A cordyceps militaris fermentation lysate, and a preparation method and application thereof

Abstract

This application discloses a cicada flower fermentation lysate, its preparation method, and its application. The preparation method includes the following steps: inoculating cicada flowers into a fermentation substrate, fermenting for 4-8 days, and sterilizing. The fermentation substrate includes edible fungi and water. This application utilizes a specific species of cicada flower to ferment edible fungi, which not only increases the content of active ingredients in the fermentation product but also enhances its cosmetic effects. The fermentation process described in this application increases the content of active ingredients in the obtained cicada flower fermentation lysate, resulting in ideal anti-aging, soothing, moisturizing, and cell energy metabolism-promoting effects. The preparation process is simple, the fermentation conditions are mild, and no organic solvents are used, achieving energy conservation and environmental protection, and demonstrating promising application prospects.

Description

Technical Field

[0001] This application belongs to the field of fermentation technology, and in particular relates to a fermentation lysate of cicada flower, its preparation method and application. Background Technology

[0002] Cicada flower (Cordycep scicadae), also known as cicada nymph grass, refers to the complex formed by the fungus Paecilomyces cicadae (belonging to the genus Cordyceps in the family Clavicipttaceus) parasitizing cicada nymphs. Cicada flower was once considered one of the "Eight Treasures of Traditional Chinese Medicine," ranking alongside Cordyceps sinensis and Cordyceps militaris as one of the three major traditional medicinal Cordyceps in my country. Furthermore, cicada flower shares similar active ingredients with Cordyceps sinensis; its mycelium contains various active components such as fungal polysaccharides, cordycepic acid, cordycepin, and adenosine, possessing health benefits such as immune regulation and anti-tumor effects.

[0003] Currently, solid-state fermentation is commonly used to increase the content of active ingredients in Cordyceps militaris. However, solid-state fermentation still has drawbacks such as long fermentation cycles and low content of active ingredients, which leads to reduced production efficiency and poor application effects. Furthermore, existing research has been reported both domestically and internationally on improving the activity of metabolites by adding appropriate amounts of traditional Chinese medicine ingredients or oily substances to the culture medium. However, there are no reports on the application of Cordyceps militaris in the fermentation of edible fungi.

[0004] Therefore, there is an urgent need for a fermentation method for edible fungi fermented from cicada flowers, which can effectively increase the content of active ingredients, improve their bioavailability and cosmetic effects. Summary of the Invention

[0005] The technical problem this application aims to solve is to overcome the shortcomings of existing technologies, such as long fermentation cycles, difficulty in controlling the fermentation process, and lack of fermentation technology, which limit the application of cicada flowers. This application provides a cicada flower fermentation lysate, its preparation method, and its application. This application utilizes a specific cicada flower strain to ferment edible fungi, which not only increases the content of active ingredients in the fermentation product but also enhances its cosmetic effects. The fermentation process described in this application results in an increased content of active ingredients in the cicada flower fermentation lysate, exhibiting ideal moisturizing, soothing, and cell energy metabolism-promoting effects. The preparation process is simple, the fermentation conditions are mild, and no organic solvents are used, achieving energy conservation and environmental protection.

[0006] This application adopts the following technical solution to solve the above-mentioned technical problems:

[0007] This application provides a method for preparing the fermentation lysate of Cordyceps militaris, which includes the following steps: inoculating Cordyceps militaris into a fermentation substrate, fermenting for 4-8 days, and sterilizing.

[0008] The fermentation substrate includes edible fungi and water.

[0009] In some embodiments, the method for preparing the fermentation substrate includes: mixing the components of the fermentation substrate until all components are completely dissolved.

[0010] In some embodiments, the edible fungi include one or more of morel mushrooms, matsutake mushrooms, silver ear fungus, and red mushrooms.

[0011] In some embodiments, the edible fungus is further subjected to a crushing and sieving process before use.

[0012] The mesh size of the sieve is 100 to 200 mesh.

[0013] In some embodiments, the mass ratio of the edible fungus to the water is 1:(200-500), preferably 0.2.

[0014] In some embodiments, the water may include purified water and / or deionized water.

[0015] In some embodiments, the fermentation substrate may further include a sterilization process before use.

[0016] The sterilization method for the fermentation substrate is generally high-temperature sterilization.

[0017] When the fermentation substrate is sterilized using the high-temperature sterilization method, the sterilization temperature can be a temperature commonly used in this type of operation in the art, preferably 110°C to 125°C, more preferably 110°C to 121°C, for example 121°C.

[0018] When the fermentation substrate is sterilized using the high-temperature sterilization method, the sterilization time can be the conventional time for this type of operation in the art, preferably 20-45 min, more preferably 25-40 min, for example 30 min.

[0019] In some embodiments, the *Cordyceps cicadae* species is deposited at the China General Microbiological Culture Collection Center (CGMCC) on August 9, 2024, with accession number CGMCC No. 41430 and classified as *Cordyceps cicadae*. The depository is located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0020] In some embodiments, the cicada flower is added in the form of cicada flower liquid.

[0021] The preparation method of the cicada flower bacterial solution includes: inoculating cicada flowers into PDA liquid culture medium, then culturing at 28°C and 180 rpm for 5 days, then spreading it onto PDA solid plates, and culturing it in a constant temperature incubator at 28°C for 7 days, then picking a single colony and culturing it in 300 ml of PDA liquid culture medium at 28°C and 180 rpm for 7 days, which is used as the cicada flower bacterial solution for subsequent use.

[0022] The concentration of live bacteria from cicada flowers in the cicada flower mycelium solution was 10. 7 CFU / mL.

[0023] In some embodiments, based on the water in the fermentation substrate, the number of *Cicada nymphs* inoculated per unit mass of the water can be 10. 5 ~10 6 CFU / mL, preferably 10 6 CFU / mL.

[0024] In some embodiments, the fermentation culture temperature may be 25-30°C, preferably 25-28°C, and more preferably 28°C.

[0025] In some embodiments, the fermentation culture time is 5 to 7 hours, preferably 5 days.

[0026] In some embodiments, the fermentation conditions and methods are conventional in the art, generally involving aerobic fermentation in a constant temperature shaking chamber.

[0027] When using the constant temperature shaking chamber for aerobic fermentation, the rotation speed of the constant temperature shaking chamber is 150-200 rpm, preferably 180 rpm.

[0028] In some embodiments, the cell wall disruption is conventional in the art, generally involving high temperature and high pressure disruption.

[0029] The temperature for cell wall breaking is 110–121°C, preferably 110°C.

[0030] The pressure for breaking the cell wall is 0.10–0.15 MPa, more preferably 0.1 MPa.

[0031] In some embodiments, the sterilization method may be a high-temperature sterilization method conventionally used in the art.

[0032] When the high-temperature sterilization method is used for sterilization, the sterilization temperature can be a temperature conventional for this type of operation in the art, preferably 100℃ to 110℃, and more preferably 110℃.

[0033] When the high-temperature sterilization method is used for sterilization, the sterilization time can be the conventional time for this type of operation in the art, preferably 20 to 45 minutes, more preferably 25 to 40 minutes, for example 30 minutes.

[0034] In some embodiments, the sterilization process may further include centrifugation, filtration, and collection of the supernatant.

[0035] The centrifugation speed can be a speed conventional for this type of operation in the art, preferably 3000-7000 rpm, more preferably 3500-6000 rpm, for example 5000 rpm.

[0036] The centrifugation time can be the conventional time for this type of operation in the art, preferably 20 to 45 minutes, more preferably 25 to 40 minutes, for example 30 minutes.

[0037] The filter membrane used for filtration has a thickness of 10–15 μm.

[0038] In some embodiments, the room temperature generally refers to 15–40°C.

[0039] This application also provides a *Cicada nymph* fermentation lysate, which is prepared by the method described above for preparing the *Cicada nymph* fermentation lysate.

[0040] This application also provides the use of the above-mentioned Cordyceps militaris fermentation lysate as a product, as an additive, or as a base in the preparation of topical skin agents.

[0041] In some embodiments, the cicada flower fermentation lysate is used as at least one of the anti-aging active ingredient, moisturizing and water-locking active ingredient, and soothing active ingredient in the topical skin agent;

[0042] The anti-aging active ingredient is either an anti-aging active ingredient that enhances lysosomal activity or a firming anti-aging active ingredient that increases intracellular ELN1 content.

[0043] The moisturizing and water-locking active ingredient is one that can increase the content of HAS in cells.

[0044] The soothing active ingredient is one that reduces the intracellular TRPV1 content.

[0045] This application also provides a topical skin agent comprising the cicada flower fermentation lysate as described above.

[0046] In some embodiments, the topical skin agent may further include active ingredients commonly used in the art, generally including at least one of moisturizing active ingredients, whitening active ingredients, anti-inflammatory active ingredients, anti-allergic active ingredients, and antioxidant active ingredients.

[0047] In some embodiments, the topical skin agent may be, in accordance with the conventions of the art, including but not limited to, face masks, serums, or toners.

[0048] In some embodiments, the cytotoxic product of cicada nymph fermentation accounts for 5% to 99% of the mass percentage of the topical skin agent, preferably 60% to 99%.

[0049] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of this application.

[0050] All reagents and raw materials used in this application are commercially available.

[0051] The positive and progressive effects of this application are as follows: This application uses Cordyceps militaris to ferment edible fungi, which can increase the content of active substances in the fermented lysate of Cordyceps militaris and also improve the cosmetic effects. After the fermentation process of this application, the content of active ingredients in the fermented lysate of Cordyceps militaris is significantly increased, and it has ideal moisturizing, soothing, firming and cell energy metabolism promotion effects. The preparation process is simple, the fermentation conditions are mild, and no organic solvents are used, thus achieving energy saving and environmental protection. Attached Figure Description

[0052] This application can be better understood by referring to the description given below in conjunction with the accompanying drawings. These drawings, together with the detailed description below, are included in and form part of this specification, and are used to further illustrate preferred embodiments of the application and explain its principles and advantages.

[0053] in:

[0054] Figure 1 The above diagram shows the comparison of intracellular lysosomal activity after the products prepared in the examples and comparative examples were treated with cells.

[0055] Figure 2 The image shows a comparison of intracellular HAS content after cells were treated with the products prepared in the examples and comparative examples.

[0056] Figure 3 The diagram shows a comparison of the intracellular TRPV1 content after the products prepared in the examples and comparative examples were treated with cells.

[0057] Figure 4 The image shows a comparison of intracellular ELN content after the products were processed into cells for both the example and comparative examples. Detailed Implementation

[0058] The present application is further illustrated below by way of embodiments, but these embodiments are not intended to limit the scope of the present application. Experimental methods not specifically described in the following embodiments are performed according to conventional methods and conditions, or as selected in accordance with the product instructions.

[0059] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0060] The *Cordyceps cicadae* species used in the following examples and comparative examples are deposited at the China General Microbiological Culture Collection Center (CGMCC) on August 9, 2024, with accession number CGMCC No. 41430. The species is classified as *Cordyceps cicadae*. The depository is located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0061] The red mushrooms in the following examples and comparative examples were purchased from Yinyun Edible Fungus Business Department in Guandu District.

[0062] The morel mushrooms used in the following examples and comparative examples were purchased from Yinyun Edible Fungus Business Department in Guandu District.

[0063] The matsutake mushrooms used in the following examples and comparative examples were purchased from Yinyun Edible Fungus Business Department in Guandu District.

[0064] The white fungus used in the following examples and comparative examples was purchased from Gutian County Gaoting Industry and Trade Co., Ltd.

[0065] The PDA liquid culture medium used in the following examples and comparative examples was purchased from Beijing Bairddi Biotechnology Co., Ltd.

[0066] Example 1

[0067] Crush red mushrooms, pass them through a 200-mesh sieve, and weigh 0.6g of red mushroom powder into 300ml of water. Mix the powder evenly with stirring, then sterilize at 121℃ for 30 minutes. After sterilization, let it stand at room temperature and cool to 25℃ to obtain the fermentation substrate.

[0068] Preparation method of Cordyceps militaris bacterial suspension: Take out the frozen bacterial suspension, thaw it at room temperature, and inoculate 300 μL of the frozen suspension into 300 ml of PDA liquid medium. Then, incubate at 28℃ and 180 rpm for 5 days. Then, spread it on PDA solid plates and incubate in a constant temperature incubator at 28℃ for 7 days. Then, pick a single colony and incubate it in 300 ml of PDA liquid medium at 28℃ and 180 rpm for 7 days. This is the Cordyceps militaris bacterial suspension for subsequent use.

[0069] Subsequently, 15g of Cordyceps militaris mycelium was inoculated into the fermentation substrate and cultured at 28℃ and 180rpm for 5 days. Then, the mycelium was disrupted at 110℃ and 0.1MPa. The mycelial residue was then removed at 5000rpm. The mixture was then finely filtered through a 13μm filter membrane, and the supernatant was collected. Finally, it was sterilized at 110℃ for 30min to obtain the Cordyceps militaris fermentation lysate.

[0070] Example 2

[0071] Compared with Example 1, the only difference is that the fermentation substrate is morel mushrooms, and the other conditions are the same as in Example 1. The specific operation is as follows:

[0072] Morel mushrooms were crushed, passed through a 200-mesh sieve, and 0.6g of morel mushroom powder was weighed and added to 300ml of water. The mixture was stirred until homogeneous, and then sterilized at 121℃ for 30 minutes. After sterilization, the mixture was allowed to stand at room temperature and cooled to 25℃ to obtain the fermentation substrate.

[0073] Preparation method of Cordyceps militaris bacterial suspension: Take out the frozen bacterial suspension, thaw it at room temperature, and inoculate 300 μL of the frozen suspension into 300 ml of PDA liquid medium. Then, incubate at 28℃ and 180 rpm for 5 days. Then, spread it on PDA solid plates and incubate in a constant temperature incubator at 28℃ for 7 days. Then, pick a single colony and incubate it in 300 ml of PDA liquid medium at 28℃ and 180 rpm for 7 days. This is the Cordyceps militaris bacterial suspension for subsequent use.

[0074] Subsequently, 15g of Cordyceps militaris mycelium was inoculated into the fermentation substrate and cultured at 28℃ and 180rpm for 5 days. Then, the mycelium was disrupted at 110℃ and 0.1MPa. The mycelial residue was then removed at 5000rpm. The mixture was then finely filtered through a 13μm filter membrane, and the supernatant was collected. Finally, it was sterilized at 110℃ for 30min to obtain the Cordyceps militaris fermentation lysate.

[0075] Example 3

[0076] Compared with Example 1, the only difference is that the fermentation substrate is matsutake mushroom, and the other conditions are the same as in Example 1. The specific operation is as follows:

[0077] The matsutake mushrooms were crushed, passed through a 200-mesh sieve, and 0.6g of matsutake powder was added to 300ml of water and mixed evenly with stirring. Then, the mixture was sterilized at 121℃ for 30 minutes. After sterilization, it was allowed to stand at room temperature and cooled to 25℃ to obtain the fermentation substrate.

[0078] Preparation method of Cordyceps militaris bacterial suspension: Take out the frozen bacterial suspension, thaw it at room temperature, and inoculate 300 μL of the frozen suspension into 300 ml of PDA liquid medium. Then, incubate at 28℃ and 180 rpm for 5 days. Then, spread it on PDA solid plates and incubate in a constant temperature incubator at 28℃ for 7 days. Then, pick a single colony and incubate it in 300 ml of PDA liquid medium at 28℃ and 180 rpm for 7 days. This is the Cordyceps militaris bacterial suspension for subsequent use.

[0079] Subsequently, 15g of Cordyceps militaris mycelium was inoculated into the fermentation substrate and cultured at 28℃ and 180rpm for 5 days. Then, the mycelium was disrupted at 110℃ and 0.1MPa. The mycelial residue was then removed at 5000rpm. The mixture was then finely filtered through a 13μm filter membrane, and the supernatant was collected. Finally, it was sterilized at 110℃ for 30min to obtain the Cordyceps militaris fermentation lysate.

[0080] Example 4

[0081] Compared with Example 1, the only difference is that the fermentation substrate is Tremella fuciformis, and the other conditions are the same as in Example 1. The specific operation is as follows:

[0082] The white fungus was crushed, passed through a 200-mesh sieve, and 0.6g of white fungus powder was added to 300ml of water and mixed evenly under stirring. Then it was sterilized at 121℃ for 30 minutes. After sterilization, it was allowed to stand at room temperature and cooled to 25℃ to obtain the fermentation substrate.

[0083] Preparation method of Cordyceps militaris bacterial suspension: Take out the frozen bacterial suspension, thaw it at room temperature, and inoculate 300 μL of the frozen suspension into 300 ml of PDA liquid medium. Then, incubate at 28℃ and 180 rpm for 5 days. Then, spread it on PDA solid plates and incubate in a constant temperature incubator at 28℃ for 7 days. Then, pick a single colony and incubate it in 300 ml of PDA liquid medium at 28℃ and 180 rpm for 7 days. This is the Cordyceps militaris bacterial suspension for subsequent use.

[0084] Subsequently, 15g of Cordyceps militaris mycelium was inoculated into the fermentation substrate and cultured at 28℃ and 180rpm for 5 days. Then, the mycelium was disrupted at 110℃ and 0.1MPa. The mycelial residue was then removed at 5000rpm. The mixture was then finely filtered through a 13μm filter membrane, and the supernatant was collected. Finally, it was sterilized at 110℃ for 30min to obtain the Cordyceps militaris fermentation lysate.

[0085] Example 5

[0086] Compared with Example 1, the only difference is the amount of red mushrooms added to the fermentation substrate; all other conditions are the same as in Example 1. The specific operation is as follows:

[0087] Crush the red mushrooms, pass them through a 200-mesh sieve, weigh 1.5g of red mushrooms and add them to 300ml of water. Mix them evenly with stirring, then sterilize them at 121℃ for 30 minutes. After sterilization, let them stand at room temperature and cool to 25℃ to obtain the fermentation substrate.

[0088] Preparation method of Cordyceps militaris bacterial suspension: Take out the frozen bacterial suspension, thaw it at room temperature, and inoculate 300 μL of the frozen suspension into 300 ml of PDA liquid medium. Then, incubate at 28℃ and 180 rpm for 5 days. Then, spread it on PDA solid plates and incubate in a constant temperature incubator at 28℃ for 7 days. Then, pick a single colony and incubate it in 300 ml of PDA liquid medium at 28℃ and 180 rpm for 7 days. This is the Cordyceps militaris bacterial suspension for subsequent use.

[0089] Subsequently, 15g of Cordyceps militaris mycelium was inoculated into the fermentation substrate and cultured at 28℃ and 180rpm for 5 days. Then, the mycelium was disrupted at 110℃ and 0.1MPa. The mycelial residue was then removed at 5000rpm. The mixture was then finely filtered through a 13μm filter membrane, and the supernatant was collected. Finally, it was sterilized at 110℃ for 30min to obtain the Cordyceps militaris fermentation lysate.

[0090] Example 6

[0091] Compared with Example 1, the only difference is the amount of cicada flower inoculated; all other conditions are the same as in Example 1. The specific operation is as follows:

[0092] Crush red mushrooms, pass them through a 200-mesh sieve, and weigh 0.6g of red mushroom powder into 300ml of water. Mix the powder evenly with stirring, and then sterilize it for 30 minutes at a temperature of 121℃ and a pressure of 0.12MPa. After sterilization, let it stand at room temperature and cool to 25℃ to obtain the fermentation substrate.

[0093] Preparation method of Cordyceps militaris bacterial suspension: Take out the frozen bacterial suspension, thaw it at room temperature, and inoculate 300 μL of the frozen suspension into 300 ml of PDA liquid medium. Then, incubate at 28℃ and 180 rpm for 5 days. Then, spread it on PDA solid plates and incubate in a constant temperature incubator at 28℃ for 7 days. Then, pick a single colony and incubate it in 300 ml of PDA liquid medium at 28℃ and 180 rpm for 7 days. This is the Cordyceps militaris bacterial suspension for subsequent use.

[0094] Subsequently, 6g of Cordyceps militaris mycelium was inoculated into the fermentation substrate and cultured at 28℃ and 180rpm for 5 days. Then, the mycelium was disrupted at 110℃ and 0.1MPa. The mycelial residue was then removed at 5000rpm. The mixture was then finely filtered through a 13μm filter membrane, and the supernatant was collected. Finally, it was sterilized at 110℃ for 30min to obtain the Cordyceps militaris fermentation lysate.

[0095] Example 7

[0096] Compared with Example 1, the only difference is the amount of cicada flower inoculated; all other conditions are the same as in Example 1. The specific operation is as follows:

[0097] Crush red mushrooms, pass them through a 200-mesh sieve, and weigh 0.6g of red mushroom powder into 300ml of water. Mix the powder evenly with stirring, and then sterilize it for 30 minutes at a temperature of 121℃ and a pressure of 0.12MPa. After sterilization, let it stand at room temperature and cool to 25℃ to obtain the fermentation substrate.

[0098] Preparation method of Cordyceps militaris bacterial suspension: Take out the frozen bacterial suspension, thaw it at room temperature, and inoculate 300 μL of the frozen suspension into 300 ml of PDA liquid medium. Then, incubate at 28℃ and 180 rpm for 5 days. Then, spread it on PDA solid plates and incubate in a constant temperature incubator at 28℃ for 7 days. Then, pick a single colony and incubate it in 300 ml of PDA liquid medium at 28℃ and 180 rpm for 7 days. This is the Cordyceps militaris bacterial suspension for subsequent use.

[0099] Subsequently, 30g of Cordyceps militaris mycelium was inoculated into the fermentation substrate and cultured at 28℃ and 180rpm for 5 days. Then, the mycelium was disrupted at 110℃ and 0.1MPa. The mycelial residue was then removed at 5000rpm. The mixture was then finely filtered through a 13μm filter membrane, and the supernatant was collected. Finally, it was sterilized at 110℃ for 30min to obtain the Cordyceps militaris fermentation lysate.

[0100] Example 8

[0101] Compared with Example 1, the only difference is that the fermentation time is 7 days, while the other conditions are the same as in Example 1. The specific operation is as follows:

[0102] Crush red mushrooms, pass them through a 200-mesh sieve, and weigh 0.6g of red mushroom powder into 300ml of water. Mix the powder evenly with stirring, and then sterilize it for 30 minutes at a temperature of 121℃ and a pressure of 0.12MPa. After sterilization, let it stand at room temperature and cool to 25℃ to obtain the fermentation substrate.

[0103] Preparation method of Cordyceps militaris bacterial suspension: Take out the frozen bacterial suspension, thaw it at room temperature, and inoculate 300 μL of the frozen suspension into 300 ml of PDA liquid medium. Then, incubate at 28℃ and 180 rpm for 5 days. Then, spread it on PDA solid plates and incubate in a constant temperature incubator at 28℃ for 7 days. Then, pick a single colony and incubate it in 300 ml of PDA liquid medium at 28℃ and 180 rpm for 7 days. This is the Cordyceps militaris bacterial suspension for subsequent use.

[0104] Subsequently, 15g of Cordyceps militaris mycelium was inoculated into the fermentation substrate and cultured at 28℃ and 180rpm for 7 days. Then, the mycelium was disrupted at 110℃ and 0.1MPa. The mycelial residue was then removed at 5000rpm. The mixture was then finely filtered through a 13μm filter membrane, and the supernatant was collected. Finally, it was sterilized at 110℃ for 30min to obtain the Cordyceps militaris fermentation lysate.

[0105] Comparative Example 1

[0106] Compared with Example 1, the only difference is that the fermentation substrate is yam, and the other conditions are the same as in Example 1. The specific operation is as follows:

[0107] The yam was crushed and passed through a 200-mesh sieve. 0.6g of yam powder was weighed and added to 300ml of water. The mixture was stirred until homogeneous. The mixture was then sterilized at 121℃ and 0.12MPa for 30 minutes. After sterilization, the mixture was allowed to stand at room temperature and cooled to 25℃ to obtain the fermentation substrate.

[0108] Preparation method of Cordyceps militaris bacterial suspension: Take out the frozen bacterial suspension, thaw it at room temperature, and inoculate 300 μL of the frozen suspension into 300 ml of PDA liquid medium. Then, incubate at 28℃ and 180 rpm for 5 days. Then, spread it on PDA solid plates and incubate in a constant temperature incubator at 28℃ for 7 days. Then, pick a single colony and incubate it in 300 ml of PDA liquid medium at 28℃ and 180 rpm for 7 days. This is the Cordyceps militaris bacterial suspension for subsequent use.

[0109] Subsequently, 15g of Cordyceps militaris mycelium was inoculated into the fermentation substrate and cultured at 28℃ and 180rpm for 5 days. Then, the mycelium was disrupted at 110℃ and 0.1MPa. The mycelial residue was then removed at 5000rpm. The mixture was then finely filtered through a 13μm filter membrane, and the supernatant was collected. Finally, it was sterilized at 110℃ for 30min to obtain the Cordyceps militaris fermentation lysate.

[0110] Comparative Example 2

[0111] Compared with Example 1, the only difference is that the fermentation substrate is PDA liquid culture medium, and the other conditions are the same as in Example 1. The specific operation is as follows:

[0112] Preparation method of Cordyceps militaris bacterial suspension: Take out the frozen bacterial suspension, thaw it at room temperature, and inoculate 300 μL of the frozen suspension into 300 ml of PDA liquid medium. Then, incubate at 28℃ and 180 rpm for 5 days. Then, spread it on PDA solid plates and incubate in a constant temperature incubator at 28℃ for 7 days. Then, pick a single colony and incubate it in 300 ml of PDA liquid medium at 28℃ and 180 rpm for 7 days. This is the Cordyceps militaris bacterial suspension for subsequent use.

[0113] 15g of Cordyceps militaris mycelium was inoculated into PDA medium and cultured at 28℃ and 180rpm for 5 days. Then, the mycelium was disrupted at 110℃ and 0.1MPa for 0.12 minutes. The mycelial residue was then removed at 5000rpm. The mixture was then finely filtered through a 13μm filter membrane, and the supernatant was collected. Finally, the mixture was sterilized at 110℃ for 30 minutes to obtain the Cordyceps militaris fermentation lysate.

[0114] Comparative Example 3

[0115] Compared with Example 1, the only difference is the fermentation time; all other conditions are the same as in Example 1. The specific operation is as follows:

[0116] Crush red mushrooms, pass them through a 200-mesh sieve, and weigh 0.6g of red mushroom powder into 300ml of water. Mix the powder evenly with stirring, and then sterilize it for 30 minutes at a temperature of 121℃ and a pressure of 0.12MPa. After sterilization, let it stand at room temperature and cool to 25℃ to obtain the fermentation substrate.

[0117] Preparation method of Cordyceps militaris bacterial suspension: Take out the frozen bacterial suspension, thaw it at room temperature, and inoculate 300 μL of the frozen suspension into 300 ml of PDA liquid medium. Then, incubate at 28℃ and 180 rpm for 5 days. Then, spread it on PDA solid plates and incubate in a constant temperature incubator at 28℃ for 7 days. Then, pick a single colony and incubate it in 300 ml of PDA liquid medium at 28℃ and 180 rpm for 7 days. This is the Cordyceps militaris bacterial suspension for subsequent use.

[0118] Subsequently, 15g of Cordyceps militaris mycelium was inoculated into the fermentation substrate and cultured at 28℃ and 180rpm for 3 days. Then, the mycelium was disrupted at 110℃ and 0.1MPa. The mycelial residue was then removed at 5000rpm. The mixture was then finely filtered through a 13μm filter membrane, and the supernatant was collected. Finally, it was sterilized at 110℃ for 30min to obtain the Cordyceps militaris fermentation lysate.

[0119] Example 1: Lysosomal Activity Test

[0120] Lysosomes can break down large molecules such as proteins, nucleic acids, and polysaccharides into smaller molecules that can be metabolized and utilized by cells, thus maintaining the cell's metabolic cycle.

[0121] HaCat cells in good logarithmic growth phase were collected, digested with trypsin, and digestion was terminated with complete culture medium. Cell counts were then performed. The cell suspension was adjusted to a concentration of 1.0 × 10⁻⁶. 4 Cells / ml were seeded into 96-well culture plates, with 100 μl of cell suspension added to each well. The edge wells were filled with sterile PBS and incubated overnight at 37°C in a 5% CO2 incubator.

[0122] When adding samples, a control group and a sample group should be set up. The negative control group contains only cell culture medium. For the sample group (cells with added cell culture medium), aspirate the culture medium and add 100 μl of 1% sample concentration to each well. For the cell control wells, add 100 μl of basal culture medium. Incubate at 37℃, 5% CO2 for 24 h. After incubation, aspirate the culture medium containing the sample, add 20 μl of neutral red staining solution and 180 μl of basal culture medium to each well, and continue incubation for 2-4 h before stopping. Add 200 μl of cell lysis buffer to each well, shake for 10 min to lyse, and measure the absorbance (OD) of each well at 540 nm. Lysosomal activity % = OD of sample group / OD of control group × 100%. Results are shown in Table 1 and... Figure 1 .

[0123] Table 1

[0124] Blank group C 100 Example 1 150 Example 2 124 Example 3 117 Example 4 138 Example 5 172 Example 6 139 Example 7 154 Example 8 142 Comparative Example 1 110 Comparative Example 2 103 Comparative Example 3 111

[0125] From Table 1 and Figure 1 The results show that after cell treatment with the products prepared in Examples 1-8 of this application, the activity of cell lysosomes was significantly increased, and the lysosome activity was significantly higher than that of Comparative Examples 1-3. Therefore, the products prepared in Examples 1-8 have a stronger ability to promote cell energy metabolism. Figure 1 In this context, *p < 0.05 indicates a statistically significant difference compared to the model group; **p < 0.01 indicates a statistically significant difference compared to the model group; and ***p < 0.001 indicates an extremely statistically significant difference compared to the model group. ### p < 0.001 indicates a highly statistically significant difference compared to the blank control group.

[0126] Example 2: Relative expression of HAS mRNA

[0127] HAS is a gene related to hyaluronic acid synthase activity. High HAS expression can promote the expression of hyaluronic acid synthase, leading to increased hyaluronic acid content in cells and thus playing a moisturizing role. Detecting the effect of samples on HAS expression can further evaluate the moisturizing effect of the samples on the skin.

[0128] Fibroblasts were seeded at a density of 300,000 cells / well (6-well plate). When the cell density reached 80%, the culture medium was aspirated. The cells were washed twice with PBS, and then 1 mL of PBS was added. The blank wells were covered with aluminum foil and placed in a UVA lamp for irradiation at 6 J / cm². 2 After irradiation, PBS was aspirated, followed by washing once with PBS. 2 mL of serum-free culture medium containing 2% sample concentration was added. The positive control was 50 μg / mL VC, and the blank control was added with serum-free culture medium. The cells were incubated at 37°C for 24 h. The supernatant was aspirated. Cells were washed twice with PBS, and RNA was extracted. After determining the concentration, cDNA was reverse transcribed, followed by qPCR. Results are shown in the table below. Figure 2 And Table 2, the specific process is as follows:

[0129] Primer information:

[0130]

[0131] The qPCR reaction system is as follows: Fast Super qPCR Master Mix Reagent Kit (Guangzhou Borren Ankang Medical Technology Co., Ltd.):

[0132]

[0133]

[0134] The three-step amplification process is as follows:

[0135]

[0136] Finally, the relative expression level is calculated. The calculation formula is as follows: Expression level ratio = 2^(-△△Ct) where: △△Ct = △Ct of the sample group - △Ct of the blank group

[0137] △Ct = Ct value of the gene to be tested - Ct value of the internal reference gene

[0138] Table 2

[0139]

[0140]

[0141] Depend on Figure 2 As shown in Table 2, after treating damaged cells with the products prepared in Examples 1-8, the intracellular HAS content increased significantly, and was significantly higher than the HAS content in Comparative Examples 1-3. This indicates that the products prepared in Examples 1-8 can promote the precipitation of elastin and effectively repair damaged cells. Figure 2In this context, *p < 0.05 indicates a statistically significant difference compared to the model group; **p < 0.01 indicates a statistically significant difference compared to the model group; and ***p < 0.001 indicates an extremely statistically significant difference compared to the model group. ## p < 0.01 indicates a statistically significant difference compared to the blank control group.

[0142] Example 3: Relative expression of TRPV1 mRNA

[0143] TRPV1 is related to human skin's perception of external stimuli. High expression of TRPV1 makes the skin more susceptible to external stimuli, causing itching, redness, and pain. This indicator is often used to assess whether a sample has a soothing effect.

[0144] Fibroblasts were seeded at a density of 300,000 cells / well (6-well plate). When the cell density reached 80%, the culture medium was aspirated. The cells were washed twice with PBS, and then 1 mL of PBS was added. The blank wells were covered with aluminum foil and placed in a UVA lamp for irradiation at 6 J / cm². 2 After irradiation, the PBS was aspirated, followed by washing once with PBS. 2 mL of serum-free culture medium containing 2% sample concentration was added. The positive control was 50 μg / mL VC, and the blank control was added with serum-free culture medium. The cells were incubated at 37°C for 24 h. The supernatant was aspirated. The cells were washed twice with PBS, and RNA was extracted from the cells. After determining the concentration, cDNA was reverse transcribed, followed by qPCR. The specific procedure is as follows:

[0145] Primer information:

[0146]

[0147] The qPCR reaction system is as follows: Fast Super qPCR Master Mix kit (Guangzhou Borren Ankang Medical Technology Co., Ltd.), see Table 3:

[0148] Table 3

[0149]

[0150]

[0151] The three-step amplification process is shown in Table 4:

[0152] Table 4

[0153]

[0154] Finally, the relative expression level is calculated. The formula is as follows: Expression level ratio = 2^(-△△Ct)

[0155] Where: △△Ct = △Ct of the sample group - △Ct of the blank group

[0156] △Ct = Ct value of the gene to be tested - Ct value of the internal reference gene. The results are shown in Table 5 and Figure 3 .

[0157] Table 5

[0158]

[0159]

[0160] Depend on Figure 3 As shown in Table 5, after treating damaged cells with the products prepared in Examples 1-8, the intracellular TRPV1 content was significantly reduced, lower than the TRPV1 content in Comparative Examples 1-3. This indicates that the products prepared in Examples 1-8 have a good soothing effect. Figure 3 In this context, *p < 0.05 indicates a statistically significant difference compared to the model group; **p < 0.01 indicates a statistically significant difference compared to the model group; and ***p < 0.001 indicates an extremely statistically significant difference compared to the model group. ### p < 0.001 indicates a highly statistically significant difference compared to the blank control group.

[0161] Example 4: Relative Expression of ELN mRNA

[0162] Elastin is an important component of the skin. Although it only accounts for 5% of the skin, it is the key to stimulating the production of collagen and elastic fibers. Collagen, which accounts for 70% of the skin, determines the skin's softness and plumpness, while elastic fibers determine the skin's firmness and elasticity. 90% of elastic fibers are composed of elastin. Detecting the gene expression level of ELN can assess whether a sample has a firming and anti-aging effect.

[0163] Fibroblast density was set at 300,000 cells / well (6-well plate). When cell density reached 80%, the culture medium was aspirated. The cells were washed twice with PBS, then 1 mL of PBS was added. The blank wells were covered with aluminum foil and placed in a UVA lamp for irradiation at 6 J / cm². After irradiation, the PBS was aspirated, and the cells were washed once with PBS. 2 mL of serum-free culture medium containing 2% sample concentration was added. The positive control was 50 μg / mL vitamin C, and the blank control was added to serum-free culture medium. The cells were incubated at 37°C for 24 h. The supernatant was aspirated. The cells were washed twice with PBS, and RNA was extracted from the cells. After determining the concentration, cDNA was reverse transcribed and then qPCR was performed. The specific procedure is as follows:

[0164] Primer information:

[0165]

[0166]

[0167] The qPCR reaction system is as follows: Fast Super qPCR Master Mix Reagent Kit (Table 6, Guangzhou Borren Ankang Medical Technology Co., Ltd.):

[0168] Table 6

[0169]

[0170] The three-step amplification process is as follows (Table 7):

[0171] Table 7

[0172]

[0173] Finally, the relative expression level is calculated. The formula is as follows: Expression level ratio = 2^(-△△Ct)

[0174] Where: △△Ct = △Ct of the sample group - △Ct of the blank group

[0175] △Ct = Ct value of the gene to be tested - Ct value of the internal reference gene. The results are shown in Table 8 and... Figure 4 .

[0176] Table 8

[0177]

[0178]

[0179] Depend on Figure 4 As shown in Table 8, after treating damaged cells with the products prepared in Examples 1-8, the intracellular ELN content significantly increased, exceeding the ELN content in Comparative Examples 1-3. This indicates that the products prepared in Examples 1-8 have good anti-aging and firming effects. Figure 4 In this context, *p < 0.05 indicates a statistically significant difference compared to the model group; **p < 0.01 indicates a statistically significant difference compared to the model group; and ***p < 0.001 indicates an extremely statistically significant difference compared to the model group. ### p < 0.001 indicates a highly statistically significant difference compared to the blank control group.

[0180] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof in this application are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0181] Although this application has been disclosed above through the description of specific embodiments, it should be understood that those skilled in the art can devise various modifications, improvements, or equivalents to this application within the spirit and scope of the appended solutions. Such modifications, improvements, or equivalents should also be considered to be included within the scope of protection claimed in this application.

Claims

1. A method for preparing a fermentation lysate product of *Cicada Flower*, characterized in that, The process includes the following steps: inoculating cicada flowers into the fermentation substrate, fermenting for 4-8 days, followed by cell wall disruption and sterilization; The fermentation substrate consists of edible fungi and water; the edible fungi are one or more of morel, matsutake, tremella, and red mushroom; the edible fungi are further crushed and sieved before use, with a sieve mesh size of 100-200 mesh; the fermentation substrate is further sterilized before use. The cell wall breaking refers to breaking the cell wall of the fermented *Cicada nymph* mycelium; the fermentation culture temperature is 25~30℃; The *Cicada Flower* specimen is deposited at the China General Microbiological Culture Collection Center (CGMCC) on August 9, 2024, with accession number CGMCC No. 41430, and is classified as *Cicada Flower* (…). Cordyceps cicadae ).

2. The method for preparing the fermentation lysate product of *Cicada Flower* as described in claim 1, characterized in that, The method for preparing the fermentation lysate of Cordyceps militaris satisfies at least one of the following conditions: The mass ratio of the edible fungus to the water is 1:(200~500). The water includes purified water and / or deionized water; The method for preparing the fermentation substrate includes: mixing the components in the fermentation substrate until all components are completely dissolved.

3. The method for preparing the fermented lysate product of *Cicada Flower* as described in claim 2, characterized in that, The method for preparing the fermentation lysate of Cordyceps militaris satisfies at least one of the following conditions: The mass ratio of the edible fungus to the water is 1:500; When the fermentation substrate is sterilized using the high-temperature sterilization method, the sterilization temperature is 110℃~125℃ and the sterilization time is 20~45min.

4. The method for preparing the fermentation lysate product of *Cicada Flower* as described in claim 3, characterized in that, When the fermentation substrate is sterilized using the high-temperature sterilization method, the sterilization temperature is 110℃~121℃ and the sterilization time is 25~40min.

5. The method for preparing the fermentation lysate product of *Cicada Flower* as described in claim 4, characterized in that, When the fermentation substrate is sterilized using the high-temperature sterilization method, the sterilization temperature is 121°C and the sterilization time is 30 minutes.

6. The method for preparing the fermentation lysate product of *Cicada Flower* as described in claim 1, characterized in that, The cicada flower was added in the form of cicada flower mycelium liquid.

7. The method for preparing the fermentation lysate product of *Cicada Flower* as described in claim 6, characterized in that, The method for preparing the fermentation lysate of Cordyceps militaris satisfies at least one of the following conditions: The preparation method of the cicada flower bacterial solution includes: inoculating cicada flowers into PDA liquid culture medium, then culturing at 28°C and 180 rpm for 5 days, then spreading it on PDA solid plates, placing it in a constant temperature incubator at 28°C for 7 days, then picking single colonies into 300 ml of PDA liquid culture medium, culturing at 28°C and 180 rpm for 7 days, which is used as the cicada flower bacterial solution for subsequent use; The concentration of live bacteria from cicada flowers in the cicada flower mycelium solution was 10. 7 CFU / mL; based on the water in the fermentation substrate, the number of *Cicada nymph* inoculated per unit mass of the water is 10. 5 ~10 6 CFU / mL.

8. The method for preparing the fermented lysate product of *Cicada Flower* as described in claim 7, characterized in that, Based on the water in the fermentation substrate, the number of *Cicada nymph* inoculated per unit mass of the water is 10. 6 CFU / mL.

9. The method for preparing the fermentation lysate product of *Cicada Flower* as described in claim 1, characterized in that, The method for preparing the fermentation lysate of Cordyceps militaris satisfies at least one of the following conditions: The fermentation culture time is 5-7 days; The fermentation culture is an aerobic fermentation; The cell wall breaking method is high-temperature and high-pressure cell wall breaking; The sterilization method is high-temperature sterilization.

10. The method for preparing the fermentation lysate product of *Cicada Flower* as described in claim 9, characterized in that, The method for preparing the fermentation lysate of Cordyceps militaris satisfies at least one of the following conditions: The fermentation culture time was 5 days; The aerobic fermentation is carried out in a constant temperature shaking chamber, and the rotation speed of the constant temperature shaking chamber is 150-200 rpm; The temperature for cell disruption is 110~121℃, and the pressure for cell disruption is 0.10~0.15Mpa; When the high-temperature sterilization method is used for sterilization, the sterilization temperature is 100℃~110℃ and the sterilization time is 20~45min.

11. The method for preparing the fermented cell lysate product of *Cicada nymph* as described in claim 10, characterized in that, The method for preparing the fermentation lysate of Cordyceps militaris satisfies at least one of the following conditions: The aerobic fermentation is carried out in a constant temperature shaking chamber, and the rotation speed of the constant temperature shaking chamber is 180 rpm; The temperature at which the cell wall is broken is 110℃, and the pressure at which the cell wall is broken is 0.1 MPa; When the high-temperature sterilization method is used for sterilization, the sterilization temperature is 110°C and the sterilization time is 25-40 minutes.

12. The method for preparing the fermentation lysate product of *Cicada Flower* as described in claim 11, characterized in that, When the high-temperature sterilization method is used for sterilization, the sterilization time is 30 minutes.

13. The method for preparing the fermentation lysate product of *Cicada Flower* as described in claim 1, characterized in that, The process after cell wall disruption also includes centrifugation, filtration, and collection of the supernatant.

14. The method for preparing the fermented cell lysate product of *Cicada nymph* as described in claim 13, characterized in that, The method for preparing the fermentation lysate of Cordyceps militaris satisfies at least one of the following conditions: The centrifuge speed is 3000~7000 rpm; The centrifugation time is 20-45 minutes; The filter membrane used for filtration has a thickness of 10~15μm.

15. The method for preparing the fermented lysate product of *Cicada Flower* as described in claim 14, characterized in that, The method for preparing the fermentation lysate of Cordyceps militaris satisfies at least one of the following conditions: The centrifuge speed is 3500~6000 rpm; The centrifugation time is 25-40 minutes.

16. The method for preparing the fermented lysate product of *Cicada Flower* as described in claim 15, characterized in that, The method for preparing the fermentation lysate of Cordyceps militaris satisfies at least one of the following conditions: The centrifuge speed is 5000 rpm; The centrifugation time was 30 minutes.

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