Phellinus igniarius mycelium fermentation product with oil control effect as well as preparation method and application of phellinus igniarius mycelium fermentation product

Abstract

The invention relates to a phellinus igniarius mycelium fermentation product with an oil control effect 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, thereby obtaining the phellinus igniarius mycelium fermentation product. The fermentation culture medium comprises the following components: tamarind fruit extract, a deep-eutectic solvent, inorganic salt and water; the deep-eutectic solvent is a ternary deep-eutectic solvent prepared from a hydrogen bond acceptor, a hydrogen bond donor and polyol; wherein the hydrogen bond receptor is selected from any one or a combination of at least two of betaine, choline chloride, glucose or fructose; the hydrogen bond donor is selected from dicarboxylic acid. According to the invention, the tamarind fruit extract and the specific ternary eutectic solvent are used as culture medium components for co-fermentation to prepare the phellinus igniarius mycelium fermentation product in one step, and the obtained fermentation product has excellent 5alpha-reductase gene expression inhibitory activity and tyrosinase inhibitory activity, and has oil control and whitening potentials.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation product technology, and relates to a fermentation product of Phellinus linteus mycelium with oil-controlling effect, 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, most Sanghuang products rely on Sanghuang fruiting bodies, and there is limited research on the liquid fermentation process of Sanghuang mycelium and its products. Therefore, developing more functional Sanghuang mycelium fermentation products would contribute to the resource development and utilization of Sanghuang. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a fermentation product of Phellinus linteus mycelium with oil-controlling effect, its preparation method and 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 a fermentation product of Phellinus linteus mycelium with oil-controlling effects, 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 tamarind fruit extract, eutectic solvent, inorganic salts, and water;

[0009] The eutectic solvent is a ternary eutectic solvent made from hydrogen bond acceptor, hydrogen bond donor and polyol;

[0010] The hydrogen bond acceptor is selected from any one or a combination of at least two of betaine, choline chloride, glucose, or fructose; the hydrogen bond donor is selected from dicarboxylic acids.

[0011] This invention creatively employs a co-fermentation process using tamarind fruit extract and a specific ternary eutectic solvent as culture medium components to prepare Phellinus linteus mycelium fermentation products in a single step. The tamarind fruit extract and the eutectic solvent jointly provide the nutrients required for Phellinus linteus fermentation, while the specific ternary eutectic solvent regulates metabolism. The fermentation products obtained using the above-mentioned culture medium combine the nutrients and effects of tamarind fruit extract, the eutectic solvent, and Phellinus linteus. The resulting Phellinus linteus mycelium fermentation products exhibit significantly increased levels of active ingredients, excellent 5α-reductase gene expression inhibition activity and tyrosinase inhibition activity, and possess potential for oil control and whitening, thus contributing to the resource development and utilization of Phellinus linteus.

[0012] The eutectic solvent in the fermentation medium used in this invention is preferably a ternary eutectic solvent prepared from a hydrogen bond acceptor, a hydrogen bond donor, and a polyol. When the hydrogen bond acceptor is selected from any one or at least two of betaine, choline chloride, glucose, or fructose, and the hydrogen bond donor is selected from dicarboxylic acids, the fermentation product obtained has superior 5α-reductase gene expression inhibition activity and tyrosinase inhibition activity.

[0013] Preferably, the mass ratio of the hydrogen bond acceptor, hydrogen bond donor and polyol is (8-12):(8-12):(10-15); wherein the specific point values ​​in (8-12) can be 8, 9, 10, 11, 12, etc., and the specific point values ​​in (10-15) can be 10, 11, 12, 13, 14, 15, etc.

[0014] Preferably, the ternary eutectic solvent is obtained by reacting hydrogen bond acceptors and hydrogen bond donors with polyols at 50-90°C (e.g., 50°C, 55°C, 60°C, 65°C, 70°C, 80°C, 90°C, etc.).

[0015] Preferably, the polyol is selected from any one or a combination of at least two of ethylene glycol, glycerol, propylene glycol, butylene glycol, or xylitol.

[0016] Preferably, the fermentation medium comprises, by mass percentage, 5-15% tamarind fruit extract, 1-10% eutectic solvent, 0.1-1% inorganic salts, and 60-95% water.

[0017] The mass percentage of tamarind fruit extract can be 5%, 6%, 8%, 10%, 12%, 13%, 15%, etc.; the mass percentage of eutectic solvent can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, etc.; the mass percentage of inorganic salt can be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, etc.; and the mass percentage of water can be 60%, 65%, 68%, 70%, 75%, 80%, 82%, 85%, 88%, 90%, 95%, etc.

[0018] Preferably, the inorganic salt is selected from sodium salts, potassium salts, magnesium salts, calcium salts, iron salts, manganese salts, sulfates, phosphates, chlorides, etc.

[0019] Preferably, the hydrogen bond donor is selected from tartaric acid.

[0020] Furthermore, compared to other types of dicarboxylic acids, tartaric acid as a hydrogen bond donor exhibits superior 5α-reductase gene expression inhibition activity and tyrosinase inhibition activity.

[0021] The tamarind 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 5α-reductase gene expression inhibition activity and tyrosinase inhibition activity.

[0022] Preferably, the tamarind fruit extract is prepared by a method comprising the following steps:

[0023] Tamarind fruit raw material was mixed with a eutectic solvent and then subjected to ultrasonic-negative pressure cavitation coupling extraction. The extract was centrifuged and filtered to obtain the tamarind fruit extract.

[0024] Preferably, the eutectic solvent is a ternary eutectic solvent prepared from a hydrogen bond acceptor, a hydrogen bond donor, and a polyol.

[0025] Preferably, the hydrogen bond acceptor is selected from any one or a combination of at least two of betaine, choline chloride, glucose or fructose; the hydrogen bond donor is selected from dicarboxylic acids.

[0026] Preferably, the mass ratio of the hydrogen bond acceptor, hydrogen bond donor and polyol is (8-12):(8-12):(10-15); wherein the specific point values ​​in (8-12) can be 8, 9, 10, 11, 12, etc., and the specific point values ​​in (10-15) can be 10, 11, 12, 13, 14, 15, etc.

[0027] Preferably, the polyol is selected from any one or a combination of at least two of ethylene glycol, glycerol, propylene glycol, butylene glycol, or xylitol.

[0028] Preferably, the hydrogen bond donor is selected from tartaric acid, and the hydrogen bond acceptor is selected from betaine.

[0029] Preferably, the mixing ratio of the tamarind fruit raw material to the eutectic solvent is 1:(8-15) g / mL, such as 1:8 g / mL, 1:9 g / mL, 1:10 g / mL, 1:11 g / mL, 1:12 g / mL, 1:13 g / mL, 1:15 g / mL, etc.

[0030] Preferably, the filtration process is followed by concentration and / or drying.

[0031] Preferably, the extraction process is carried out at a temperature of 40-60℃ (e.g., 40℃, 45℃, 50℃, 55℃, 60℃, etc.) and for a time of 30-70 min (e.g., 30 min, 40 min, 50 min, 60 min, 70 min, etc.).

[0032] Preferably, the pressure of the negative pressure cavitation is -0.03 to -0.10 MPa, such as -0.03 MPa, -0.04 MPa, -0.05 MPa, -0.06 MPa, -0.07 MPa, -0.08 MPa, -0.09 MPa, -0.10 MPa, etc.

[0033] Preferably, the power of the ultrasonic wave is 100-400 W (e.g., 100 W, 150 W, 200 W, 250 W, 300 W, 350 W, 400 W, etc.).

[0034] 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.

[0035] The seed liquid of Phellinus linteus mycelium can be obtained using existing techniques in this field.

[0036] For example, it can be obtained using the following process:

[0037] 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.

[0038] 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.

[0039] Preferably, the culture temperature is 20-30℃ (e.g., 20℃, 22℃, 24℃, 25℃, 26℃, 28℃, 30℃, etc.), and the culture time is 3-8 days (e.g., 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, etc.).

[0040] 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 0.05-0.25 MPa (e.g., 0.05 MPa, 0.1 MPa, 0.15 MPa, 0.2 MPa, 0.25 MPa, etc.) and stirring.

[0041] 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.).

[0042] Preferably, the clear liquid is passed through a 0.10-0.22 μm organic membrane.

[0043] Preferably, the clarified liquid is further concentrated and / or dried after membrane filtration and sterilization.

[0044] 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.

[0045] 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.

[0046] 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 whitening effects.

[0047] Fourthly, the present invention provides the use of the fermentation product of Phellinus linteus mycelium according to the second aspect in the preparation of tyrosinase inhibitors.

[0048] Fifthly, 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 oil-controlling effects.

[0049] 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 5α-reductase gene expression inhibitor.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] This invention creatively employs a co-fermentation process using tamarind fruit extract and a specific ternary eutectic solvent as culture medium components to prepare Phellinus linteus mycelium fermentation products in a single step. The tamarind fruit extract and the eutectic solvent jointly provide the nutrients required for Phellinus linteus fermentation, while the specific ternary eutectic solvent regulates metabolism. The fermentation products obtained using the above-mentioned culture medium combine the nutrients and effects of tamarind fruit extract, the eutectic solvent, and Phellinus linteus. The resulting Phellinus linteus mycelium fermentation products exhibit significantly increased levels of active ingredients, excellent 5α-reductase gene expression inhibition activity and tyrosinase inhibition activity, and possess potential for oil control and whitening, thus contributing to the resource development and utilization of Phellinus linteus. Detailed Implementation

[0052] 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.

[0053] Preparation Example 1-1

[0054] This preparation example provides a eutectic solvent, and the preparation method is as follows:

[0055] Betaine and tartaric acid are mixed and reacted at 60°C until the system becomes a clear and transparent liquid. Then, it is mixed with glycerol for 30 minutes to obtain the final product. The mass ratio of betaine to tartaric acid to glycerol is 8:12:15.

[0056] Preparation Examples 1-2

[0057] This preparation example provides a eutectic solvent, and the preparation method is as follows:

[0058] Choline chloride and tartaric acid are mixed and reacted at 60°C until the system becomes a clear and transparent liquid. Then, it is mixed with propylene glycol for 30 min to obtain the final product. The mass ratio of choline chloride to tartaric acid to propylene glycol is 8:10:13.

[0059] Preparation Examples 1-3

[0060] This preparation example provides a eutectic solvent, and the preparation method is as follows:

[0061] Glucose and tartaric acid are mixed and reacted at 60°C until the system becomes a clear and transparent liquid. Then, it is mixed with ethylene glycol for 30 min to obtain the final product. The mass ratio of glucose to tartaric acid to ethylene glycol is 8:8:12.

[0062] Preparation Examples 1-4

[0063] This preparation example provides a eutectic solvent, and the preparation method is as follows:

[0064] Fructose and tartaric acid are mixed and reacted at 60°C until the system becomes a clear and transparent liquid. Then, it is mixed with butanediol for 30 min to obtain the final product. The mass ratio of fructose to tartaric acid to butanediol is 10:10:14.

[0065] Preparation Examples 1-5

[0066] This preparation example provides a eutectic solvent. The preparation method differs from that of Preparation Example 1-1 only in that tartaric acid is replaced by succinic acid in equal mass, while other conditions remain unchanged.

[0067] Preparation Examples 1-6

[0068] This preparation example provides a eutectic solvent. The preparation method differs from that of Preparation Example 1-1 only in that tartaric acid is replaced by lactic acid in equal mass, while other conditions remain unchanged.

[0069] Preparation Examples 1-7

[0070] This preparation example provides a eutectic solvent. The difference between the preparation method and that of Preparation Example 1-1 is that the ternary eutectic solvent is replaced with a binary eutectic solvent: betaine and tartaric acid are mixed in a mass ratio of 2:3 and reacted at 60°C until the system becomes a clear and transparent liquid.

[0071] Preparation Examples 1-8

[0072] This preparation example provides a eutectic solvent. The difference between the preparation method and that of Preparation Example 1-1 is that the ternary eutectic solvent is replaced with a binary eutectic solvent: betaine and glycerol are mixed in a mass ratio of 1:3 and reacted at 60°C until the system is a clear and transparent liquid.

[0073] Preparation Example 2-1

[0074] This preparation example provides a tamarind fruit extract, prepared by the following method:

[0075] After crushing the tamarind fruit, it was mixed with the eutectic solvent prepared in Example 1-1 at a ratio of 1:10 g / mL, and subjected to ultrasonic-negative pressure cavitation coupled extraction to obtain the extract. The treatment temperature was 50℃, the time was 50 min, the negative pressure cavitation pressure was -0.05 MPa, and the ultrasonic power was 250 W. The extract was centrifuged, filtered, and the filtrate was concentrated under reduced pressure and freeze-dried to obtain the tamarind fruit extract.

[0076] Preparation Example 2-2

[0077] This preparation example provides a tamarind fruit extract, prepared by the following method:

[0078] After crushing the tamarind fruit, it was mixed with the eutectic solvent prepared in Preparation Examples 1-2 at a ratio of 1:8 g / mL, and subjected to ultrasonic-negative pressure cavitation coupled extraction to obtain the extract. The treatment temperature was 40℃, the time was 70 min, the negative pressure cavitation pressure was -0.03 MPa, and the ultrasonic power was 400 W. The extract was centrifuged, filtered, and the filtrate was concentrated under reduced pressure and freeze-dried to obtain the tamarind fruit extract.

[0079] Preparation Examples 2-3

[0080] This preparation example provides a tamarind fruit extract, prepared by the following method:

[0081] After crushing the tamarind fruit, it was mixed with the eutectic solvent prepared in Examples 1-3 at a ratio of 1:12 g / mL, and subjected to ultrasonic-negative pressure cavitation coupled extraction to obtain the extract. The treatment temperature was 55℃, the time was 40 min, the negative pressure cavitation pressure was -0.07 MPa, and the ultrasonic power was 300 W. The extract was centrifuged, filtered, and the filtrate was concentrated under reduced pressure and freeze-dried to obtain the tamarind fruit extract.

[0082] Preparation Examples 2-4

[0083] This preparation example provides a tamarind fruit extract, prepared by the following method:

[0084] After crushing the tamarind fruit, it was mixed with the eutectic solvent prepared in Examples 1-4 at a ratio of 1:15 g / mL, and subjected to ultrasonic-negative pressure cavitation coupled extraction to obtain the extract. The treatment temperature was 60℃, the time was 30 min, the negative pressure cavitation pressure was -0.10 MPa, and the ultrasonic power was 200 W. The extract was centrifuged, filtered, and the filtrate was concentrated under reduced pressure and freeze-dried to obtain the tamarind fruit extract.

[0085] Preparation Examples 2-5

[0086] This preparation example provides a tamarind fruit 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 1-5, while other conditions remain unchanged.

[0087] Preparation Examples 2-6

[0088] This preparation example provides a tamarind fruit 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 1-6, while other conditions remain unchanged.

[0089] Preparation Examples 2-7

[0090] This preparation example provides a tamarind fruit 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 1-7, while other conditions remain unchanged.

[0091] Preparation Examples 2-8

[0092] This preparation example provides a tamarind fruit 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 1-8, while other conditions remain unchanged.

[0093] Preparation Examples 2-9

[0094] This preparation example provides a tamarind fruit extract, prepared by the following method:

[0095] The tamarind fruit was pulverized and mixed with the eutectic solvent prepared in Example 1-1 at a ratio of 1:10 g / mL. The mixture was then subjected to a hot extraction treatment under ultrasonic assistance to obtain an extract. The treatment temperature was 50℃, the time was 50 min, and the ultrasonic power was 250 W. The extract was centrifuged, filtered, and the filtrate was concentrated under reduced pressure and freeze-dried to obtain the tamarind fruit extract.

[0096] Example 1

[0097] This embodiment provides a fermentation product of Phellinus linteus mycelium, prepared by the following method:

[0098] (1) Preparation of culture medium: 10% of the tamarind extract of Preparation Example 2-1, 5% of the eutectic solvent of Preparation Example 1-1, 0.2% of potassium dihydrogen phosphate, 0.2% of magnesium sulfate, and the remainder water; sterilize the culture medium at 121℃ for 30 min.

[0099] (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 6 days to obtain the first-level seed liquid of Sanghuang mycelium.

[0100] (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 25℃ and 200rpm for 4 days to obtain the secondary seed liquid of Sanghuang mycelium.

[0101] (4) Inoculate the secondary seed liquid of Sanghuang mycelium into the fermenter containing the culture medium of step (1) at an inoculation rate of 10% (v / v). The culture temperature is 25℃, 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. Then, the fermentation liquid is harvested from the tank.

[0102] (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.

[0103] Example 2

[0104] This embodiment provides a fermentation product of Phellinus linteus mycelium, prepared by the following method:

[0105] (1) Preparation of culture medium: 15% of the tamarind extract of Preparation Example 2-2, 3% of the eutectic solvent of Preparation Example 1-2, 0.1% of potassium dihydrogen phosphate, 0.3% of magnesium sulfate, and the remainder water; sterilize the culture medium at 121℃ for 30 min.

[0106] (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 28°C and 200 rpm for 5 days to obtain the first-level seed liquid of Sanghuang mycelium.

[0107] (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 28℃ and 200rpm for 3 days to obtain the secondary seed liquid of Sanghuang mycelium.

[0108] (4) Inoculate the secondary seed liquid of Sanghuang mycelium into the fermenter containing the culture medium of step (1) at an inoculation rate of 10% (v / v). The culture temperature is 28℃, the aeration rate is 80 L, the stirring rate is 100 r / min, the pressure is 0.1 MPa, and the culture is carried out for 7 days. Then, the fermentation liquid is harvested from the fermenter.

[0109] (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.

[0110] Example 3

[0111] This embodiment provides a fermentation product of Phellinus linteus mycelium, prepared by the following method:

[0112] (1) Preparation of culture medium: 8% of the tamarind fruit extract of preparation examples 2-3, 10% of the eutectic solvent of preparation examples 1-3, 0.3% of potassium dihydrogen phosphate, 0.1% of magnesium sulfate, and the remainder water; sterilize the culture medium at 121℃ for 30 min.

[0113] (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 23°C and 200 rpm for 8 days to obtain the first-level seed liquid of Sanghuang mycelium.

[0114] (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 23℃ and 200rpm for 7 days to obtain the secondary seed liquid of Sanghuang mycelium.

[0115] (4) Inoculate the secondary seed liquid of Sanghuang mycelium into the fermenter containing the culture medium of step (1) at an inoculation rate of 10% (v / v). The culture temperature is 23℃, the aeration rate is 40 L, the stirring rate is 100 r / min, the pressure is 0.1 MPa, and the culture is carried out for 8 days. Then, the fermentation liquid is harvested from the fermentation tank.

[0116] (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.

[0117] Examples 4-9

[0118] 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 extract of tamarind fruit from Preparation Example 2-1 is replaced in equal amounts with the extracts of tamarind fruit from Preparation Examples 2-4, 2-5, 2-6, 2-7, 2-8, and 2-9 in the culture medium, while other components remain unchanged.

[0119] Comparative Examples 1-2

[0120] This comparative example provides two fermentation products of Phellinus linteus mycelium. The difference between the preparation method and 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 1-7 and Preparation Example 1-8 in the culture medium, while other components remain unchanged.

[0121] Comparative Example 3

[0122] This comparative example provides a fermentation product of Phellinus linteus mycelium. The preparation method is different from that of Example 1 only in step (1) in which 10% of the tamarind fruit extract of Preparation Example 2-1 is replaced with 4% glucose, 2% yeast powder and 4% peptone in the culture medium, while the other components remain unchanged.

[0123] Comparative Example 4

[0124] 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.

[0125] Test Example 1

[0126] The oil-controlling effects of the fermentation products obtained in Examples 1-9 and Comparative Examples 1-4 were evaluated:

[0127] (1) Preparation of test samples: The fermentation broths obtained in Examples 1-9 and Comparative Examples 1-4 were dried and then prepared with water to prepare test samples with a concentration of 10%.

[0128] (2) Evaluation method:

[0129] (2.1) Wild-type AB strain zebrafish were randomly selected and transferred to 24-well plates, with 12 zebrafish and 2.5 mL of test solution in each well. The plates were incubated at 28 °C for 24 h. The 12 zebrafish from each well were collected into 1.5 mL tubes, the solution was removed, 0.5 mL of RNAlater solution was added, and the plates were frozen for storage.

[0130] (2.2) Discard the RNAlater, wash three times with PBS; discard PBS on ice, add 500 μL TRIzol reagent. Homogenize the embryos with the granules, incubate on ice for 10 min; add 100 μL chloroform, vortex for 1 min, incubate on ice for 5 min. Centrifuge for 20 min, transfer the supernatant to a 1.5 mL centrifuge tube, add 250 μL isopropanol, vortex for 1 min, incubate on ice for 10 min, then centrifuge for 20 min. Discard the supernatant, add 500 μL ethanol, vortex for 1 min, centrifuge for 5 min, incubate on ice for 10 min, repeat three times. Discard the ethanol, centrifuge for 5 min, air dry in a fume hood for 10 min; add 10 μL enzyme-free water, heat at 55℃ for 15 min.

[0131] (2.3) Dilute RNA with enzyme-free water to 1000 ng / 7 μL; reverse transcribe cDNA using the Takara PrimerScriptRT kit containing gDNA Eraser and store frozen.

[0132] (2.4) Prepare primer mixture and real-time PCR master mixture; dilute cDNA 10 times with enzyme-free water; add 9 µL of PCR master mixture + 1 µL of diluted cDNA to each well of a 96-well plate; seal the plate with optical gel film, centrifuge for 5 min, and perform real-time PCR amplification.

[0133] (2.5) Collect real-time PCR data. Ct is used as the amplification result, and the amplification amount of β-actin gene is used as the housekeeping gene. The relative expression levels of 5α-reductase genes (srd5a1, srd5a2a and srd5a2b) are calculated as the test results.

[0134]

[0135] In the formula: T is the relative expression level of 5α-reductase gene in zebrafish in the test group; C is the relative expression level of 5α-reductase gene in zebrafish in the blank control group.

[0136] The results for each group are shown in Table 1.

[0137] Table 1

[0138]

[0139] As shown in Table 1, compared with the fermentation products obtained in Comparative Examples 1-4, the fermentation product of *Sanghuang* mycelium prepared by the method involved in this invention, namely, the co-fermentation method using *Tamarindus edulis* extract and a specific ternary eutectic solvent as a culture medium component, exhibits significantly superior 5α-reductase gene expression inhibition activity, i.e., it has better oil control potential. Furthermore, comparing the data from Examples 1 and 4-9 shows that the preparation process of *Tamarindus edulis* extract in the fermentation medium also affects the above-mentioned efficacy to some extent.

[0140] Test Example 2

[0141] The skin-whitening effects of the fermentation products obtained in Examples 1-9 and Comparative Examples 1-4 were evaluated:

[0142] (1) Preparation of test samples: The fermentation broths obtained in Examples 1-9 and Comparative Examples 1-4 were dried and dissolved in dimethyl sulfoxide (DMSO), and diluted to 800 µg / mL with 0.05M phosphate buffer (pH=6.8); ready for use.

[0143] (2) Evaluation method:

[0144] Mix 60 µL of sample with 30 µL of tyrosinase solution (100 U / mL, phosphate buffer) and incubate at 37°C for 10 minutes. Then add 120 µL of L-DOPA (1 mg / mL) and incubate at room temperature for 5 minutes. The blank control group was prepared using 30 µL of buffer instead of the tyrosinase solution. The control sample group used DMSO instead of the analyte. In the blank control group, 30 µL of buffer was used instead of the enzyme solution. Absorbance was measured at 475 nm. The experiment was repeated three times. The tyrosinase inhibition rate was calculated using the following formula:

[0145]

[0146] In the formula: As is the absorbance of the sample, Abs is the absorbance of the blank group of the sample, Ac is the absorbance of the control sample group, and Abc is the absorbance of the blank group of the control sample.

[0147] The results for each group are shown in Table 2.

[0148] Table 2

[0149]

[0150] As shown in Table 2, compared with the fermentation products obtained in Comparative Examples 1-4, the fermentation product of *Sanghuang* mycelium prepared by the method involved in this invention, namely, the co-fermentation method using *Tamarindus edulis* extract and a specific ternary eutectic solvent as a culture medium component, exhibits significantly superior tyrosinase inhibitory activity, indicating better whitening potential. Furthermore, comparing the data from Examples 1 and 4-9 shows that the preparation process of the *Tamarindus edulis* extract in the fermentation medium also affects the aforementioned efficacy to some extent.

[0151] Test Example 3

[0152] The cell safety of the fermentation products obtained in Examples 1-9 was evaluated:

[0153] (1) Preparation of test samples: The fermentation broth obtained in Examples 1-9 was dried and then prepared into a 10% concentration test sample using culture medium.

[0154] (2) Evaluation method:

[0155] (2.1) The human melanocyte suspension was seeded into a 96-well plate at 100 μL per well and incubated for 24 h;

[0156] (2.2) Discard the culture medium and add 100 μL of the test sample solution (experimental group) or culture medium (control group) to each well.

[0157] (2.3) After culturing for 24 h, add 10 μL of MTT solution to each well and incubate in an incubator for 4 h;

[0158] (2.4) Discard the culture medium, add 150 μL of DMSO to each well, shake for 10 min, and then measure the absorbance (OD) at 490 nm. 490nm The results were compared with those of the control sample that was not treated with fermentation products and the results were calculated.

[0159]

[0160] Where: Test OD 490 nm The average OD of the experimental group 490nm ;Neg OD 490nm The average OD of the control group 490 nm .

[0161] The results for each group are shown in Table 3.

[0162] Table 3

[0163]

[0164] As shown in Table 3, the fermentation products of Phellinus linteus mycelium prepared by the preparation method of this invention have high cell safety.

[0165] 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.

[0166] 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.

[0167] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable way 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 a fermentation product of Phellinus linteus mycelium with oil-controlling effects, 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 tamarind fruit extract, eutectic solvent, inorganic salts, and water; The eutectic solvent is a ternary eutectic solvent made from hydrogen bond acceptor, hydrogen bond donor and polyol; The hydrogen bond acceptor is selected from any one or a combination of at least two of betaine, choline chloride, glucose, or fructose; the hydrogen bond donor is selected from dicarboxylic acids.

2. The preparation method according to claim 1, characterized in that, The mass ratio of the hydrogen bond acceptor, hydrogen bond donor and polyol is (8-12):(8-12):(10-15); The polyol is selected from any one or a combination of at least two of ethylene glycol, glycerol, propylene glycol, butylene glycol, or xylitol.

3. The preparation method according to claim 1, characterized in that, The fermentation medium comprises, by mass percentage, 5-15% tamarind fruit extract, 1-10% eutectic solvent, 0.1-1% inorganic salts, and 60-95% water.

4. The preparation method according to claim 1, characterized in that, The hydrogen bond donor is selected from tartaric acid.

5. The preparation method according to claim 1, characterized in that, The tamarind fruit extract is prepared by a method comprising the following steps: Tamarind fruit raw material was mixed with a eutectic solvent and then subjected to ultrasonic-negative pressure cavitation coupling extraction. The extract was centrifuged and filtered to obtain the tamarind fruit extract. The eutectic solvent is a ternary eutectic solvent made from hydrogen bond acceptor, hydrogen bond donor and polyol; The hydrogen bond acceptor is selected from any one or a combination of at least two of betaine, choline chloride, glucose, or fructose; the hydrogen bond donor is selected from dicarboxylic acids.

6. The preparation method according to claim 5, characterized in that, The mass ratio of the hydrogen bond acceptor, hydrogen bond donor and polyol is (8-12):(8-12):(10-15); The polyol is selected from any one or a combination of at least two of ethylene glycol, glycerol, propylene glycol, butylene glycol, or xylitol; The hydrogen bond donor is selected from tartaric acid, and the hydrogen bond acceptor is selected from betaine; The mixing ratio of the tamarind fruit raw material to the eutectic solvent is 1:(8-15) 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 40-60℃ for 30-70 minutes. The pressure of the negative pressure cavitation is -0.03 to -0.10 MPa; The power of the ultrasonic wave is 100-400 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 3-8 days; The culture was carried out under conditions of aeration rate of 40-80 L, pressure of 0.05-0.25 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 products with oil-controlling effects.

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