Preparation method of schizophyllum commune mycelium fermentation liquor as well as product and application of schizophyllum commune mycelium fermentation liquor

Abstract

The invention belongs to the technical field of fermentation, and relates to a preparation method of schizophyllum commune mycelium fermentation liquor and a product and application of the schizophyllum commune mycelium fermentation liquor, the preparation method comprises the following steps: inoculating schizophyllum commune into a culture medium, and standing and culturing to obtain primary seed liquor; the first-stage seed solution is inoculated into a culture medium and cultured, a second-stage seed solution is obtained, and culture comprises a standing culture stage and a stirring culture stage which are alternately carried out; and inoculating the secondary seed solution into a culture medium, fermenting, carrying out filter pressing on the obtained fermentation liquid, and collecting filtrate, thereby obtaining the product. According to the method, more schizophyllum commune mycelia are obtained by controlling the preparation mode of the seed liquid, the obtaining of schizophyllum commune polysaccharide during fermentation is promoted, the heavy metals possibly existing due to the heavy metal enrichment capacity of macro fungi are reduced by using the schizophyllum commune mycelia for fermentation, the quality of each batch of products is better controlled, the cost is remarkably reduced, and the method is suitable for industrial production. And the damage to the environment is reduced to a certain extent.

Description

Technical Field

[0001] This invention belongs to the field of fermentation technology and relates to a method for preparing fermentation broth of Schizophyllum commune mycelium, its products, and applications. Background Technology

[0002] Schizophyllum commune, a fungus with both edible and medicinal value, produces fermentation products rich in various natural active ingredients such as Schizophyllum commune polysaccharides, bioactive peptides, and terpenoids, exhibiting potential anti-inflammatory and immunomodulatory activities in the biopharmaceutical field. With the continuous growth in demand for Schizophyllum commune fermentation products, improving fermentation efficiency and increasing the yield of active ingredients has become a core technological requirement for industrial production.

[0003] The quality of the seed culture is a crucial prerequisite for the effectiveness of microbial fermentation. Among the indicators, cell density, as a core parameter of the seed culture, directly affects the cell growth rate, metabolite synthesis efficiency, and fermentation cycle in the subsequent fermentation process. Seed cultures with high cell density can rapidly form a dominant microbial community in the early stages of fermentation, shorten the cell adaptation period, improve the utilization rate of nutrients such as carbon and nitrogen sources, reduce the risk of contamination by other microorganisms, and ultimately achieve efficient synthesis and large-scale production of active ingredients.

[0004] Therefore, developing a method with high fermentation efficiency has become an urgent problem to be solved in this field. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a method for preparing fermentation broth of Schizophyllum commune mycelium, as well as its products and applications.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a method for preparing a fermentation broth of Schizophyllum commune mycelium, the method comprising:

[0008] (1) Inoculate the Schizophyllum commune into the culture medium and let it stand to obtain the primary seed culture;

[0009] (2) Inoculate the primary seed solution into the culture medium and culture it to obtain the secondary seed solution;

[0010] (3) Inoculate the secondary seed liquid into the culture medium, ferment, and then filter the resulting fermentation liquid and collect the filtrate to obtain the product;

[0011] The culture in step (2) includes alternating static culture and agitated culture phases.

[0012] This invention controls the preparation method of the seed liquid to obtain more Schizophyllum commune mycelium, promotes the acquisition of Schizophyllum commune polysaccharides during fermentation, and reduces the potential presence of heavy metals due to the ability of macrofungi to accumulate heavy metals during fermentation. This allows for better control of the quality of each batch of products, significantly reduces costs, and to some extent reduces environmental damage.

[0013] Preferably, the static culture in step (1) is carried out at a temperature of 25-32°C for 96-144 h.

[0014] Temperatures can be selected from 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, etc., and time can be selected from 96h, 100h, 104h, 108h, 112h, 116h, 120h, 124h, 128h, 132h, 136h, 140h, 144h, etc. Other specific point values ​​within the above ranges can also be selected, which will not be elaborated here.

[0015] Preferably, the culture temperature during the static culture stage is 25-32℃, and the culture time for a single culture is 6-10 h.

[0016] Temperatures can be selected from 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, etc., and time can be selected from 6h, 6.5h, 7h, 7.5h, 8h, 8.5h, 9h, 9.5h, 10h, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0017] Preferably, the culture temperature during the stirring culture stage is 25-32℃, and the culture time for a single culture is 10-50 min.

[0018] Temperatures can be selected from 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, etc., and time can be selected from 10min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, etc. Other specific values ​​within the above range can also be selected, which will not be elaborated here.

[0019] Preferably, the total culture time in step (2) is 24-96 h, such as 24 h, 28 h, 32 h, 36 h, 40 h, 44 h, 48 h, 52 h, 56 h, 60 h, 64 h, 68 h, 72 h, 76 h, 80 h, 84 h, 88 h, 92 h, 96 h, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0020] Preferably, the stirred culture also includes aeration, and the aeration volume for a single stirred culture is 10-30 L, such as 10 L, 15 L, 20 L, 25 L, 30 L, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0021] Preferably, the stirring and culture is carried out at a speed of 20-100 rpm and a pressure of 0.02-0.08 MPa.

[0022] The rotational speed can be selected from 20 rpm, 30 rpm, 40 rpm, 50 rpm, 60 rpm, 70 rpm, 80 rpm, 90 rpm, 100 rpm, etc., and the pressure can be selected from 0.02 MPa, 0.03 MPa, 0.04 MPa, 0.05 MPa, 0.06 MPa, 0.07 MPa, 0.08 MPa, etc. Other specific values ​​within the above range can also be selected, which will not be elaborated here.

[0023] Preferably, the fermentation temperature is 25-32℃ and the time is 24-120 h.

[0024] Temperatures can be selected from 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, etc., and time can be selected from 24h, 30h, 40h, 50h, 60h, 70h, 80h, 90h, 100h, 110h, 120h, etc. Other specific values ​​within the above range can also be selected, which will not be elaborated here.

[0025] Preferably, the fermentation speed is 120-180 rpm, the pressure is 0.1-0.2 MPa, and the aeration rate is 50-130 L.

[0026] The rotational speed can be selected from 120 rpm, 130 rpm, 140 rpm, 150 rpm, 160 rpm, 170 rpm, 180 rpm, etc., the pressure can be selected from 0.1 MPa, 0.12 MPa, 0.14 MPa, 0.16 MPa, 0.18 MPa, 0.2 MPa, etc., and the ventilation volume can be selected from 50 L, 60 L, 70 L, 80 L, 90 L, 100 L, 110 L, 120 L, 130 L, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0027] Preferably, the culture medium comprises, by weight percentage, 1-3% glucose, 0.1-0.6% malt extract, 0.01-0.06% KH2PO4, 0.01-0.06% MgSO4·7H2O, and water.

[0028] The mass percentage of glucose can be selected from 1%, 1.2%, 1.5%, 1.8%, 2%, 2.2%, 2.5%, 2.8%, 3%, etc.; the mass percentage of malt extract can be selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, etc.; the mass percentage of KH2PO4 can be selected from 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, etc.; the mass percentage of MgSO4·7H2O can be selected from 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, etc. Other specific values ​​within the above ranges can be selected, and will not be elaborated here.

[0029] Preferably, the inoculation amount in steps (2) and (3) is independently 5-15 vt%, such as 5vt%, 8vt%, 10vt%, 12vt%, 15vt%, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0030] Preferably, the filtrate in step (3) further includes centrifugation to obtain the supernatant, with a centrifugation speed of 5000-15000 rpm and a time of 15-45 min.

[0031] The engine speed can be selected from 5000 rpm, 6000 rpm, 7000 rpm, 8000 rpm, 9000 rpm, 10000 rpm, 11000 rpm, 12000 rpm, 13000 rpm, 14000 rpm, 15000 rpm, etc., and the time can be selected from 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, etc. Other specific values ​​within the above range can also be selected, which will not be described in detail here.

[0032] In a second aspect, the present invention provides a *Schizophyllum commune* mycelial fermentation broth prepared according to the method for preparing *Schizophyllum commune* mycelial fermentation broth as described in the first aspect.

[0033] Thirdly, the present invention provides an application of the fermentation broth of Schizophyllum commune mycelium according to the second aspect in the preparation of products with antioxidant, soothing, moisturizing and repairing effects.

[0034] Preferably, the product includes cosmetics or skincare products.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] This invention controls the preparation method of the seed liquid to obtain more Schizophyllum commune mycelium, promotes the acquisition of Schizophyllum commune polysaccharides during fermentation, and reduces the potential presence of heavy metals due to the ability of macrofungi to accumulate heavy metals during fermentation. This allows for better control of the quality of each batch of products, significantly reduces costs, and to some extent reduces environmental damage. Detailed Implementation

[0037] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0038] The *Schizophyllum commune* strain used in the following examples is GDMCC NO.5.46.

[0039] Preparation Example 1

[0040] This preparation example provides a culture medium comprising, by weight percentage, 2% glucose, 0.3% malt extract, 0.03% KH2PO4, 0.03% MgSO4·7H2O, and the balance being water.

[0041] The preparation method is as follows: the raw materials are physically mixed and sterilized at 115℃ for 30 minutes.

[0042] Preparation Example 2

[0043] This preparation example provides a culture medium comprising, by weight percentage, 1% glucose, 0.6% malt extract, 0.01% KH2PO4, 0.06% MgSO4·7H2O, and the balance being water.

[0044] The preparation method is the same as in Preparation Example 1.

[0045] Preparation Example 3

[0046] This preparation example provides a culture medium comprising, by weight percentage, 3% glucose, 0.1% malt extract, 0.06% KH2PO4, 0.01% MgSO4·7H2O, and the balance being water.

[0047] The preparation method is the same as in Preparation Example 1.

[0048] Preparation Example 4

[0049] This preparation example provides a culture medium that differs from Preparation Example 1 only in that malt extract powder is replaced with peptone in equal amounts, while other components and contents remain unchanged.

[0050] The preparation method is the same as in Preparation Example 1.

[0051] Preparation Example 5

[0052] This preparation example provides a culture medium that differs from Preparation Example 1 only in that malt extract powder is replaced with yeast powder in equal amounts, while other components and contents remain unchanged.

[0053] The preparation method is the same as in Preparation Example 1.

[0054] Preparation Example 6

[0055] This preparation example provides a culture medium that differs from Preparation Example 1 only in that malt extract powder is replaced with potato extract powder in equal amounts, while other components and contents remain unchanged.

[0056] The preparation method is the same as in Preparation Example 1.

[0057] Example 1

[0058] This embodiment provides a method for preparing fermentation broth of Schizophyllum commune mycelium, the preparation method comprising:

[0059] (1) Under aseptic conditions, a 9mm punch was used to take a piece of mycelium from the plate, chop it up and inoculate it into the culture medium prepared in Preparation Example 1. The culture was incubated at 28°C for 5 days to obtain a primary seed culture.

[0060] (2) The primary seed culture was inoculated into the culture medium prepared in Preparation Example 1 at an inoculation rate of 10 vt%, and cultured at 28°C for 2 days. The culture included alternating 8-hour static culture and 30-minute shaking culture. The shaking culture was carried out at a speed of 50 rpm and a pressure of 0.05 MPa. Aeration was carried out at 20 L per shaking culture to obtain the secondary seed culture.

[0061] (3) The secondary seed liquid was inoculated into the culture medium prepared in Preparation Example 1. The inoculation amount was 10 vt%, the fermentation temperature was 28℃, the aeration rate was 100 L, the stirring speed was 150 rpm, the pressure was 0.15 MPa, and the culture time was 3 days.

[0062] (4) After fermentation, the fermentation liquid is filtered using a plate and frame filter press, the filtrate is centrifuged again, and the supernatant is collected.

[0063] Example 2

[0064] This embodiment provides a method for preparing fermentation broth of Schizophyllum commune mycelium, the preparation method comprising:

[0065] (1) Under aseptic conditions, a 9mm punch was used to take a piece of mycelium from the plate, chop it up, and inoculate it into the culture medium prepared in Preparation Example 2. The culture was incubated at 25°C for 6 days to obtain a primary seed culture.

[0066] (2) The primary seed culture was inoculated into the culture medium prepared in Preparation Example 2 at an inoculation rate of 5 vt%, and cultured at 25°C for 4 days. The culture included alternating 6-hour static culture and 10-min shaking culture. The shaking culture was carried out at a speed of 30 rpm and a pressure of 0.02 MPa. Aeration was carried out at 10 L per shaking culture to obtain the secondary seed culture.

[0067] (3) The secondary seed liquid was inoculated into the culture medium prepared in Preparation Example 2. The inoculation amount was 5 vt%, the fermentation temperature was 25℃, the aeration rate was 50 L, the stirring speed was 120 rpm, the pressure was 0.1 MPa, and the culture time was 5 days.

[0068] (4) After fermentation, the fermentation liquid is filtered using a plate and frame filter press, the filtrate is centrifuged again, and the supernatant is collected.

[0069] Example 3

[0070] This embodiment provides a method for preparing fermentation broth of Schizophyllum commune mycelium, the preparation method comprising:

[0071] (1) Under aseptic conditions, a 9mm punch was used to take a piece of mycelium from the plate, chop it up and inoculate it into the culture medium prepared in Preparation Example 3. The culture was incubated at 32°C for 4 days to obtain the first-stage seed culture.

[0072] (2) The primary seed culture was inoculated into the culture medium prepared in Preparation Example 3 at an inoculation rate of 15 vt%, and cultured at 32°C for 1 day. The culture included alternating 10-hour static culture and 50-minute shaking culture. The shaking culture was carried out at a speed of 100 rpm and a pressure of 0.08 MPa. Aeration was carried out at 30 L per shaking culture to obtain the secondary seed culture.

[0073] (3) The secondary seed liquid was inoculated into the culture medium prepared in Preparation Example 3. The inoculation amount was 15 vt%, the fermentation temperature was 32℃, the aeration rate was 130 L, the stirring speed was 180 rpm, the pressure was 0.2 MPa, and the culture time was 2 days.

[0074] (4) After fermentation, the fermentation liquid is filtered using a plate and frame filter press, the filtrate is centrifuged again, and the supernatant is collected.

[0075] Example 4

[0076] This embodiment provides a method for preparing fermentation broth of Schizophyllum commune mycelium. The only difference between this embodiment and Example 1 is that the culture medium used is different. The culture medium used in steps (1)-(3) of this application is the culture medium prepared in Example 4. Other operations remain unchanged.

[0077] Example 5

[0078] This embodiment provides a method for preparing fermentation broth of Schizophyllum commune mycelium. The only difference between this embodiment and Example 1 is that the culture medium used is different. The culture medium used in steps (1)-(3) of this application is the culture medium prepared in Example 5. Other operations remain unchanged.

[0079] Example 6

[0080] This embodiment provides a method for preparing fermentation broth of Schizophyllum commune mycelium. The only difference between this embodiment and Example 1 is that the culture medium used is different. The culture medium used in steps (1)-(3) of this application is the culture medium prepared in Example 6. Other operations remain unchanged.

[0081] Comparative Example 1

[0082] This comparative example provides a method for preparing fermentation broth of Schizophyllum commune mycelium. The only difference between the other examples 1 and the one in example 1 is that step (2) is to "inoculate the primary seed liquid into the culture medium prepared in Example 1 at an inoculation rate of 10 vt% and incubate it at 28°C for 2 days". Other operations remain unchanged.

[0083] Comparative Example 2

[0084] This comparative example provides a method for preparing fermentation broth of Schizophyllum commune mycelium. The only difference between the other examples is that step (2) is to "inoculate the primary seed liquid into the culture medium prepared in Example 1 at an inoculation rate of 10 vt%, and culture it at 28°C with shaking for 2 days. The shaking speed is 50 rpm, the pressure is 0.05 MPa, and 20 L of aeration is provided for each shaking culture to obtain the secondary seed liquid". Other operations remain unchanged.

[0085] Comparative Example 3

[0086] This comparative example provides a method for preparing *Schizophyllum commune* mycelial fermentation broth. The only difference between the other examples is that step (1) is "under aseptic conditions, a mycelial block is taken from the plate using a 9mm punch, chopped, and inoculated into the culture medium prepared in Example 1, and cultured at 28°C with shaking for 5 days at a shaking speed of 200 rpm to obtain a primary seed culture," and step (2) is "the primary seed culture is inoculated into the culture medium prepared in Example 1 at an inoculation rate of 10 vt%, and cultured statically at 28°C for 2 days." All other operations remain unchanged.

[0087] Comparative Example 4

[0088] This comparative example provides a method for preparing fermentation broth of Schizophyllum commune mycelium. The only difference between the other examples is that step (1) is "under aseptic conditions, a 9mm punch is used to take a piece of mycelium from the plate, chop it, and inoculate it into the culture medium prepared in Example 1. The culture is shaken at 28°C for 5 days at a shaking speed of 200 rpm to obtain a primary seed culture". Step (2) is "the primary seed culture is inoculated into the culture medium prepared in Example 1 at an inoculation rate of 10 vt%, and the culture is shaken at 28°C for 2 days at a shaking speed of 50 rpm and a pressure of 0.05 MPa. Aeration is carried out for 20 L per shake culture to obtain a secondary seed culture". All other operations remain unchanged.

[0089] Comparative Example 5

[0090] This comparative example provides a method for preparing fermentation broth of Schizophyllum commune mycelium. The only difference between the other examples is that step (1) is "under aseptic conditions, a 9mm punch is used to take a piece of mycelium from the plate, chop it, and inoculate it into the culture medium prepared in Example 1. The culture is shaken at 28°C for 5 days, and the shaking speed is 200 rpm to obtain the first-stage seed liquid". Other operations remain unchanged.

[0091] Test Example 1

[0092] Security test

[0093] Test samples: Examples 1-6

[0094] Test method:

[0095] Each product group was diluted with PBS to a 10% test sample solution. Forty eligible volunteers aged 20-45 years were selected. The product was placed in the small chamber of the plaque applicator, while the control well was left untreated. The plaque applicator containing the test substance was applied to the inner forearm of the subject using hypoallergenic adhesive tape, and the palm was gently pressed to ensure even application to the skin for 24 hours. Skin reactions were observed according to the criteria in Table 1 at 30 min, 24 h, and 48 h after removing the plaque applicator.

[0096] Table 1

[0097]

[0098] Table 2

[0099]

[0100] The results showed that the products of this invention all had negative reactions after trial use, indicating that the fermentation products of Schizophyllum commune mycelium involved in this invention are safe and mild and will not cause adverse reactions such as skin irritation or sensitization.

[0101] Test Example 2

[0102] Evaluation of antioxidant effects

[0103] 1. DPPH free radical scavenging experiment

[0104] 1.1 Preparation of DPPH solution: Weigh 5.0 mg of DPPH, dissolve it in an appropriate amount of anhydrous ethanol, sonicate it in the dark until fully dissolved, and then dilute to 100 mL with anhydrous ethanol to prepare a 50 μg / mL DPPH solution. This solution should be prepared and used immediately.

[0105] 1.2 Setting up experimental group A S : 100 μL of sample solution was mixed with 300 μL of DPPH solution; Control group Ac: 100 μL of sample solution was mixed with 300 μL of anhydrous ethanol; Blank group A b Mix 300 μL of DPPH solution (50.0 μg / mL) with 100 μL of sample solvent solution. React at room temperature in the dark for 0.5 h, and measure the absorbance at 517 nm to calculate the scavenging rate.

[0106] 1.3 Formula for calculating DPPH free radical scavenging rate:

[0107] Clearance rate (%) = [1-(A)] s -A c ) / A b ]×100%

[0108] Among them, A s A represents the absorbance value of the experimental group; c The absorbance value is for the control group; A b This represents the absorbance value of the blank group.

[0109] 2. ABTS free radical scavenging experiment:

[0110] 2.1 Preparation of ABTS stock solution: Dissolve ABTS in water to prepare a 7 mmol / L solution, and prepare a 2.45 mmol / L solution with potassium persulfate. Then mix the two solutions at a volume ratio of 1:1 and let them react at 25°C in the dark for 14 hours.

[0111] Preparation of ABTS working solution: Dilute the ABTS stock solution with phosphate buffer (PBS, pH 7.4) to obtain an absorbance of 0.70±0.02 at a wavelength of 734 nm.

[0112] 2.2 Setting up Experimental Group A s Mix 0.4 mL of sample solution with 3.6 mL of ABTS working solution; Blank group A bMix 0.4 mL of sample solvent solution with 3.6 mL of ABTS working solution. React at 25°C in the dark for 5 min, and measure the absorbance at 734 nm. Calculate the clearance rate.

[0113] 2.3 Formula for calculating ABTS free radical scavenging rate:

[0114] Clearance rate (%) = [1-(A)] b -A s ) / A b ]×100%

[0115] Among them, A s A represents the absorbance value of the experimental group; b This represents the absorbance value of the blank group.

[0116] 3 Hydroxyl radical scavenging experiment

[0117] 3.1 Solution preparation:

[0118] 6 mmol / L FeSO4: Dissolve FeSO4 in water to prepare 6 mmol / L FeSO4.

[0119] 6 mmol / L H2O2: Prepare 6 mmol / L H2O2 using 30% H2O2.

[0120] 6 mmol / L salicylic acid solution: Prepare a 6 mmol / L salicylic acid solution using anhydrous ethanol.

[0121] 3.2 Setting up Experimental Group A s 100 μL of sample solution was mixed with 100 μL of FeSO4, 100 μL of H2O2, and 100 μL of salicylic acid solution; blank group A b 100 μL of sample solution was mixed with 100 μL of FeSO4, 100 μL of water, and 100 μL of salicylic acid solution; control group A c Mix 100 μL FeSO4, 100 μL H2O2, 100 μL sample solution, and 100 μL solvent. React in a water bath at 37℃ in the dark for 30 min, and measure the absorbance at 510 nm. Calculate the scavenging rate.

[0122] 3.3 Formula for calculating hydroxyl radical scavenging rate:

[0123] Clearance rate (%) = [1-(A)] s -A c ) / A b ]×100%

[0124] 4. Experimental Results

[0125] The scavenging effects of each embodiment on DPPH free radicals, ABTS free radicals, and hydroxyl free radicals are shown in Table 3.

[0126] Table 3

[0127]

[0128] The results are shown in Table 3. The fermentation broth of Schizophyllum commune mycelium prepared in this invention has good DPPH, ABTS and hydroxyl radical scavenging effects, and the preparation method of the fermentation broth and the selection of the culture medium will affect the above effects.

[0129] Test Example 3

[0130] Evaluation of soothing effect

[0131] (1) Prepare the test sample.

[0132] (2) Experimental procedure: Test tubes A and B (0.5 mL of sample solution and 0.5 mL of hyaluronidase (500 U / mL)), test tubes C and D (0.5 mL of distilled water and 0.5 mL of acetate buffer solution), incubated at 37°C for 20 min; 0.1 mL (2.5 mmol / L) CaCl2 solution was added, and incubated at 37°C for 20 min; 0.5 mL of sodium hyaluronate (0.5 mg / mL) was added to test tubes A and C, and 0.5 mL of acetate buffer solution was added to test tubes B and D, and incubated at 37°C for 40 min; 0.5 mL of acetylacetone solution, 0.1 mL of NaOH solution (5 mol / L) and 0.5 mL of distilled water were added, and the mixture was incubated in a boiling water bath for 15 min, immediately cooled in ice water for 5 min, 1 mL of P-DAB colorimetric reagent was added, and after thorough shaking, 3.5 mL of anhydrous ethanol was added, and the mixture was allowed to stand for 30 min for color development. The color was then developed at 530°C. The OD values ​​of the four test tubes A, B, C, and D were measured at nm.

[0133] The formula for calculating the hyaluronidase activity inhibition rate is:

[0134] Hyaluronidase inhibition rate (%) = [(CD) - (AB)] / (CD) × 100%

[0135] Table 4

[0136]

[0137] The results are shown in Table 4. The fermentation broth of Schizophyllum commune mycelium prepared by the present invention has a good hyaluronidase inhibition effect, and the preparation method of the fermentation broth and the selection of the culture medium will affect the above effect.

[0138] Test Example 4

[0139] Moisturizing and repairing effect evaluation test:

[0140] 1. Test Methods

[0141] (1) Subjects should not use any products (cosmetics or topical medications) on the test site 2-3 days before the visit, and should not let the site come into contact with water for 1-3 hours. Clean the inside of the forearms of both hands (clean by wiping with a dry tissue);

[0142] (2) The sample application area should be randomly distributed in the marked areas of the left and right arms, with each test area having an area of ​​3 cm × 3 cm;

[0143] (3) Before the formal test, you should sit quietly in a standard room for at least 20 minutes without drinking water or beverages. Your forearm should be exposed and placed in the test position, and you should remain relaxed.

[0144] (4) Test the blank value of the smeared area, i.e., T0.

[0145] (5) Fermentation broth of Schizophyllum commune mycelium at 2 mg / cm³ 2 Apply the sample in a single application using a latex finger cot, and spread the sample evenly over the test area.

[0146] (6) Use a stratum corneum hydration meter to measure at the fourth hour, i.e., T4. Each area should be measured in parallel at least three times.

[0147] The moisture content of the stratum corneum of the skin is shown in Table 5.

[0148] Table 5

[0149]

[0150] The results are shown in Table 5. The fermentation broth of Schizophyllum commune mycelium prepared by the present invention has a good moisturizing effect, and the preparation method of the fermentation broth and the selection of the culture medium will affect the above effect.

[0151] The above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A method for preparing a fermentation broth of Schizophyllum frondosum mycelium, characterized by, The preparation method comprises: (1) inoculating Schizophyllum to a culture medium, standing and culturing to obtain a first seed liquid; (2) inoculating the first seed liquid to a culture medium, culturing to obtain a second seed liquid; (3) inoculating the second seed liquid to a culture medium, fermenting, and collecting the filtrate after pressure filtration of the obtained fermentation liquid, to obtain the Schizophyllum mycelium fermentation liquid; The culturing of step (2) comprises a standing and culturing stage and a stirring and culturing stage alternately.

2. The preparation method of Schizophyllum frondesum mycelium fermentation broth according to claim 1, characterized in that, The standing and culturing of step (1) is at a temperature of 25-32℃ for 96-144 h.

3. The method of preparing a Schizophyllum frondesiforme mycelium fermentation broth according to claim 1 or 2, characterized by, The standing and culturing stage is at a temperature of 25-32℃ for 6-10 h each time; Preferably, the stirring and culturing stage is at a temperature of 25-32℃ for 10-50 min each time.

4. The process for the preparation of Schizophyllum frondes- sum mycelium broth according to any one of claims 1 to 3, characterized in that, The total culturing time of step (2) is 24-96 h.

5. The process for the preparation of Schizophyllum frondes- sum mycelium broth according to any one of claims 1 to 4, characterized in that, The stirring and culturing further comprises aeration, and the aeration amount of each stirring and culturing is 10-30 L; Preferably, the stirring and culturing is at a rotation speed of 20-100 rpm and a pressure of 0.02-0.08 MPa.

6. The process for the preparation of Schizophyllum frondes- sum mycelium broth according to any one of claims 1 to 5, characterized in that, The fermentation is at a temperature of 25-32℃ for 24-120 h; Preferably, the fermentation is at a rotation speed of 120-180 rpm, a pressure of 0.1-0.2 MPa, and an aeration amount of 50-130 L.

7. The method of producing a Schizophyllum frondesiforme mycelium fermentation broth according to any one of claims 1 to 6, characterized in that, The culture medium comprises, in terms of mass percentage, 1-3% of glucose, 0.1-0.6% of malt extract powder, 0.01-0.06% of KH2PO4, 0.01-0.06% of MgSO4.7H2O, and water.

8. The process for the preparation of Schizophyllum frondes- sum mycelium broth according to any one of claims 1 to 7, characterized in that, The inoculation of steps (2) and (3) is independently at an inoculation amount of 5-15 vt%; Preferably, the filtrate of step (3) further comprises centrifuging to obtain supernatant, and the centrifuging is at a rotation speed of 5000-15000 rpm for 15-45 min.

9. The Schizophyllum mycelium fermentation liquid prepared by the preparation method of the Schizophyllum mycelium fermentation liquid according to any one of claims 1-8.

10. The use of the Schizophyllum mycelium fermentation liquid according to claim 9 in the preparation of a product having the effects of antioxidation, soothing, moisturizing, and repair.

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