A phellinus igniarius mycelium fermentation product, and a preparation method and application thereof

Abstract

The present application relates to a kind of phellinus igniarius mycelium fermentation product and its preparation method and application, the preparation method includes the following steps: phellinus igniarius mycelium seed liquid is inoculated in sterile fermentation medium and is cultured, solid-liquid separation, clear liquid is filtered and sterilized, and the phellinus igniarius mycelium fermentation product is obtained;The component of the fermentation medium includes beet extract, eutectic solvent and water.The present application creatively uses beet extract and eutectic solvent as medium component to prepare phellinus igniarius mycelium fermentation product, wherein beet extract and eutectic solvent provide the nutrition required for phellinus fermentation together, while using eutectic solvent to regulate metabolism, compared with the product obtained by traditional medium fermentation, the active ingredient content in the fermentation product obtained by the medium with the above formula is significantly improved, and the uniformity of fermentation product is good and stability is high, appearance is clear liquid, without abnormal odor, with slight phellinus characteristic odor.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fermentation products of strains, and relates to a fermentation product of Phellinus baumii mycelium and a preparation method and application thereof. BACKGROUND

[0002] Phellinus baumii (Latin name Sanghuangporus) is a traditional medicinal fungus, which is named because it usually grows on mulberry plants and its fruiting bodies are yellowish brown. As a traditional Chinese medicine, it has been recorded to be used for the treatment of gonorrhea, spleen deficiency diarrhea, sore accumulation, and metrorrhagia and vaginal bleeding. In recent years, people have gradually found that it has good effects in anti-tumor and immune regulation. Wild Phellinus baumii fruiting bodies are rare, and overexploitation leads to a sharp decrease in resources. Artificially cultivated Phellinus baumii has a long growth cycle and cannot meet the needs of large-scale production. In comparison, liquid fermentation of Phellinus baumii mycelium has the advantages of sterility, controllable production conditions, automatic control, and large-scale production, and can obtain a large amount of cell products and metabolites in a short time.

[0003] At present, there is little research on the liquid fermentation product of Phellinus baumii mycelium in the prior art. Therefore, the functional Phellinus baumii mycelium fermentation product is a direction worth exploring, which is helpful for the development and utilization of Phellinus baumii resources. SUMMARY

[0004] In view of the deficiencies of the prior art, the purpose of the present application is to provide a fermentation product of Phellinus baumii mycelium and a preparation method and application thereof, in particular to provide a fermentation product of Phellinus baumii mycelium with antioxidant, moisturizing and soothing and repairing effects and a preparation method and application thereof.

[0005] To achieve the purpose of the present application, the following technical solutions are adopted:

[0006] In a first aspect, the present application provides a preparation method of a fermentation product of Phellinus baumii (Latin name Sanghuangporus) mycelium, which comprises the following steps:

[0007] Inoculating Phellinus baumii mycelium seed liquid into sterile fermentation medium for culture, solid-liquid separation, membrane filtration of the supernatant and sterilization to obtain the fermentation product of Phellinus baumii mycelium;

[0008] The components of the fermentation medium include beet extract, deep eutectic solvent and water.

[0009] The application creatively uses beet extract and a deep eutectic solvent as medium components for co-extraction and fermentation to prepare a Phellinus igniarius mycelium fermentation product in one step, wherein the beet extract and the deep eutectic solvent together provide the nutrients required for Phellinus igniarius fermentation, while the deep eutectic solvent is used to regulate metabolism. Compared with the product obtained by traditional medium fermentation, the fermentation product obtained by using the above-mentioned medium has a significantly improved content of active ingredients, good uniformity and high stability, and the appearance is a clear liquid without abnormal odor, with a slight characteristic odor of Phellinus igniarius.

[0010] The application combines beet extract, a deep eutectic solvent and Phellinus igniarius by biotechnology, and comprehensively uses the nutrients and effects of the three, so that the obtained Phellinus igniarius mycelium fermentation product has excellent antioxidant effect and significantly improves the expression effect of AQP3 and FLG genes of cells, can efficiently promote the expression of aquaporin and barrier protein genes, realizes efficient skin moisturizing and soothing repair, and makes the application of Phellinus igniarius have a wide prospect.

[0011] Preferably, the components of the fermentation medium include beet extract 5-20%, deep eutectic solvent 1-10% and water 65-94% by mass percentage.

[0012] The mass percentage of the beet extract can be 5%, 6%, 8%, 10%, 12%, 13%, 15%, 16%, 17%, 18%, 20%, etc.; the mass percentage of the deep eutectic solvent can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, etc.; and the mass percentage of the water can be 65%, 68%, 70%, 75%, 80%, 82%, 85%, 88%, 90%, 94%, etc.

[0013] Preferably, the components of the fermentation medium further include inorganic salt 0.1-1% by mass percentage, such as 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, etc.

[0014] Preferably, the inorganic salt is selected from sodium salt, potassium salt, magnesium salt, calcium salt, iron salt, manganese salt, sulfate salt, phosphate salt, chloride salt, etc.

[0015] Preferably, the deep eutectic solvent is obtained by reacting a hydrogen bond acceptor and a hydrogen bond donor at 50-90℃ (such as 50℃, 55℃, 60℃, 65℃, 70℃, 80℃, 90℃, etc.) until the liquid is clear and transparent; the hydrogen bond acceptor is selected from choline chloride and / or betaine; and the hydrogen bond donor is selected from any one or a combination of at least two of glycerol, propylene glycol, butanediol, xylitol or sorbitol.

[0016] The eutectic solvent in the fermentation medium is preferably prepared by reacting choline chloride and / or betaine with a polyol, and when the hydrogen bond acceptor is betaine and the hydrogen bond donor is glycerol, the fermentation product has more excellent antioxidant efficacy, moisturizing efficacy and soothing and repairing efficacy.

[0017] Preferably, the hydrogen bond acceptor is betaine and the hydrogen bond donor is glycerol.

[0018] Preferably, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:(5-7), such as 1:5, 1:5.5, 1:6, 1:6.5, 1:7, etc.

[0019] The beet extract in the fermentation medium can be prepared by using the prior art extraction process, and more preferably by using the following specific preparation process, and the fermentation product prepared by using the process has more excellent antioxidant efficacy, moisturizing efficacy and soothing and repairing efficacy.

[0020] Preferably, the beet extract is prepared by a preparation method comprising the following steps:

[0021] The beet raw material is mixed with the eutectic solvent and then subjected to microwave-ultrasonic combined extraction treatment, and the extraction liquid is centrifuged and filtered to obtain the beet extract.

[0022] Preferably, the eutectic solvent is obtained by reacting a hydrogen bond acceptor with a hydrogen bond donor at 50-90°C (such as 50°C, 55°C, 60°C, 65°C, 70°C, 80°C, 90°C, etc.); the hydrogen bond acceptor is selected from choline chloride and / or betaine; and the hydrogen bond donor is selected from any one or a combination of at least two of glycerol, propylene glycol, butanediol, xylitol or sorbitol.

[0023] Preferably, the mixing ratio of the beet raw material to the eutectic solvent is 1:(5-20) g / mL, such as 1:5 g / mL, 1:8 g / mL, 1:10 g / mL, 1:12 g / mL, 1:15 g / mL, 1:17 g / mL, 1:20 g / mL, etc.

[0024] Preferably, the extraction treatment is carried out at a temperature of 40-70°C (such as 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, etc.) for a time of 2-5 h (such as 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, etc.).

[0025] Preferably, the microwave treatment has a power of 300-500 W (e.g. 300 W, 340 W, 400 W, 450 W, 500 W, etc.), and the ultrasonic treatment has a power of 50-300 W (e.g. 50 W, 100 W, 150 W, 200 W, 250 W, 300 W, etc.).

[0026] Preferably, the filtration is followed by a concentration treatment and / or a drying treatment.

[0027] Preferably, the inoculation volume of the P. sylvestris mycelium seed liquid is 5-15% of the volume of the sterile fermentation medium, e.g. 5%, 6%, 7%, 8%, 10%, 11%, 12%, 13%, 14%, 15%, etc.

[0028] The P. sylvestris mycelium seed liquid can be prepared using methods known in the art.

[0029] For example, the P. sylvestris mycelium seed liquid can be prepared using the following process:

[0030] Under sterile conditions, a piece of the slant culture is inoculated into the medium, and cultured at 20-30°C with 100-200 rpm shaking for 3-10 days; or the seed liquid after culturing is inoculated into the medium again at an inoculation volume of 5-15%, and cultured at 20-30°C with 100-200 rpm shaking for 3-10 days.

[0031] The medium used in the preparation of the seed liquid can be a conventional seed culture medium (e.g. beef extract peptone medium), or can have the same formulation as the fermentation medium described above: 5-20% of beet extract, 1-10% of deep eutectic solvent, 65-94% of water, and optionally 1-10% of inorganic salt, by mass percentage.

[0032] Preferably, the culture is carried out at a temperature of 20-30°C (e.g. 20°C, 22°C, 24°C, 25°C, 26°C, 28°C, 30°C, etc.) for a time period of 3-10 days (e.g. 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, etc.).

[0033] Preferably, the culture is carried out under aeration of 40-80 L (e.g. 40 L, 50 L, 60 L, 70 L, 80 L, etc.), pressure of 0.05-0.3 MPa (e.g. 0.05 MPa, 0.1 MPa, 0.15 MPa, 0.2 MPa, 0.25 MPa, 0.3 MPa, etc.), and stirring.

[0034] Preferably, the solid-liquid separation comprises centrifugation at 10000-15000 rpm (e.g. 10000 rpm, 11000 rpm, 12000 rpm, 13000 rpm, 14000 rpm, 15000 rpm, etc.) for 30-60 min (e.g. 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, etc.).

[0035] Preferably, the supernatant is filtered through a 0.10-0.22 μm organic membrane.

[0036] Preferably, the supernatant is further subjected to concentration and / or drying after being filtered and sterilized.

[0037] All other specific point values not listed in the above-mentioned numerical ranges can be selected, and are within the protection scope of the present application, and are not repeated here in view of brief expression.

[0038] In a second aspect, the present application provides a Phellinus igniarius mycelium fermentation product prepared by the preparation method according to the first aspect.

[0039] In a third aspect, the present application provides use of the Phellinus igniarius mycelium fermentation product according to the second aspect in preparation of a product with antioxidant efficacy.

[0040] In a fourth aspect, the present application provides use of the Phellinus igniarius mycelium fermentation product according to the second aspect in preparation of a product with free radical scavenging efficacy.

[0041] In a fifth aspect, the present application provides use of the Phellinus igniarius mycelium fermentation product according to the second aspect in preparation of a product with moisturizing efficacy.

[0042] In a sixth aspect, the present application provides use of the Phellinus igniarius mycelium fermentation product according to the second aspect in preparation of a product for improving expression level of AQP3 of human keratinocytes.

[0043] In a seventh aspect, the present application provides use of the Phellinus igniarius mycelium fermentation product according to the second aspect in preparation of a product with soothing and repairing efficacy.

[0044] In an eighth aspect, the present application provides use of the Phellinus igniarius mycelium fermentation product according to the second aspect in preparation of a product for improving expression level of FLG of human keratinocytes.

[0045] Compared with the prior art, the present application has the following beneficial effects:

[0046] The application creatively uses beet extract and a deep eutectic solvent as medium components to prepare Phellinus igniarius mycelium fermentation products, wherein the beet extract and the deep eutectic solvent jointly provide the nutrients required for Phellinus igniarius fermentation, and at the same time, the deep eutectic solvent is used to regulate metabolism. Compared with the products obtained by traditional medium fermentation, the fermentation products obtained by the medium with the above formula have a significantly improved content of active ingredients, good uniformity and high stability, and the appearance is a clear liquid without abnormal odor, and has a slight characteristic odor of Phellinus igniarius.

[0047] The application combines beet extract, deep eutectic solvent and Phellinus igniarius by biotechnology, and comprehensively uses the nutrients and effects of the three, so that the obtained Phellinus igniarius mycelium fermentation product has excellent antioxidant effect and obvious effect on the expression of AQP3 and FLG genes of cells, can efficiently promote the expression of aquaporin and barrier protein genes, realizes efficient skin moisturizing and soothing repair, and makes the application of Phellinus igniarius have a wide prospect. DETAILED DESCRIPTION

[0048] In order to further illustrate the technical means adopted by the application and its effects, the technical solutions of the application will be further described below in combination with preferred embodiments of the application, but the application is not limited in the scope of the embodiments.

[0049] Preparation Example 1-1

[0050] The preparation example provides a deep eutectic solvent, and the preparation method is as follows:

[0051] Beet alkali and glycerol are mixed at a molar ratio of 1:6, and the reaction is carried out at 60 DEG C until the system is a clear transparent liquid.

[0052] Preparation Example 1-2

[0053] The preparation example provides a deep eutectic solvent, and the preparation method is as follows:

[0054] Choline chloride and glycerol are mixed at a molar ratio of 1:6, and the reaction is carried out at 60 DEG C until the system is a clear transparent liquid.

[0055] Preparation Example 1-3

[0056] The preparation example provides a deep eutectic solvent, and the preparation method is as follows:

[0057] Beet alkali and propylene glycol are mixed at a molar ratio of 1:6, and the reaction is carried out at 60 DEG C until the system is a clear transparent liquid.

[0058] Preparation Example 1-4

[0059] The preparation example provides a deep eutectic solvent, and the preparation method is as follows:

[0060] Mix betaine and sorbitol in a molar ratio of 1:6, and carry out the reaction at 60°C until the system is a clear transparent liquid, to obtain the product.

[0061] Preparation Example 2-1

[0062] The present preparation example provides a beet extract, and the preparation method is as follows:

[0063] After the beet is crushed, it is mixed with the eutectic solvent prepared in Preparation Example 1-1 at a ratio of 1:10 g / mL, and subjected to microwave-ultrasonic combined treatment to obtain an extract; the treatment temperature is 55°C, the treatment time is 4 h, the microwave treatment power is 400 W, and the ultrasonic treatment power is 150 W; the extract is centrifuged and filtered, the filtrate is concentrated under reduced pressure and freeze-dried to obtain the beet extract.

[0064] Preparation Example 2-2

[0065] The present preparation example provides a beet extract, and the preparation method is as follows:

[0066] After the beet is crushed, it is mixed with the eutectic solvent prepared in Preparation Example 1-1 at a ratio of 1:15 g / mL, and subjected to microwave-ultrasonic combined treatment to obtain an extract; the treatment temperature is 65°C, the treatment time is 3 h, the microwave treatment power is 300 W, and the ultrasonic treatment power is 250 W; the extract is centrifuged and filtered, the filtrate is concentrated under reduced pressure and freeze-dried to obtain the beet extract.

[0067] Preparation Example 2-3

[0068] The present preparation example provides a beet extract, and the preparation method is as follows:

[0069] After the beet is crushed, it is mixed with the eutectic solvent prepared in Preparation Example 1-1 at a ratio of 1:8 g / mL, and subjected to microwave-ultrasonic combined treatment to obtain an extract; the treatment temperature is 45°C, the treatment time is 4.5 h, the microwave treatment power is 500 W, and the ultrasonic treatment power is 100 W; the extract is centrifuged and filtered, the filtrate is concentrated under reduced pressure and freeze-dried to obtain the beet extract.

[0070] Preparation Example 2-4

[0071] The present preparation example provides a beet extract, and the preparation method is as follows:

[0072] Preparation Example 2-5

[0073] The preparation example provides a beet extract, and the preparation method is only different from that of the preparation example 2-1 in that the low eutectic solvent prepared in the preparation example 1-1 is replaced by the low eutectic solvent prepared in the preparation example 1-3 in equal volume, and other conditions are kept unchanged.

[0074] Preparation example 2-6

[0075] The preparation example provides a beet extract, and the preparation method is as follows.

[0076] The beet is crushed, mixed with the low eutectic solvent prepared in the preparation example 1-1 at a ratio of 1:10 g / mL, and subjected to microwave treatment to obtain an extract; the treatment temperature is 55 DEG C, the treatment time is 4.5 h, and the microwave treatment power is 500 W; the extract is centrifuged and filtered, the filtrate is concentrated under reduced pressure and freeze-dried to obtain the beet extract.

[0077] Preparation example 2-7

[0078] The preparation example provides a beet extract, and the preparation method is as follows.

[0079] The beet is crushed, mixed with the low eutectic solvent prepared in the preparation example 1-1 at a ratio of 1:10 g / mL, and subjected to ultrasonic treatment to obtain an extract; the treatment temperature is 55 DEG C, the treatment time is 4.5 h, and the ultrasonic treatment power is 250 W; the extract is centrifuged and filtered, the filtrate is concentrated under reduced pressure and freeze-dried to obtain the beet extract.

[0080] Preparation example 2-8

[0081] The preparation example provides a beet extract, and the preparation method is as follows.

[0082] The beet is crushed, mixed with water at a ratio of 1:10 g / mL, and subjected to microwave-ultrasonic combined treatment to obtain an extract; the treatment temperature is 55 DEG C, the treatment time is 4 h, the microwave treatment power is 400 W, and the ultrasonic treatment power is 150 W; the extract is centrifuged and filtered, the filtrate is concentrated under reduced pressure and freeze-dried to obtain the beet extract.

[0083] Preparation example 2-9

[0084] The preparation example provides a beet extract, and the preparation method is as follows.

[0085] The beet is crushed, mixed with 70% ethanol aqueous solution at a ratio of 1:10 g / mL, and subjected to microwave-ultrasonic combined treatment to obtain an extract; the treatment temperature is 55 DEG C, the treatment time is 4 h, the microwave treatment power is 400 W, and the ultrasonic treatment power is 150 W; the extract is centrifuged and filtered, the filtrate is concentrated under reduced pressure and freeze-dried to obtain the beet extract.

[0086] Example 1

[0087] This example provides a Phellinus igniarius mycelium fermentation product, and the preparation method is as follows:

[0088] (1) Preparation of culture medium: beet extract of Preparation Example 2-1 10%, deep eutectic solvent of Preparation Example 1-1 5%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.2%, and the rest is water; sterilize the culture medium at 121°C for 30 min.

[0089] (2) Under sterile conditions, take the bacterial block from the slope and inoculate into the shake flask containing the culture medium of step (1), and culture at 27°C, 200 rpm shaking bed for 7 days to obtain the Phellinus igniarius mycelium primary seed liquid.

[0090] (3) According to the inoculation amount of 10% (v / v), inoculate the Phellinus igniarius mycelium primary seed liquid into the shake flask containing the culture medium of step (1), and culture at 27°C, 200 rpm shaking bed for 5 days to obtain the Phellinus igniarius mycelium secondary seed liquid;

[0091] (4) According to the inoculation amount of 10% (v / v), inoculate the Phellinus igniarius mycelium secondary seed liquid into the fermentation tank containing the culture medium of step (1), and culture at 27°C, with aeration amount of 60 L, stirring speed of 100 r / min, and pressure of 0.1 MPa for 7 days, and then harvest the fermentation liquid.

[0092] (5) Centrifuge the harvested fermentation liquid at 12000 r / min for 30 min, and then pass the supernatant through a 0.22 μm organic membrane, and sterilize at 121°C for 30 min to obtain the Phellinus igniarius mycelium fermentation liquid.

[0093] Example 2

[0094] This example provides a Phellinus igniarius mycelium fermentation product, and the preparation method is as follows:

[0095] (1) Preparation of culture medium: beet extract of Preparation Example 2-2 15%, deep eutectic solvent of Preparation Example 1-1 5%, potassium dihydrogen phosphate 0.3%, magnesium sulfate 0.2%, and the rest is water; sterilize the culture medium at 121°C for 30 min.

[0096] (2) Under sterile conditions, take the bacterial block from the slope and inoculate into the shake flask containing the culture medium of step (1), and culture at 25°C, 200 rpm shaking bed for 7 days to obtain the Phellinus igniarius mycelium primary seed liquid.

[0097] (3) According to the inoculation amount of 5% (v / v), inoculate the Phellinus igniarius mycelium primary seed liquid into the shake flask containing the culture medium of step (1), and culture at 25°C, 200 rpm shaking bed for 8 days to obtain the Phellinus igniarius mycelium secondary seed liquid;

[0098] (4) According to the inoculation amount of 5% (v / v), the second-level Phellinus igniarius mycelium seed liquid is inoculated into the fermenter containing the culture medium of step (1), the culture temperature is 25℃, the ventilation amount is 80 L, the stirring speed is 150 r / min, the pressure is 0.1 MPa, the culture is carried out for 8 days, and the fermentation liquid is harvested after the fermenter is discharged.

[0099] (5) The harvested fermentation liquid is centrifuged at 15000 r / min for 30 min, the supernatant is filtered through a 0.22 μm organic membrane, and the Phellinus igniarius mycelium fermentation liquid is obtained after sterilization at 121℃ for 30 min.

[0100] Example 3

[0101] The present example provides a Phellinus igniarius mycelium fermentation product, and the preparation method is as follows:

[0102] (1) Preparation of culture medium: beet extract of Preparation Example 2-3 is 8%, deep eutectic solvent of Preparation Example 1-1 is 10%, potassium dihydrogen phosphate is 0.3%, magnesium sulfate is 0.2%, and the rest is water; the culture medium is sterilized at 121℃ for 30 min.

[0103] (2) Under sterile conditions, the bacterial block is taken from the slope and inoculated into the shake flask containing the culture medium of step (1), and the culture is carried out at 30℃ and 100 rpm of shaking bed for 7 days to obtain the first-level Phellinus igniarius mycelium seed liquid.

[0104] (3) According to the inoculation amount of 15% (v / v), the first-level Phellinus igniarius mycelium seed liquid is inoculated into the shake flask containing the culture medium of step (1), the culture temperature is 30℃, the culture is carried out by 100 rpm of shaking bed for 4 days to obtain the second-level Phellinus igniarius mycelium seed liquid.

[0105] (4) According to the inoculation amount of 15% (v / v), the second-level Phellinus igniarius mycelium seed liquid is inoculated into the fermenter containing the culture medium of step (1), the culture temperature is 30℃, the ventilation amount is 40 L, the stirring speed is 150 r / min, the pressure is 0.1 MPa, the culture is carried out for 10 days, and the fermentation liquid is harvested after the fermenter is discharged.

[0106] (5) The harvested fermentation liquid is centrifuged at 10000 r / min for 40 min, the supernatant is filtered through a 0.22 μm organic membrane, and the Phellinus igniarius mycelium fermentation liquid is obtained after sterilization at 121℃ for 30 min.

[0107] Examples 4-9

[0108] The present example provides six kinds of Phellinus igniarius mycelium fermentation products, and the preparation method is different from that of Example 1 only in that the beet extract of Preparation Example 2-1 in the culture medium of step (1) is replaced by the beet extract of Preparation Example 2-4, Preparation Example 2-5, Preparation Example 2-6, Preparation Example 2-7, Preparation Example 2-8, and Preparation Example 2-9 respectively in equal amount, and other components remain unchanged.

[0109] Examples 10-12

[0110] This example provides three kinds of Phellinus igniarius mycelium fermentation products, the preparation method is different from example 1 only in that step (1) the low eutectic solvent of preparation example 1-1 is replaced by the low eutectic solvent of preparation example 1-2, preparation example 1-3, preparation example 1-4 in the culture medium, and other components remain unchanged.

[0111] Comparative example 1

[0112] This comparative example provides a kind of Phellinus igniarius mycelium fermentation product, the preparation method is different from example 1 only in that step (1) the 10% beet extract of preparation example 2-1 is replaced by 4% glucose, 2% yeast powder and 4% peptone in the culture medium, and other components remain unchanged.

[0113] Comparative example 2

[0114] This comparative example provides a kind of Phellinus igniarius mycelium fermentation product, the preparation method is different from example 1 only in that step (1) the 10% beet extract of preparation example 2-1 is replaced by 1% glucose, 9% potato filtrate in the culture medium, and other components remain unchanged.

[0115] Comparative example 3

[0116] This comparative example provides a kind of Phellinus igniarius mycelium fermentation product, the preparation method is different from example 1 only in that step (1) the low eutectic solvent is absent in the culture medium, and the mass is supplemented by water, and other components remain unchanged.

[0117] Test example 1

[0118] The appearance, uniformity and odor of the fermentation products prepared in examples 1-12 and comparative examples 1-3 are evaluated, as shown in table 1:

[0119] Table 1

[0120]

[0121] As shown in the results in table 1, the Phellinus igniarius mycelium fermentation filtrate prepared by the preparation method involved in the present application is a light yellow transparent liquid at 25℃, the system is uniform without stratification, and shows the characteristic odor of Phellinus igniarius; while the Phellinus igniarius mycelium fermentation filtrate prepared by other culture medium in comparative example 1-2 is a brown turbid liquid at 25℃, the system is unstable, has stratification phenomenon, and shows abnormal odor.

[0122] Test example 2

[0123] The antioxidant efficacy of the fermentation products prepared in examples 1-12 and comparative examples 1-3 is evaluated:

[0124] (1) Test sample preparation: The fermentation broth prepared in Examples 1-12 and Comparative Examples 1-3 was dried and then prepared into a test sample with a concentration of 10% for standby.

[0125] (2) Evaluation method:

[0126] (2.1) Preparation of DPPH solution: 5.0 mg of DPPH was weighed and dissolved in a suitable amount of anhydrous ethanol, and then ultrasonically dissolved in the dark. The solution was then diluted with anhydrous ethanol to 100 mL to prepare a DPPH solution with a concentration of 50 μg / mL. The solution should be prepared and used immediately.

[0127] (2.2) Sample group setting: 100 μL of sample solution was mixed with 100 μL of DPPH solution (50.0 μg / mL); sample blank group: 100 μL of sample solution was mixed with 100 μL of anhydrous ethanol; control group: 100 μL of DPPH solution (50.0 μg / mL) was mixed with 100 μL of anhydrous ethanol. The reaction was carried out at room temperature in the dark for 0.5 h, and the absorbance was measured at 517 nm. The clearance rate was calculated.

[0128] (2.3) DPPH free radical clearance rate calculation formula:

[0129] Clearance rate (%) = [1-(Ai-Aj) / Ac] x 100%

[0130] Wherein, Aj is the absorbance value of the sample blank group; Ai is the absorbance value of the sample group; and Ac is the absorbance value of the control group.

[0131] The results of each group are shown in Table 2:

[0132] Table 2

[0133]

[0134] As shown by the results in Table 2, compared with the fermentation products prepared in Comparative Examples 1-3, the Phellinus igniarius mycelium fermentation products prepared by the preparation method of the present application have excellent DPPH clearance capacity, i.e. excellent antioxidant performance. As shown by the data results of Comparative Example 1 and Examples 4-12, the type of eutectic solvent in the fermentation medium and the preparation process of the beet extract also affect the above-mentioned effects to some extent.

[0135] Test Example 3

[0136] The moisturizing effect of the fermentation products prepared in Examples 1-12 and Comparative Examples 1-3 was evaluated:

[0137] (1) Test sample preparation: The fermentation broth prepared in Examples 1-12 and Comparative Examples 1-3 was dried and then prepared into a test sample with a concentration of 10% for standby.

[0138] (2) Evaluation method:

[0139] (2.1) Cell inoculation and culture: Collect HaCaT cells in the logarithmic growth phase, inoculate 2x10 5 cells / well into a 12-well plate, and incubate in an incubator (37°C, 5% CO2) overnight.

[0140] (2.2) Modeling: aspirate the complete culture medium in the well, add PBS solvent, and irradiate under UVB lamp for 7.5 min.

[0141] (2.3) Grouping and sample addition: aspirate the PBS solution, add the test sample to the sample group, add the complete culture medium to the model group, and culture for another 48 h.

[0142] (2.4) Detection and data analysis: collect the cells and extract RNA, and after reverse transcription to cDNA, perform fluorescence quantitative PCR to detect the expression amount of AQP3 in each sample. Calculate the relative expression improvement rate (%) of AQP3 gene = (sample group expression amount - model group expression amount) / model group expression amount x 100%.

[0143] The results of each group are shown in Table 3:

[0144] Table 3

[0145]

[0146] As shown by the results in Table 3, compared with the fermentation products prepared by Comparative Examples 1-3, the Phellinus igniarius mycelium fermentation product prepared by the preparation method involved in the present application has excellent effect of improving the expression level of AQP3 gene, i.e., has excellent moisturizing potential. And as shown by the data results of Comparative Example 1 and Examples 4-12, the type selection of the deep eutectic solvent in the fermentation medium and the preparation process of the sugar beet extract also affect the above-mentioned effect to some extent.

[0147] Test Example 4

[0148] The soothing and repairing effects of the fermentation products prepared by Examples 1-12 and Comparative Examples 1-3 were evaluated.

[0149] (1) Preparation of test sample: dry the fermentation liquids prepared by Examples 1-12 and Comparative Examples 1-3, and then prepare a test sample with a concentration of 10% for standby.

[0150] (2) Evaluation method:

[0151] (2.1) Cell inoculation and culture: Collect HaCaT cells in the logarithmic growth phase, inoculate 2x10 5 cells / well into a 12-well plate, and incubate in an incubator (37°C, 5% CO2) overnight.

[0152] (2.2) Molding: the complete medium in the hole was sucked out, PBS solvent was added, and exposed to UVB lamp for 7.5 min.

[0153] (2.3) Grouping and sample addition: the PBS solution was sucked out, the sample group was added with the test sample, the model group was added with complete medium, and then cultured for 48 h.

[0154] (2.4) Detection and data analysis: the cells were collected and RNA was extracted, after reverse transcription to cDNA, the expression amount of FLG in each sample was detected by fluorescence quantitative PCR. The relative expression promotion rate (%) of FLG gene = (sample group expression amount - model group expression amount) / model group expression amount x 100%.

[0155] The results of each group are shown in Table 4:

[0156] Table 4

[0157]

[0158] It can be seen from the results recorded in Table 4 that, compared with the fermentation products prepared by Comparative Examples 1-3, the Phellinus igniarius mycelium fermentation product prepared by the preparation method involved in the present application has excellent effect of promoting the expression level of FLG gene, that is, excellent soothing and repairing potential. And from the data results of Comparative Example 1 and Examples 4-12, it can be seen that the type selection of the deep eutectic solvent in the fermentation medium and the preparation process of the sugar beet extract also affect the above-mentioned effect to a certain extent.

[0159] The applicant declares that the technical solutions of the present application are illustrated by the above examples, but the present application is not limited to the above examples, that is, it does not mean that the present application must rely on the above examples to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific modes, etc. fall within the protection scope and disclosure scope of the present application.

[0160] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above embodiments, and within the technical concept scope of the present application, the technical solutions of the present application can be subjected to various simple modifications, and these simple modifications all belong to the protection scope of the present application.

[0161] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined by any suitable means without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combination manners.

Claims

1. A method for preparing a fermentation product of Phellinus igniarius mycelium, characterized by, The preparation method comprises the following steps: Inoculating the Phellinus igniarius mycelium seed liquid into a sterile fermentation medium for culture, solid-liquid separation, membrane filtration and sterilization of the clear liquid to obtain the Phellinus igniarius mycelium fermentation product; The culture temperature is 20-30℃, and the culture time is 3-10 days; The culture is carried out under the conditions of aeration amount 40-80 L, pressure 0.05-0.3 MPa and stirring state; The fermentation medium comprises, in terms of mass percentage, beet extract 5-20%, deep eutectic solvent 1-10%, inorganic salt 0.1-1% and water 65-94%; The deep eutectic solvent is obtained by reacting a hydrogen bond acceptor and a hydrogen bond donor at a molar ratio of 1:(5-7) at 50-90℃; The hydrogen bond acceptor is selected from choline chloride and / or betaine; and the hydrogen bond donor is selected from any one or a combination of at least two of glycerol, propylene glycol, butanediol, xylitol or sorbitol.

2. The production method according to claim 1, characterized by, The hydrogen bond acceptor is betaine, and the hydrogen bond donor is glycerol.

3. The preparation method according to claim 1, characterized in that, The beet extract is obtained by a preparation method comprising the following steps: After mixing the beet raw material and the deep eutectic solvent, microwave-ultrasonic combined extraction treatment is carried out, and the extraction liquid is centrifuged and filtered to obtain the beet extract; The deep eutectic solvent is obtained by reacting a hydrogen bond acceptor and a hydrogen bond donor at 50-90℃, the hydrogen bond acceptor is selected from choline chloride and / or betaine, and the hydrogen bond donor is selected from any one or a combination of at least two of glycerol, propylene glycol, butanediol, xylitol or sorbitol.

4. The production method according to claim 3, characterized by, The mixing ratio of the beet raw material and the deep eutectic solvent is 1:(5-20) g / mL; The filtration is further followed by concentration treatment and / or drying treatment.

5. The preparation method according to claim 3, characterized in that, The extraction treatment temperature is 40-70℃, and the time is 2-5 h; The microwave treatment power is 300-500 W, and the ultrasonic treatment power is 50-300 W.

6. The method of claim 1, wherein, The inoculation volume of the Phellinus igniarius mycelium seed liquid is 5-15% of the volume of the sterile fermentation medium; The solid-liquid separation comprises centrifugation at 10000-15000 rpm for 30-60 min; The membrane filtration of the clear liquid is 0.10-0.22 μm organic membrane; The clear liquid is further subjected to concentration treatment and / or drying treatment after the membrane filtration and sterilization.

7. The Phellinus igniarius mycelium fermentation product prepared by the preparation method according to any one of claims 1-6.

8. Use of the Phellinus igniarius mycelium fermentation product according to claim 7 in the preparation of a product having any one of the following functions or a combination of at least two functions: 1) antioxidant function; 2) moisturizing function; 3) soothing and repairing function.

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