Phellinus igniarius mycelium fermentation product as well as preparation method and application thereof

Abstract

The invention relates to a phellinus igniarius mycelium fermentation product and a preparation method and application thereof, and the preparation method comprises the following steps: inoculating a phellinus igniarius mycelium seed solution into a sterile fermentation culture medium for culturing, carrying out solid-liquid separation, filtering a clear liquid through a membrane and sterilizing to obtain the phellinus igniarius mycelium fermentation product, the fermentation medium comprises the following components: a beet extract, a deep eutectic solvent and water. The beet extract and the deep-eutectic solvent are creatively adopted as culture medium components to prepare the phellinus igniarius mycelium fermentation product, the beet extract and the deep-eutectic solvent jointly provide nutrients needed by phellinus igniarius fermentation, meanwhile, the deep-eutectic solvent is used for regulating metabolism, and compared with a product obtained through fermentation of a traditional culture medium, the phellinus igniarius mycelium fermentation product has the advantages that the fermentation efficiency is high; the content of active ingredients in a fermentation product obtained by adopting the culture medium with the formula is remarkably increased, and the fermentation product is good in uniformity and high in stability, is a clear liquid in appearance, has no abnormal odor and has slight phellinus igniarius characteristic odor.

Description

Technical Field

[0001] This invention belongs to the field of strain fermentation product technology, and relates to a fermentation product of Phellinus linteus mycelium, its preparation method and application. Background Technology

[0002] Sanghuang (Latin name Sanghuangporus) is a traditional medicinal fungus, named for its yellowish-brown fruiting body, which typically grows on plants of the genus *Morus*. As a traditional Chinese medicine, it has been recorded in ancient times as being used to treat gonorrhea, spleen deficiency diarrhea, sores, metrorrhagia, and leukorrhea. In recent years, its effects on anti-tumor and immune regulation have been gradually discovered. Wild Sanghuang fruiting bodies are scarce, and over-exploitation has led to a sharp decline in resources. Artificially cultivated Sanghuang has a long growth cycle, which cannot meet the needs of large-scale production. In contrast, liquid fermentation of Sanghuang mycelium offers advantages such as sterility, controllable production conditions, rapid production of large quantities of cell products and metabolites, automation, and large-scale production, making it suitable for large-scale industrial cultivation of Sanghuang-related products.

[0003] Currently, there is limited research on liquid fermentation products of Phellinus linteus mycelium using existing technologies. Therefore, functional Phellinus linteus mycelium fermentation products are a worthwhile area of ​​exploration, which would contribute to the resource development and utilization of Phellinus linteus. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a fermentation product of Phellinus linteus mycelium, its preparation method, and its application, particularly a fermentation product of Phellinus linteus mycelium with antioxidant, moisturizing, and soothing repair effects, its preparation method, and its application.

[0005] To achieve this objective, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a method for preparing fermentation products of Sanghuangporus mycelium, the preparation method comprising the following steps:

[0007] The seed liquid of Phellinus linteus mycelium was inoculated into a sterile fermentation medium for culture, and the solid-liquid separation, the clear liquid was filtered through a membrane and sterilized to obtain the fermentation product of Phellinus linteus mycelium.

[0008] The fermentation medium comprises beet extract, eutectic solvent, and water.

[0009] This invention creatively employs beet extract and eutectic solvent as culture medium components for co-extraction and fermentation in a one-step process to prepare Phellinus linteus mycelium fermentation products. The beet extract and eutectic solvent together provide the nutrients required for Phellinus linteus fermentation, while the eutectic solvent is used to regulate metabolism. Compared with products obtained by fermentation using traditional culture media, the fermentation products obtained using the above-formulated culture medium have significantly increased content of active ingredients, good uniformity and high stability, and are clear liquids with no abnormal odor and a slight characteristic odor of Phellinus linteus.

[0010] This invention combines beet extract, eutectic solvent and Phellinus linteus using biotechnology, integrating the nutrients and effects of the three. The resulting Phellinus linteus mycelium fermentation product has excellent antioxidant effects and significantly enhances the expression of AQP3 and FLG genes in cells. It can efficiently promote the expression of hydration protein and barrier protein genes, achieving efficient skin moisturizing and soothing repair, making the application of Phellinus linteus promising.

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

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

[0013] Preferably, the fermentation medium further comprises 0.1-1% inorganic salt 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 salts, potassium salts, magnesium salts, calcium salts, iron salts, manganese salts, sulfates, phosphates, chlorides, etc.

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

[0016] The eutectic solvent in the fermentation medium used in this invention is preferably prepared by reacting choline chloride and / or betaine with a polyol. When the hydrogen bond acceptor is selected from betaine and the hydrogen bond donor is selected from glycerol, the fermentation product obtained has better antioxidant, moisturizing and soothing repair effects.

[0017] Preferably, the hydrogen bond acceptor is selected from betaine, and the hydrogen bond donor is selected from 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 used in this invention can be obtained using existing extraction techniques, but is more preferably obtained using the following specific preparation process, resulting in fermentation products with superior antioxidant, moisturizing, and soothing / repairing effects.

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

[0021] The beet raw material was mixed with a eutectic solvent and then subjected to microwave-ultrasonic extraction. The extract was 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 (e.g., 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; the hydrogen bond donor is selected from any one or a combination of at least two of glycerol, propylene glycol, butylene glycol, xylitol or sorbitol.

[0023] Preferably, the mixing ratio of the beet raw material and the eutectic solvent is 1:(5-20) g / mL, for example, 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 process is carried out at a temperature of 40-70℃ (e.g., 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, etc.) for a time of 2-5 h (e.g., 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, etc.).

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

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

[0027] Preferably, the inoculation volume of the Phellinus linteus mycelium seed liquid is 5-15% of the volume of the sterile fermentation medium, such as 5%, 6%, 7%, 8%, 10%, 11%, 12%, 13%, 14%, 15%, etc.

[0028] The prepared *Sanghuang* mycelium seed solution can be obtained using existing techniques in this field.

[0029] For example, it can be obtained using the following process:

[0030] Under aseptic conditions, take bacterial blocks from the slant and inoculate them into the culture medium, then culture them at 20-30℃ and 100-200 rpm for 3-10 days; or inoculate the cultured seed liquid into the culture medium at an inoculation rate of 5-15%, and culture it at 20-30℃ and 100-200 rpm for 3-10 days.

[0031] The culture medium used in preparing the seed culture can be a conventional seed culture medium (e.g., beef extract peptone medium) or the same formulation as the aforementioned fermentation medium: comprising, by weight percentage, 5-20% beet extract, 1-10% eutectic solvent, 65-94% water, and optionally 1-10% inorganic salts.

[0032] Preferably, the culture temperature is 20-30℃ (e.g., 20℃, 22℃, 24℃, 25℃, 26℃, 28℃, 30℃, etc.), and the culture time is 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 conditions 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 includes centrifugation at 10,000-15,000 rpm (e.g., 10,000 rpm, 11,000 rpm, 12,000 rpm, 13,000 rpm, 14,000 rpm, 15,000 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 clarified liquid is passed through a 0.10-0.22 μm organic membrane.

[0036] Preferably, the clarified liquid is further concentrated and / or dried after membrane filtration and sterilization.

[0037] All other specific point values ​​not listed above within the numerical ranges mentioned above can be selected and are all within the protection scope of this invention. For the sake of brevity, they will not be described in detail here.

[0038] In a second aspect, the present invention provides a fermentation product of Phellinus linteus mycelium prepared according to the preparation method described in the first aspect.

[0039] Thirdly, the present invention provides the application of the fermentation product of Phellinus linteus mycelium according to the second aspect in the preparation of products with antioxidant effects.

[0040] Fourthly, the present invention provides the application of the fermentation product of Phellinus linteus mycelium according to the second aspect in the preparation of products with free radical scavenging effects.

[0041] Fifthly, the present invention provides the use of the fermentation product of Phellinus linteus mycelium according to the second aspect in the preparation of a product with moisturizing effect.

[0042] In a sixth aspect, the present invention provides the use of the fermentation product of Phellinus linteus mycelium according to the second aspect in the preparation of a product that enhances the expression level of AQP3 in human keratinocytes.

[0043] In a seventh aspect, the present invention provides the use of the fermentation product of Phellinus linteus mycelium according to the second aspect in the preparation of products with soothing and repairing effects.

[0044] Eighthly, the present invention provides the use of the fermentation product of Phellinus linteus mycelium according to the second aspect in the preparation of a product that enhances the expression level of FLG in human keratinocytes.

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

[0046] This invention creatively uses beet extract and eutectic solvent as culture medium components to prepare fermentation products of Phellinus linteus mycelium. The beet extract and eutectic solvent together provide the nutrients required for Phellinus linteus fermentation, while the eutectic solvent is used to regulate metabolism. Compared with products obtained by fermentation in traditional culture media, the fermentation products obtained by using the above-formulated culture medium have significantly increased content of active ingredients, and the fermentation products have good uniformity and high stability. The appearance is a clear liquid with no abnormal odor and a slight characteristic odor of Phellinus linteus.

[0047] This invention combines beet extract, eutectic solvent and Phellinus linteus using biotechnology, integrating the nutrients and effects of the three. The resulting Phellinus linteus mycelium fermentation product has excellent antioxidant effects and significantly enhances the expression of AQP3 and FLG genes in cells. It can efficiently promote the expression of hydration protein and barrier protein genes, achieving efficient skin moisturizing and soothing repair, making the application of Phellinus linteus promising. Detailed Implementation

[0048] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0049] Preparation Example 1-1

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

[0051] Betaine and glycerol are mixed in a molar ratio of 1:6 and reacted at 60°C until the system becomes a clear and transparent liquid.

[0052] Preparation Examples 1-2

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

[0054] Choline chloride and glycerol are mixed in a molar ratio of 1:6 and reacted at 60°C until the system becomes a clear and transparent liquid.

[0055] Preparation Examples 1-3

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

[0057] Betaine and propylene glycol are mixed in a molar ratio of 1:6 and reacted at 60°C until the system becomes a clear and transparent liquid.

[0058] Preparation Examples 1-4

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

[0060] Betaine and sorbitol are mixed in a molar ratio of 1:6 and reacted at 60°C until the system becomes a clear and transparent liquid.

[0061] Preparation Example 2-1

[0062] This preparation example provides a beet extract, prepared by the following method:

[0063] After crushing the beet, it was mixed with the eutectic solvent prepared in 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 was 55℃ and the time was 4 h. The power of the microwave treatment was 400 W and the power of the ultrasonic treatment was 150 W. The extract was centrifuged, filtered, and the filtrate was concentrated under reduced pressure and freeze-dried to obtain beet extract.

[0064] Preparation Example 2-2

[0065] This preparation example provides a beet extract, prepared by the following method:

[0066] After crushing the beet, it was mixed with the eutectic solvent prepared in 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 was 65℃ and the time was 3 h. The power of the microwave treatment was 300 W and the power of the ultrasonic treatment was 250 W. The extract was centrifuged, filtered, and the filtrate was concentrated under reduced pressure and freeze-dried to obtain beet extract.

[0067] Preparation Examples 2-3

[0068] This preparation example provides a beet extract, prepared by the following method:

[0069] After crushing the beet, it was mixed with the eutectic solvent prepared in 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 was 45℃ and the time was 4.5 h. The power of the microwave treatment was 500 W and the power of the ultrasonic treatment was 100 W. The extract was centrifuged, filtered, and the filtrate was concentrated under reduced pressure and freeze-dried to obtain beet extract.

[0070] Preparation Examples 2-4

[0071] This preparation example provides a beet extract. The preparation method differs from that of Preparation Example 2-1 only in that the eutectic solvent obtained in Preparation Example 1-1 is replaced by an equal volume of the eutectic solvent obtained in Preparation Example 1-2, while other conditions remain unchanged.

[0072] Preparation Examples 2-5

[0073] This preparation example provides a beet extract. The preparation method differs from that of Preparation Example 2-1 only in that the eutectic solvent obtained in Preparation Example 1-1 is replaced by an equal volume of the eutectic solvent obtained in Preparation Example 1-3, while other conditions remain unchanged.

[0074] Preparation Examples 2-6

[0075] This preparation example provides a beet extract, prepared by the following method:

[0076] After crushing the beet, it was mixed with the eutectic solvent prepared in Example 1-1 at a ratio of 1:10 g / mL, and microwaved to obtain an extract. The treatment temperature was 55℃, the time was 4.5 h, and the microwave power was 500 W. The extract was centrifuged, filtered, and the filtrate was concentrated under reduced pressure and freeze-dried to obtain beet extract.

[0077] Preparation Examples 2-7

[0078] This preparation example provides a beet extract, prepared by the following method:

[0079] After crushing the beet, it was mixed with the eutectic solvent prepared in Example 1-1 at a ratio of 1:10 g / mL, and the mixture was subjected to ultrasonic treatment to obtain an extract. The treatment temperature was 55℃ and the time was 4.5 h. The ultrasonic power was 250 W. The extract was centrifuged, filtered, and the filtrate was concentrated under reduced pressure and freeze-dried to obtain beet extract.

[0080] Preparation Examples 2-8

[0081] This preparation example provides a beet extract, prepared by the following method:

[0082] After crushing the beets, they were mixed with water at a ratio of 1:10 g / mL and subjected to microwave-ultrasound combined treatment to obtain an extract. The treatment temperature was 55℃ and the time was 4 h. The power of the microwave treatment was 400 W and the power of the ultrasonic treatment was 150 W. The extract was centrifuged, filtered, and the filtrate was concentrated under reduced pressure and freeze-dried to obtain the beet extract.

[0083] Preparation Examples 2-9

[0084] This preparation example provides a beet extract, prepared by the following method:

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

[0086] Example 1

[0087] This embodiment provides a fermentation product of Phellinus linteus mycelium, prepared by the following method:

[0088] (1) Preparation of culture medium: 10% beet extract of preparation example 2-1, 5% eutectic solvent of preparation example 1-1, 0.2% potassium dihydrogen phosphate, 0.2% magnesium sulfate, and the remainder water; sterilize the culture medium at 121℃ for 30 min.

[0089] (2) Under aseptic conditions, take the mycelium block from the slant and inoculate it into a shake flask containing the culture medium of step (1). Shake the flask at 27°C and 200 rpm for 7 days to obtain the first-level seed liquid of Sanghuang mycelium.

[0090] (3) Inoculate the primary seed liquid of Sanghuang mycelium into a shake flask containing the culture medium of step (1) at an inoculation amount of 10% (v / v), and culture at 27℃ and 200rpm for 5 days to obtain the secondary seed liquid of Sanghuang mycelium.

[0091] (4) Inoculate the secondary seed liquid of Phellinus linteus mycelium into the fermenter containing the culture medium of step (1) at an inoculation rate of 10% (v / v). The culture temperature is 27℃, the aeration rate is 60 L, the stirring rate is 100 r / min, the pressure is 0.1 MPa, and the culture is carried out for 7 days. The fermentation liquid is then harvested from the fermenter.

[0092] (5) The harvested fermentation broth was centrifuged at 12000 r / min for 30 min, the supernatant was passed through a 0.22 μm organic membrane, and sterilized at 121℃ for 30 min to obtain the fermentation broth of Sanghuang mycelium.

[0093] Example 2

[0094] This embodiment provides a fermentation product of Phellinus linteus mycelium, prepared by the following method:

[0095] (1) Preparation of culture medium: 15% beet extract of preparation example 2-2, 5% eutectic solvent of preparation example 1-1, 0.3% potassium dihydrogen phosphate, 0.2% magnesium sulfate, and the remainder water; sterilize the culture medium at 121℃ for 30 min.

[0096] (2) Under aseptic conditions, take the mycelium block from the slant and inoculate it into a shake flask containing the culture medium of step (1). Shake the flask at 25°C and 200 rpm for 7 days to obtain the first-level seed liquid of Sanghuang mycelium.

[0097] (3) At an inoculation rate of 5% (v / v), the primary seed liquid of Sanghuang mycelium was inoculated into a shake flask containing the culture medium of step (1), and cultured at 25℃ and 200rpm for 8 days to obtain the secondary seed liquid of Sanghuang mycelium.

[0098] (4) Inoculate the secondary seed liquid of Phellinus linteus mycelium into the fermenter containing the culture medium of step (1) at an inoculation rate of 5% (v / v). The culture temperature is 25℃, the aeration rate is 80 L, the stirring rate is 150 r / min, the pressure is 0.1 MPa, and the culture is carried out for 8 days. The fermentation liquid is then harvested from the fermenter.

[0099] (5) The harvested fermentation broth was centrifuged at 15000 r / min for 30 min, the supernatant was passed through a 0.22 μm organic membrane, and sterilized at 121℃ for 30 min to obtain the fermentation broth of Sanghuang mycelium.

[0100] Example 3

[0101] This embodiment provides a fermentation product of Phellinus linteus mycelium, prepared by the following method:

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

[0103] (2) Under aseptic conditions, take the mycelium block from the slant and inoculate it into a shake flask containing the culture medium of step (1). Shake the flask at 30°C and 100 rpm for 7 days to obtain the first-level seed liquid of Sanghuang mycelium.

[0104] (3) At an inoculation rate of 15% (v / v), the primary seed liquid of Sanghuang mycelium was inoculated into a shake flask containing the culture medium of step (1), and cultured at 30℃ and 100rpm for 4 days to obtain the secondary seed liquid of Sanghuang mycelium.

[0105] (4) Inoculate the secondary seed liquid of Sanghuang mycelium into the fermenter containing the culture medium of step (1) at an inoculation rate of 15% (v / v). The culture temperature is 30℃, the aeration rate is 40 L, the stirring rate is 150 r / min, the pressure is 0.1 MPa, and the culture is carried out for 10 days. The fermentation liquid is then harvested.

[0106] (5) The harvested fermentation broth was centrifuged at 10,000 r / min for 40 min, the supernatant was passed through a 0.22 μm organic membrane, and sterilized at 121℃ for 30 min to obtain the fermentation broth of Sanghuang mycelium.

[0107] Examples 4-9

[0108] This embodiment provides six fermentation products of Phellinus linteus mycelium. The only difference between the preparation method and that of Example 1 is that in step (1), the beet extract of Preparation Example 2-1 is replaced in equal amounts with the beet extracts 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 in the culture medium, while the other components remain unchanged.

[0109] Examples 10-12

[0110] This embodiment provides three fermentation products of Phellinus linteus mycelium. The difference between the preparation method and that of Example 1 is that in step (1), the eutectic solvent of Preparation Example 1-1 is replaced in equal amounts with the eutectic solvent of Preparation Example 1-2, Preparation Example 1-3 and Preparation Example 1-4 in the culture medium, while other components remain unchanged.

[0111] Comparative Example 1

[0112] This comparative example provides a fermentation product of Phellinus linteus mycelium. The difference between the preparation method and Example 1 is that in step (1), 10% of the beet extract of Example 2-1 is replaced with 4% glucose, 2% yeast powder and 4% peptone in the culture medium, while the other components remain unchanged.

[0113] Comparative Example 2

[0114] This comparative example provides a fermentation product of Phellinus linteus mycelium. The difference between the preparation method and Example 1 is that in step (1), 10% of the beet extract of Example 2-1 is replaced with 1% glucose and 9% potato filtrate in the culture medium, while the other components remain unchanged.

[0115] Comparative Example 3

[0116] This comparative example provides a fermentation product of Phellinus linteus mycelium. The difference between the preparation method and Example 1 is that the eutectic solvent is missing in the culture medium in step (1), and its mass is made up by water, while other components remain unchanged.

[0117] Test Example 1

[0118] The appearance, uniformity, and odor of the fermentation products obtained in Examples 1-12 and Comparative Examples 1-3 were evaluated, as shown in Table 1:

[0119] Table 1

[0120]

[0121] As shown in Table 1, the fermentation filtrate of Phellinus linteus mycelium prepared by the preparation method of the present invention is a pale yellow transparent liquid at 25°C, the system is homogeneous and does not separate into layers, and exhibits the characteristic odor of Phellinus linteus; while the fermentation filtrate of Phellinus linteus mycelium prepared by Comparative Examples 1-2 using other culture media is a brown turbid liquid at 25°C, the system is unstable, has a separation phenomenon, and exhibits an abnormal odor.

[0122] Test Example 2

[0123] The antioxidant effects of the fermentation products obtained in Examples 1-12 and Comparative Examples 1-3 were evaluated:

[0124] (1) Preparation of test samples: The fermentation broths obtained in Examples 1-12 and Comparative Examples 1-3 were dried and then prepared into test samples with a concentration of 10% for later use.

[0125] (2) Evaluation method:

[0126] (2.1) 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.

[0127] (2.2) Sample group: 100 μL of sample solution was mixed with 100 μL of DPPH solution (50.0 μg / mL); 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 mixture was reacted at room temperature in the dark for 0.5 h, and the absorbance was measured at 517 nm. The scavenging rate was calculated.

[0128] (2.3) Formula for calculating DPPH free radical scavenging rate:

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

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

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

[0132] Table 2

[0133]

[0134] As shown in Table 2, compared with the fermentation products obtained in Comparative Examples 1-3, the fermentation product of *Sanghuang* mycelium prepared by the method involved in this invention has excellent DPPH scavenging ability, that is, excellent antioxidant properties. Furthermore, comparing the data from Examples 1 and 4-12, it can be seen that the choice of the type of eutectic solvent in the fermentation medium and the preparation process of the beet extract also affect the above-mentioned efficacy to some extent.

[0135] Test Example 3

[0136] The moisturizing effects of the fermentation products obtained in Examples 1-12 and Comparative Examples 1-3 were evaluated:

[0137] (1) Preparation of test samples: The fermentation broths obtained in Examples 1-12 and Comparative Examples 1-3 were dried and then prepared into test samples with a concentration of 10% for later use.

[0138] (2) Evaluation method:

[0139] (2.1) Cell seeding and culture: HaCaT cells in logarithmic growth phase were collected and cultured at a rate of 2 × 10⁻⁶ cells / year. 5 Inoculate one cell per well into a 12-well plate and incubate overnight in an incubator (37°C, 5% CO2).

[0140] (2.2) Modeling: The complete culture medium in the well was aspirated, PBS solvent was added, and the well was exposed to UVB lamp for 7.5 min.

[0141] (2.3) Grouping and sample administration: The PBS solution was aspirated, the test sample was added to the sample group, and the complete culture medium was added to the model group. The samples were then incubated for 48 h.

[0142] (2.4) Detection and data analysis: Cells were collected and RNA was extracted. After reverse transcription to cDNA, the expression level of AQP3 in each sample was detected by real-time quantitative PCR. The relative expression enhancement rate of AQP3 gene (%) was calculated as follows: (Expression level in sample group - Expression level in model group) / Expression level in model group × 100%.

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

[0144] Table 3

[0145]

[0146] As shown in Table 3, compared with the fermentation products obtained in Comparative Examples 1-3, the fermentation product of *Sanghuang* mycelium prepared by the method of this invention has a superior effect on enhancing the expression level of the AQP3 gene, i.e., it has excellent moisturizing potential. Furthermore, comparing the data from Examples 1 and 4-12, it can be seen that the choice of the eutectic solvent type in the fermentation medium and the preparation process of the beet extract also affect the above-mentioned effects to some extent.

[0147] Test Example 4

[0148] The soothing and repairing effects of the fermentation products obtained in Examples 1-12 and Comparative Examples 1-3 were evaluated:

[0149] (1) Preparation of test samples: The fermentation broths obtained in Examples 1-12 and Comparative Examples 1-3 were dried and then prepared into test samples with a concentration of 10% for later use.

[0150] (2) Evaluation method:

[0151] (2.1) Cell seeding and culture: HaCaT cells in logarithmic growth phase were collected and cultured at a rate of 2 × 10⁻⁶ cells / year. 5 Inoculate one cell per well into a 12-well plate and incubate overnight in an incubator (37°C, 5% CO2).

[0152] (2.2) Modeling: The complete culture medium in the well was aspirated, PBS solvent was added, and the well was exposed to UVB lamp for 7.5 min.

[0153] (2.3) Grouping and sample administration: The PBS solution was aspirated, the test sample was added to the sample group, and the complete culture medium was added to the model group. The samples were then incubated for 48 h.

[0154] (2.4) Detection and data analysis: Cells were collected and RNA was extracted. After reverse transcription to cDNA, the expression level of FLG in each sample was detected by real-time quantitative PCR. The relative expression enhancement rate of FLG gene (%) was calculated as follows: (Expression level of sample group - Expression level of model group) / Expression level of model group × 100%.

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

[0156] Table 4

[0157]

[0158] As shown in Table 4, compared with the fermentation products obtained in Comparative Examples 1-3, the fermentation product of *Sanghuang* mycelium prepared by the method involved in this invention has a superior effect on increasing the expression level of the FLG gene, that is, it has excellent soothing and repair potential. Furthermore, comparing the data from Examples 1 and 4-12, it can be seen that the choice of the eutectic solvent type in the fermentation medium and the preparation process of the beet extract also affect the above-mentioned efficacy to some extent.

[0159] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0160] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0161] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A method for preparing fermentation products of Phellinus linteus mycelium, characterized in that, The preparation method includes the following steps: The seed liquid of Phellinus linteus mycelium was inoculated into a sterile fermentation medium for culture, and the solid-liquid separation, the clear liquid was filtered through a membrane and sterilized to obtain the fermentation product of Phellinus linteus mycelium. The fermentation medium comprises beet extract, eutectic solvent, and water.

2. The preparation method according to claim 1, characterized in that, The fermentation medium comprises, by mass percentage, 5-20% beet extract, 1-10% eutectic solvent, and 65-94% water; The eutectic solvent is obtained by reacting a hydrogen bond acceptor with a hydrogen bond donor at 50-90°C; The hydrogen bond acceptor is selected from choline chloride and / or betaine; the hydrogen bond donor is selected from any one or a combination of at least two of glycerol, propylene glycol, butylene glycol, xylitol or sorbitol.

3. The preparation method according to claim 2, characterized in that, The hydrogen bond acceptor is selected from betaine, and the hydrogen bond donor is selected from glycerol; The molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:(5-7).

4. The preparation method according to claim 1, characterized in that, The fermentation medium also includes 0.1-1% inorganic salts by mass percentage.

5. The preparation method according to claim 1, characterized in that, The beet extract is prepared by a method comprising the following steps: The beet raw material was mixed with a eutectic solvent and then subjected to microwave-ultrasonic extraction. The extract was centrifuged and filtered to obtain the beet extract.

6. The preparation method according to claim 5, characterized in that, The eutectic solvent is obtained by reacting a hydrogen bond acceptor with a hydrogen bond donor at 50-90°C; The hydrogen bond acceptor is selected from choline chloride and / or betaine; the hydrogen bond donor is selected from any one or a combination of at least two of glycerol, propylene glycol, butylene glycol, xylitol or sorbitol; The mixing ratio of the beet raw material to the eutectic solvent is 1:(5-20) g / mL; The filtered material is then further concentrated and / or dried.

7. The preparation method according to claim 5, characterized in that, The extraction process is carried out at a temperature of 40-70℃ for 2-5 hours. The power of the microwave treatment is 300-500 W, and the power of the ultrasonic treatment is 50-300 W.

8. The preparation method according to claim 1, characterized in that, The inoculation volume of the Phellinus linteus mycelium seed solution is 5-15% of the volume of the aseptic fermentation medium; The culture temperature is 20-30℃, and the culture time is 3-10 days; The culture was carried out under conditions of aeration rate of 40-80 L, pressure of 0.05-0.3 MPa, and stirring. The solid-liquid separation includes centrifugation at 10,000-15,000 rpm for 30-60 min; The clarified liquid is passed through a 0.10-0.22 μm organic membrane; The clarified liquid is further concentrated and / or dried after membrane filtration and sterilization.

9. The fermentation product of Phellinus linteus mycelium prepared by any one of claims 1-8.

10. The application of the fermentation product of Phellinus linteus mycelium according to claim 9 in the preparation of a product having any one of the following effects or a combination of at least two of the following effects: 1) Antioxidant effect; 2) Moisturizing effect; 3) Soothing and repairing effect.

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