238] Experimental Example 4

[0239] Cell repair capacity

[0240] In this experiment, the SPG sample of Example 1 was tested for its BJ cell repair capacity. The SPG sample was treated with a 0.22 μm microporous filter and stored for use.

[0241] The specific experimental steps are as follows:

[0242] The wound healing four-hole insert was placed in a 12-well plate, and BJ cells in the logarithmic growth phase were inoculated into the 12-well plate. 110 μL of cell suspension was added to each hole of the four-hole insert, and the inoculation density was 1*105 cells / hole. The 12-well plate was placed in a 37°C carbon dioxide incubator for 24 h. After the cells were basically full-grown, the wound healing four-hole insert was gently pulled out, then washed once with PBS, 110 μL of 1% serum medium containing 5% SPG sample was added to each hole of the four-hole insert, and a photograph was taken. The 12-well plate was placed in a 37°C carbon dioxide incubator for incubation until the observation time set in the experiment. At the set time, the width of the scratch was observed and measured under a microscope and a photograph was taken.

[0243]

[0244] In the formula:

[0245] A1: 24 h scratch width;

[0246] B1: 0 h scratch width;

[0247] The test results are shown in the attached Figure 3 .

[0248] From Figure 3It can be seen that in the BJ cell scratch experiment, the healing rate of BJ cells treated with 5% SPG was 64.31%, which was better than that treated with 1% serum, indicating that SPG has good cell repair ability.

[0249] Experimental Example 5

[0250] This experiment tests the moisturizing and soothing ability of the SPG sample of Example 1

[0251] Thirty-two volunteers were recruited and used a cream containing 0.5% SPG for 28 days. The change in skin stratum corneum water content and the response to lactic acid pain score at 0 days and the 29th day were detected to evaluate the moisturizing performance and soothing effect of SPG.

[0252] Cream base formula: 0.5% SPG, 7% caprylic / capric triglyceride, 5% liquid paraffin, 4% glycerol, 3% propylene glycol, 3% ethylhexyl palmitate, 3% vaseline, 2% ceteareth-21, 2% dimethicone, 2% cetearyl alcohol, 1.5% ceteareth-2, 1.5% glyceryl monostearate, 0.2% nipagin methyl ester, 0.2% carbomer, 0.2% triethanolamine, 0.1% nipagin ethyl ester, and the balance is water (64.8%).

[0253] Table 8 Age information of the subjects

[0254]

[0255] The trend chart of the change in the stratum corneum water content of the volunteers before and after using the sample is shown in the specification Figure 4 .

[0256] From Figure 4 It can be seen that after using the cream containing 0.5% SPG, the stratum corneum water content of the volunteers was significantly increased by 31.75% compared with before use, indicating that SPG has good moisturizing performance and can be applied in moisturizing and hydrating products.

[0257] The change chart of the red area color of the facial skin of the volunteers before and after using the sample is shown in the specification Figure 5 .

[0258] The change chart of the facial color score and the lactic acid pain score of the volunteers before and after using the sample is shown in the specification Figure 6 .

[0259] From Figure 5 and Figure 6 It can be seen that after using the cream containing 0.5% SPG, the overall red area color of the facial skin of the volunteers became lighter, the a* value was significantly reduced by 3.33% compared with before use, and the lactic acid pain score value was significantly decreased by 56.2% compared with before use, indicating that SPG can effectively improve the skin and has a soothing effect.

[0260] The above descriptions are only the preferred embodiments of the present application, and are not intended to limit the present application. The present application can have various changes and modifications for those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A process for the preparation of β-(1,3)-(1,6)-glucan, characterized in that, The preparation method comprises the following steps: inoculating the fermentation seed liquid of Schizophyllum to the culture medium to perform fermentation; the culture medium is a mixture of a first component and a second component; the first component comprises, in percentage by mass, 1-5% of glucose, 0.5-5% of tryptone, 0.1-1% of yeast extract powder, 0.01-0.1% of magnesium sulfate, 0.05-0.5% of potassium dihydrogen phosphate, and the rest is water; the second component is vitamin B1 and folic acid; the concentration of the vitamin B1 in the culture medium is 7.5-15 mg / L, and the concentration of the folic acid is 2.5-5 mg / L; the fermentation of the Schizophyllum seed liquid to the culture medium comprises a first fermentation stage and a second fermentation stage; the first fermentation stage comprises: inoculating the Schizophyllum seed liquid to the culture medium to perform fermentation at 25-30 ℃ and 150-200 r / min for 24-100 hours; the second fermentation stage comprises: adding a promoting factor to the culture medium to perform fermentation at 25-30 ℃ and 150-200 r / min for 24-72 hours; the promoting factor is litchi wood chips; the weight of the promoting factor added is (0.5-1.5) : (98.5-99.5) of the volume of the culture medium.

2. The preparation method of claim 1, wherein the first component comprises, in percentage by mass, 2-5% of glucose, 1-5% of tryptone, 0.25-0.5% of yeast extract powder, 0.05-0.1% of magnesium sulfate, 0.1-0.5% of potassium dihydrogen phosphate, and the rest is water.

3. The preparation method of claim 1, wherein the volume of the Schizophyllum seed liquid added is (5-10) : (90-95) of the volume of the culture medium during the first fermentation stage. ​ ​

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