Cordyceps sobolifera fermentation lysate as well as preparation method and application thereof

Abstract

The invention discloses a cordyceps sobolifera fermentation lysate as well as a preparation method and application thereof, and the preparation method comprises the following steps: inoculating cordyceps sobolifera into a fermentation substrate, fermenting and culturing for 4-8 days, and sterilizing, so as to obtain the cordyceps sobolifera fermentation lysate, wherein the fermentation substrate comprises edible fungi and water. According to the invention, the edible fungi are fermented by using a specific variety of cordyceps sobolifera, so that the content of active ingredients in the fermentation product is increased, the beautifying effect can be improved, the content of active ingredients in the cordyceps sobolifera fermentation lysate prepared by the fermentation process is increased, and the cordyceps sobolifera fermentation lysate has ideal effects of resisting aging, relieving, moisturizing, promoting cell energy metabolism and the like; the preparation process is simple, the fermentation condition is mild, no organic solvent is used, energy conservation and environmental protection are achieved, and the method has good application prospects.

Description

Technical Field

[0001] The present application belongs to the field of fermentation technology, and in particular relates to a cicada fungus fermentation lysate, and a preparation method and application thereof. Background Art

[0002] Cicada fungus (Cordycep scicadae), also known as cicada pupa grass, refers to the complex formed by the fungus cicada fungus (Paecilomyces cicadaae) of the genus Cordyceps of the family Clavicipttaceus parasitizing on cicada nymphs. Cicada fungus was once regarded as one of the "eight treasures of Chinese medicine". It is as famous as Cordyceps sinensis and Cordyceps militaris, and is one of the three traditional medicinal cordyceps in my country. The active ingredients of cicada fungus and Cordyceps sinensis are similar. Its mycelium contains fungal polysaccharides, cordycepic acid, cordycepin, adenosine and other active ingredients, and has medical and health functions, such as regulating immunity and anti-tumor.

[0003] At present, in order to increase the content of active ingredients in cicada fungus, people generally adopt solid fermentation, but solid fermentation still has defects such as long fermentation cycle and low content of active ingredients, which leads to reduced production efficiency and poor application effect. Existing research has been reported many times at home and abroad on adding appropriate amounts of traditional Chinese medicine ingredients or oily substances to the culture medium to increase the activity of metabolites, but there has been no report on the application of cicada fungus in the fermentation of edible fungi.

[0004] Therefore, there is an urgent need for a fermentation method for fermenting edible fungi from cicada fungus, so as to effectively increase the active content of effective ingredients and improve their bioavailability and beauty effects. Summary of the invention

[0005] The technical problem to be solved by the present application is to overcome the defects of the prior art such as the long fermentation cycle of cicada fungus, the difficulty in grasping the fermentation process, the lack of cicada fungus fermentation process, and the limited application of cicada fungus, and to provide a cicada fungus fermentation lysate, and its preparation method and application. The present application utilizes a specific cicada fungus species to ferment edible fungi, which not only increases the active ingredient content of the fermentation product, but also improves the beauty effect. After the fermentation process of the present application, the active ingredient content in the obtained cicada fungus fermentation lysate is increased, and it has ideal moisturizing, soothing, and promoting cell energy metabolism. The preparation process is simple, the fermentation conditions are mild, and no organic solvents are used, achieving energy saving and environmental protection.

[0006] This application adopts the following technical solutions to solve the above technical problems:

[0007] The present application provides a method for preparing a cicada fungus fermentation lysate, which comprises the following steps: inoculating cicada fungus into a fermentation substrate, fermenting and culturing for 4 to 8 days, and sterilizing;

[0008] Wherein, the fermentation substrate includes edible fungi and water.

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

[0010] In some embodiments, the edible fungi include one or more of morels, matsutake mushrooms, tremella, and red mushrooms.

[0011] In some embodiments, the edible fungi are further crushed and sieved before use.

[0012] Wherein, the mesh number of the sieving is 100 to 200 meshes.

[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 be further sterilized before use.

[0016] Wherein, the method for sterilizing the fermentation substrate can generally be a high temperature sterilization method.

[0017] When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization temperature may be a conventional temperature for such operations in the art, preferably 110°C to 125°C, more preferably 110°C to 121°C, for example 121°C.

[0018] When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization time can be the conventional time for such operation in the art, preferably 20 to 45 minutes, more preferably 25 to 40 minutes, for example 30 minutes.

[0019] In some embodiments, the cicada fungus is preserved in the General Microbiology Center of China Microorganism Culture Collection Administration, the preservation date is August 9, 2024, the preservation number is CGMCC No.41430, the classification name is Cordyceps cicadae, the name of the preservation unit is the General Microbiology Center of China Microorganism Culture Collection Administration (referred to as CGMCC), and the address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101.

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

[0021] Among them, the preparation method of the cicada fungus liquid includes: inoculating cicada fungus into PDA liquid culture medium, then culturing at 28°C and 180rpm for 5 days, then coating it on a PDA solid plate, placing it in a 28°C constant temperature incubator for 7 days, then picking a single colony into 300ml PDA liquid culture medium, culturing at 28°C and 180rpm for 7 days, and using it as cicada fungus liquid for subsequent use.

[0022] The concentration of live bacteria of Cicadae in the Cicadae fungus liquid is 10 7 CFU / mL.

[0023] In some embodiments, based on the water in the fermentation substrate, the number of cicada fungi inoculated per unit mass of the water may 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 conditions and methods of the fermentation culture are conventional in the art, and aerobic fermentation is generally performed in a constant temperature shaking box.

[0027] Wherein, when the constant temperature oscillation box is used for the aerobic fermentation, the rotation speed of the constant temperature oscillation box is 150-200 rpm, preferably 180 rpm.

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

[0029] Wherein, the wall breaking temperature is 110-121°C, preferably 110°C.

[0030] The wall breaking pressure is 0.10-0.15 MPa, 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 the sterilization, the sterilization temperature can be a conventional temperature for such operations in the art, preferably 100° C. to 110° C., more preferably 110° C.

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

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

[0035] The centrifugal rotation speed may be a conventional rotation speed for such operation in the art, preferably 3000-7000 rpm, more preferably 3500-6000 rpm, for example 5000 rpm.

[0036] The centrifugation time may be the conventional time for such operation in the art, preferably 20 to 45 min, more preferably 25 to 40 min, for example 30 min.

[0037] Wherein, the filter membrane of the filtration is 10 to 15 μm.

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

[0039] The present application also provides a cicada fungus fermentation lysate, which is prepared by the preparation method of the cicada fungus fermentation lysate as described above.

[0040] The present application also provides a use of the cicada fungus fermentation lysate as described above directly as a product, as an additive or as a base in the preparation of a skin external preparation.

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

[0042] Wherein, the anti-aging active ingredient is an anti-aging active ingredient having the ability to increase lysosomal activity or a firming anti-aging active ingredient having the ability to increase the intracellular ELN1 content;

[0043] Wherein, the moisturizing and water-locking active ingredient is a moisturizing and water-locking active ingredient that has the function of increasing the HAS content in cells.

[0044] Wherein, the soothing active ingredient is a soothing active ingredient that can reduce the intracellular TRPV1 content.

[0045] The present application also provides a skin external preparation, which includes the Cordyceps sinensis fermentation lysate as described above.

[0046] In some embodiments, the skin topical preparation may further include active ingredients conventionally 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 preparation may include but is not limited to a facial mask, an essence or a toner according to conventional methods in the art.

[0048] In some embodiments, the mass percentage of the Cicadae Convolvulus fermentation lysate in the skin external preparation may be 5% to 99%, preferably 60% to 99%.

[0049] On the basis of conforming to the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present application.

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

[0051] The positive progressive effect of the present application is that the present application uses cicada fungus to ferment edible fungi, which can increase the content of active substances in the cicada fungus fermentation lysate and improve the beauty effect. After the fermentation process of the present application, the active ingredient content of the obtained cicada fungus fermentation lysate is significantly increased, and it has ideal moisturizing, soothing, firming, and promoting cell energy metabolism effects. The preparation process is simple, the fermentation conditions are mild, and no organic solvents are used, thereby achieving energy saving and environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0053] in:

[0054] Figure 1 This is a comparison chart of intracellular lysosomal activity after cells were treated with the products obtained in the embodiment and the comparative example;

[0055] Figure 2 This is a comparison chart of HAS content in cells after the cells were treated with the products obtained in the embodiment and the comparative example;

[0056] Figure 3 This is a comparison chart of the intracellular TRPV1 content after the cells were treated with the products prepared in the embodiment and the comparative example;

[0057] Figure 4 This is a comparison chart of the ELN content in cells after the cells were treated with the products prepared in Example and Comparative Example. DETAILED DESCRIPTION

[0058] The present application is further described below by way of examples, but the present application is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are selected according to conventional methods and conditions, or according to the product instructions.

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

[0060] The cicada fungus in the following embodiments and comparative examples is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, with a deposit date of August 9, 2024, a deposit number of CGMCC No.41430, a classification name of cicada fungus (Cordyceps cicadae), and a depository name of the General Microbiological Center of China Microorganism Culture Collection Administration (referred to as CGMCC), with an address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and a zip code of 100101.

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

[0062] The morels in the following examples and comparative examples were purchased from Yinyun Edible Fungi Business Department in Guandu District.

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

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

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

[0066] Example 1

[0067] Crush the red mushroom, pass it through a 200-mesh sieve, weigh 0.6 g of the red mushroom powder, add it into 300 ml of water, and mix it evenly under stirring, then sterilize it at 121° C. for 30 minutes, let it stand to room temperature after sterilization, and wait for it to cool to 25° C. to obtain a fermentation substrate;

[0068] The preparation method of cicada fungus liquid is as follows: taking out the frozen liquid, thawing it at room temperature, taking out 300 μL of the frozen liquid and inoculating it into 300 mL PDA liquid culture medium, then culturing it at 28° C. and 180 rpm for 5 days, then coating it on a PDA solid plate, placing it in a 28° C. constant temperature incubator and culturing it for 7 days, then picking out a single colony and placing it in 300 mL PDA liquid culture medium, culturing it at 28° C. and 180 rpm for 7 days, and using it as cicada fungus liquid for subsequent use;

[0069] Subsequently, 15 g of cicada fungus liquid was inoculated into the fermentation substrate, and cultured at 28°C and 180 rpm for 5 days. The mycelium was then broken at 110°C and 0.1 MPa. The mycelium residues were then removed at 5000 rpm. The mixture was finely filtered through a 13 μm filter membrane, the supernatant was taken, and finally sterilized at 110°C for 30 min to obtain the cicada fungus fermentation lysate.

[0070] Example 2

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

[0072] The morels were crushed and passed through a 200-mesh sieve, and 0.6 g of the morel powder was weighed and added into 300 ml of water, and mixed evenly under stirring, and then sterilized at 121° C. for 30 minutes, and allowed to stand at room temperature after sterilization, and cooled to 25° C. to obtain a fermentation substrate;

[0073] The preparation method of cicada fungus liquid is as follows: taking out the frozen liquid, thawing it at room temperature, taking out 300 μL of the frozen liquid and inoculating it into 300 mL PDA liquid culture medium, then culturing it at 28° C. and 180 rpm for 5 days, then coating it on a PDA solid plate, placing it in a 28° C. constant temperature incubator and culturing it for 7 days, then picking out a single colony and placing it in 300 mL PDA liquid culture medium, culturing it at 28° C. and 180 rpm for 7 days, and using it as cicada fungus liquid for subsequent use;

[0074] Subsequently, 15 g of cicada fungus liquid was inoculated into the fermentation substrate, and cultured at 28°C and 180 rpm for 5 days. The mycelium was then broken at 110°C and 0.1 MPa. The mycelium residues were then removed at 5000 rpm. The mixture was finely filtered through a 13 μm filter membrane, the supernatant was taken, and finally sterilized at 110°C for 30 min to obtain the cicada fungus fermentation lysate.

[0075] Example 3

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

[0077] The matsutake mushrooms were crushed and passed through a 200-mesh sieve, and 0.6 g of matsutake mushroom powder was weighed and added to 300 ml of water, and mixed evenly under stirring, and then sterilized at 121° C. for 30 minutes, and allowed to stand at room temperature after sterilization, and cooled to 25° C. to obtain a fermentation substrate;

[0078] The preparation method of cicada fungus liquid is as follows: taking out the frozen liquid, thawing it at room temperature, taking out 300 μL of the frozen liquid and inoculating it into 300 mL PDA liquid culture medium, then culturing it at 28° C. and 180 rpm for 5 days, then coating it on a PDA solid plate, placing it in a 28° C. constant temperature incubator and culturing it for 7 days, then picking out a single colony and placing it in 300 mL PDA liquid culture medium, culturing it at 28° C. and 180 rpm for 7 days, and using it as cicada fungus liquid for subsequent use;

[0079] Subsequently, 15 g of cicada fungus liquid was inoculated into the fermentation substrate, and cultured at 28°C and 180 rpm for 5 days. The mycelium was then broken at 110°C and 0.1 MPa. The mycelium residues were then removed at 5000 rpm. The mixture was finely filtered through a 13 μm filter membrane, the supernatant was taken, and finally sterilized at 110°C for 30 min to obtain the cicada fungus fermentation lysate.

[0080] Example 4

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

[0082] The Tremella fuciformis was crushed and passed through a 200-mesh sieve, and 0.6 g of Tremella fuciformis powder was weighed and added into 300 ml of water, and mixed evenly under stirring, and then sterilized at 121° C. for 30 minutes, and allowed to stand at room temperature after sterilization, and cooled to 25° C. to obtain a fermentation substrate;

[0083] The preparation method of cicada fungus liquid is as follows: taking out the frozen liquid, thawing it at room temperature, taking out 300 μL of the frozen liquid and inoculating it into 300 mL PDA liquid culture medium, then culturing it at 28° C. and 180 rpm for 5 days, then coating it on a PDA solid plate, placing it in a 28° C. constant temperature incubator and culturing it for 7 days, then picking out a single colony and placing it in 300 mL PDA liquid culture medium, culturing it at 28° C. and 180 rpm for 7 days, and using it as cicada fungus liquid for subsequent use;

[0084] Subsequently, 15 g of cicada fungus liquid was inoculated into the fermentation substrate, and cultured at 28°C and 180 rpm for 5 days. The mycelium was then broken at 110°C and 0.1 MPa. The mycelium residues were then removed at 5000 rpm. The mixture was finely filtered through a 13 μm filter membrane, the supernatant was taken, and finally sterilized at 110°C for 30 min to obtain the cicada fungus fermentation lysate.

[0085] Example 5

[0086] Compared with Example 1, the only difference is that the amount of red mushroom added to the fermentation substrate is different, and other conditions are the same as those in Example 1. The specific operations are as follows:

[0087] Crush the red mushrooms, pass through a 200-mesh sieve, weigh 1.5 g of the red mushrooms, add them into 300 ml of water, and mix them evenly under stirring, then sterilize them at 121° C. for 30 minutes, let them stand to room temperature after sterilization, and wait for them to cool to 25° C. to obtain a fermentation substrate;

[0088] The preparation method of cicada fungus liquid is as follows: taking out the frozen liquid, thawing it at room temperature, taking out 300 μL of the frozen liquid and inoculating it into 300 mL PDA liquid culture medium, then culturing it at 28° C. and 180 rpm for 5 days, then coating it on a PDA solid plate, placing it in a 28° C. constant temperature incubator and culturing it for 7 days, then picking out a single colony and placing it in 300 mL PDA liquid culture medium, culturing it at 28° C. and 180 rpm for 7 days, and using it as cicada fungus liquid for subsequent use;

[0089] Subsequently, 15 g of cicada fungus liquid was inoculated into the fermentation substrate, and cultured at 28°C and 180 rpm for 5 days. The mycelium was then broken at 110°C and 0.1 MPa. The mycelium residues were then removed at 5000 rpm. The mixture was finely filtered through a 13 μm filter membrane, the supernatant was taken, and finally sterilized at 110°C for 30 min to obtain the cicada fungus fermentation lysate.

[0090] Example 6

[0091] Compared with Example 1, the only difference is that the inoculation amount of Cicadae confusa is different, and other conditions are the same as those in Example 1. The specific operations are as follows:

[0092] The red mushrooms were crushed and passed through a 200-mesh sieve, and 0.6 g of the red mushroom powder was weighed and added into 300 ml of water, and mixed evenly under stirring, and then sterilized at a temperature of 121° C. and a pressure of 0.12 MPa for 30 minutes, and then allowed to stand at room temperature after sterilization, and cooled to 25° C. to obtain a fermentation substrate;

[0093] The preparation method of cicada fungus liquid is as follows: taking out the frozen liquid, thawing it at room temperature, taking out 300 μL of the frozen liquid and inoculating it into 300 mL PDA liquid culture medium, then culturing it at 28° C. and 180 rpm for 5 days, then coating it on a PDA solid plate, placing it in a 28° C. constant temperature incubator and culturing it for 7 days, then picking out a single colony and placing it in 300 mL PDA liquid culture medium, culturing it at 28° C. and 180 rpm for 7 days, and using it as cicada fungus liquid for subsequent use;

[0094] Subsequently, 6 g of cicada fungus liquid was inoculated into the fermentation substrate, and cultured at 28°C and 180 rpm for 5 days. The mycelium was then broken at 110°C and 0.1 MPa. The mycelium residues were then removed at 5000 rpm, and then finely filtered with a 13 μm filter membrane, the supernatant was taken, and finally sterilized at 110°C for 30 min to obtain the cicada fungus fermentation lysate.

[0095] Example 7

[0096] Compared with Example 1, the only difference is that the inoculation amount of Cicadae confusa is different, and other conditions are the same as those in Example 1. The specific operations are as follows:

[0097] The red mushrooms were crushed and passed through a 200-mesh sieve, and 0.6 g of the red mushroom powder was weighed and added into 300 ml of water, and mixed evenly under stirring, and then sterilized at a temperature of 121° C. and a pressure of 0.12 MPa for 30 minutes, and then allowed to stand at room temperature after sterilization, and cooled to 25° C. to obtain a fermentation substrate;

[0098] The preparation method of cicada fungus liquid is as follows: taking out the frozen liquid, thawing it at room temperature, taking out 300 μL of the frozen liquid and inoculating it into 300 mL PDA liquid culture medium, then culturing it at 28° C. and 180 rpm for 5 days, then coating it on a PDA solid plate, placing it in a 28° C. constant temperature incubator and culturing it for 7 days, then picking out a single colony and placing it in 300 mL PDA liquid culture medium, culturing it at 28° C. and 180 rpm for 7 days, and using it as cicada fungus liquid for subsequent use;

[0099] Subsequently, 30 g of cicada fungus liquid was inoculated into the fermentation substrate, and cultured at 28°C and 180 rpm for 5 days. The mycelium was then broken at 110°C and 0.1 MPa. The mycelium residues were then removed at 5000 rpm, and then finely filtered with a 13 μm filter membrane, the supernatant was taken, and finally sterilized at 110°C for 30 min to obtain the cicada fungus fermentation lysate.

[0100] Example 8

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

[0102] The red mushrooms were crushed and passed through a 200-mesh sieve, and 0.6 g of the red mushroom powder was weighed and added into 300 ml of water, and mixed evenly under stirring, and then sterilized at a temperature of 121° C. and a pressure of 0.12 MPa for 30 minutes, and then allowed to stand at room temperature after sterilization, and cooled to 25° C. to obtain a fermentation substrate;

[0103] The preparation method of cicada fungus liquid is as follows: taking out the frozen liquid, thawing it at room temperature, taking out 300 μL of the frozen liquid and inoculating it into 300 mL PDA liquid culture medium, then culturing it at 28° C. and 180 rpm for 5 days, then coating it on a PDA solid plate, placing it in a 28° C. constant temperature incubator and culturing it for 7 days, then picking out a single colony and placing it in 300 mL PDA liquid culture medium, culturing it at 28° C. and 180 rpm for 7 days, and using it as cicada fungus liquid for subsequent use;

[0104] Subsequently, 15 g of cicada fungus liquid was inoculated into the fermentation substrate, and cultured at 28°C and 180 rpm for 7 days. The mycelium was then broken at 110°C and 0.1 MPa. The mycelium residues were then removed at 5000 rpm, and then finely filtered with a 13 μm filter membrane, the supernatant was taken, and finally sterilized at 110°C for 30 min to obtain the cicada fungus fermentation lysate.

[0105] Comparative Example 1

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

[0107] The yam was crushed and passed through a 200-mesh sieve, and 0.6 g of the yam powder was weighed and added to 300 ml of water, and mixed evenly under stirring, and then sterilized at a temperature of 121° C. and a pressure of 0.12 MPa for 30 minutes, and then allowed to stand at room temperature after sterilization, and cooled to 25° C. to obtain a fermentation substrate;

[0108] The preparation method of cicada fungus liquid is as follows: taking out the frozen liquid, thawing it at room temperature, taking out 300 μL of the frozen liquid and inoculating it into 300 mL PDA liquid culture medium, then culturing it at 28° C. and 180 rpm for 5 days, then coating it on a PDA solid plate, placing it in a 28° C. constant temperature incubator and culturing it for 7 days, then picking out a single colony and placing it in 300 mL PDA liquid culture medium, culturing it at 28° C. and 180 rpm for 7 days, and using it as cicada fungus liquid for subsequent use;

[0109] Subsequently, 15 g of cicada fungus liquid was inoculated into the fermentation substrate, and cultured at 28°C and 180 rpm for 5 days. The mycelium was then broken at 110°C and 0.1 MPa. The mycelium residues were then removed at 5000 rpm. The mixture was finely filtered through a 13 μm filter membrane, the supernatant was taken, and finally sterilized at 110°C for 30 min to obtain the cicada fungus 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 other conditions are the same as those in Example 1. The specific operations are as follows:

[0112] The preparation method of cicada fungus liquid is as follows: taking out the frozen liquid, thawing it at room temperature, taking out 300 μL of the frozen liquid and inoculating it into 300 mL PDA liquid culture medium, then culturing it at 28° C. and 180 rpm for 5 days, then coating it on a PDA solid plate, placing it in a 28° C. constant temperature incubator and culturing it for 7 days, then picking out a single colony and placing it in 300 mL PDA liquid culture medium, culturing it at 28° C. and 180 rpm for 7 days, and using it as cicada fungus liquid for subsequent use;

[0113] Take 15g of cicada fungus liquid and inoculate it into PDA culture medium, culture it at 28℃ and 180rpm for 5 days, then break the mycelium at a temperature of 110℃ and a pressure of 0.1MPa for 0.12min, then remove the mycelium residues at 5000rpm, then finely filter it with a 13μm filter membrane, take the supernatant, and finally sterilize it at 110℃ for 30min to obtain the cicada fungus fermentation lysate.

[0114] Comparative Example 3

[0115] Compared with Example 1, the only difference is that the fermentation time is different, and other conditions are the same as those in Example 1. The specific operations are as follows:

[0116] The red mushrooms were crushed and passed through a 200-mesh sieve, and 0.6 g of the red mushroom powder was weighed and added into 300 ml of water, and mixed evenly under stirring, and then sterilized at a temperature of 121° C. and a pressure of 0.12 MPa for 30 minutes, and then allowed to stand at room temperature after sterilization, and cooled to 25° C. to obtain a fermentation substrate;

[0117] The preparation method of cicada fungus liquid is as follows: taking out the frozen liquid, thawing it at room temperature, taking out 300 μL of the frozen liquid and inoculating it into 300 mL PDA liquid culture medium, then culturing it at 28° C. and 180 rpm for 5 days, then coating it on a PDA solid plate, placing it in a 28° C. constant temperature incubator and culturing it for 7 days, then picking out a single colony and placing it in 300 mL PDA liquid culture medium, culturing it at 28° C. and 180 rpm for 7 days, and using it as cicada fungus liquid for subsequent use;

[0118] Subsequently, 15 g of cicada fungus liquid was inoculated into the fermentation substrate, and cultured at 28°C and 180 rpm for 3 days. The mycelium was then broken at 110°C and 0.1 MPa. The mycelium residues were then removed at 5000 rpm, and then finely filtered with a 13 μm filter membrane, the supernatant was taken, and finally sterilized at 110°C for 30 min to obtain the cicada fungus fermentation lysate.

[0119] Effect Example 1 Lysosomal activity test

[0120] Lysosomes can degrade macromolecules such as proteins, nucleic acids, and polysaccharides into small molecules that are 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, digested with complete medium, and counted; the concentration of the cell suspension was adjusted to 1.0×10 4 cells / ml, inoculated into 96-well culture plates, added 100 μl of cell suspension to each well, filled the edge wells with sterile PBS, and cultured in a 37°C, 5% CO2 incubator overnight.

[0122] When adding samples, a control group and a sample group should be set up. Among them, the negative control group only has cell culture medium, and the sample group (cell culture medium test sample is added to the cells) draws the culture medium, adds 100μl of 1% concentration sample to each well, and adds 100μl of basic culture medium to the cell control well, and cultured in a 37°C, 5% CO2 incubator for 24h; at the end of the culture, draw the culture medium containing the sample, add 20μl neutral red dye solution and 180μl basic culture medium to each well, continue to culture for 2-4h and then terminate the culture; add 200μl cell lysis solution to each well, shake and lyse for 10min, and measure the absorbance value OD of each well at a wavelength of 540nm. Lysosomal activity % = OD sample group / OD control group × 100%. The results are shown in Tables 1 and Figure 1 .

[0123] Table 1

[0124] Group Lysosomal activity (%) 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 the products prepared in Examples 1 to 8 of the present application were used to treat cells, the activity of the cell lysosomes was significantly increased, and the lysosome activity was significantly higher than that of Comparative Examples 1 to 3. Therefore, it can be seen that the products prepared in Examples 1 to 8 have a stronger ability to promote cell energy metabolism. Figure 1 In the table, *p<0.05 indicates that there is a statistical difference compared with the model group; **p<0.01 indicates that there is a significant statistical difference compared with the model group; ***p<0.001 indicates that there is an extremely significant statistical difference compared with the model group; ### p<0.001, indicating that there is an extremely significant statistical difference compared with the blank control group.)

[0126] Effect Example 2 HAS mRNA relative expression

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

[0128] The density of fibroblasts was 300,000 / well (6-well plate). When the cell density reached 80%, the culture medium was discarded; the cells were washed twice with PBS, and then 1 mL of PBS was added. After the blank wells were covered with tin foil, they were placed in a UVA light box and irradiated at 6 J / cm 2 ; After irradiation, discard PBS, then wash once with PBS, add 2mL of sample serum-free medium with a concentration of 2%, the positive control is 50μg / mL VC, and the blank group is added with serum-free medium; put in a 37℃ incubator and incubate for 24h; discard the supernatant; wash twice with PBS, collect cells to extract RNA, determine the concentration and reverse transcribe cDNA, and then perform qPCR. The results are shown in 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 Kit (Guangzhou Boren Ankang Medical Technology Co., Ltd.):

[0132]

[0133]

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

[0135]

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

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

[0138] Table 2

[0139]

[0140]

[0141] Depend on Figure 2 As shown in the results of Table 2, after the damaged cells were treated with the products prepared in Examples 1 to 8, the HAS content in the cells increased significantly, which was significantly higher than the HAS content in Comparative Examples 1 to 3. It can be seen that the products prepared in Examples 1 to 8 can promote the precipitation of elastin and effectively repair damaged cells. Figure 2In the table, *p<0.05 indicates that there is a statistical difference compared with the model group; **p<0.01 indicates that there is a significant statistical difference compared with the model group; ***p<0.001 indicates that there is an extremely significant statistical difference compared with the model group; ## p<0.01, indicating a significant statistical difference compared with the blank control group.

[0142] Effect Example 3 TRPV1 mRNA relative expression

[0143] TRPV1 is related to the 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 evaluate whether the sample has a soothing effect.

[0144] The density of fibroblasts was 300,000 / well (6-well plate). When the cell density reached 80%, the culture medium was discarded; the cells were washed twice with PBS, and then 1 mL of PBS was added. After the blank wells were covered with tin foil, they were placed in a UVA light box and irradiated at 6 J / cm 2 ; After irradiation, discard PBS, then wash once with PBS, add 2mL of sample serum-free medium with a concentration of 2%, the positive control is 50μg / mL VC, and the blank group is added with serum-free medium; put in a 37℃ incubator and incubate for 24h; discard the supernatant; wash twice with PBS, collect cells to extract RNA, determine the concentration and reverse transcribe cDNA, and then perform qPCR. The specific process is as follows:

[0145] Primer Information:

[0146]

[0147] The qPCR reaction system is as follows: Fast Super qPCR Master Mix Kit (Guangzhou Boren 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, calculate the relative expression level. The calculation formula is as follows: Expression ratio = 2^(-△△Ct)

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

[0156] △Ct = Ct value of the gene to be tested - Ct value of the 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 the results of Table 5, after the damaged cells were treated with the products prepared in Examples 1 to 8, the intracellular TRPV1 content was significantly reduced, which was lower than the TRPV1 content in Comparative Examples 1 to 3. It can be seen that the products prepared in Examples 1 to 8 have a good soothing effect. Figure 3 In the table, *p<0.05 indicates that there is a statistical difference compared with the model group; **p<0.01 indicates that there is a significant statistical difference compared with the model group; ***p<0.001 indicates that there is an extremely significant statistical difference compared with the model group; ### p<0.001, indicating that there is an extremely significant statistical difference compared with the blank control group.)

[0161] Effect Example 4 ELN mRNA relative expression

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

[0163] The density of the plated fibroblasts was 300,000 / well (6-well plate). When the cell density reached 80%, the culture medium was aspirated and discarded; washed twice with PBS, and then 1 mL of PBS was added. After covering the blank well with tin foil, the well was placed in a UVA light box and irradiated at 6 J / cm2; after irradiation, the PBS was aspirated and discarded, and then washed once with PBS, 2 mL of serum-free medium containing a sample with a concentration of 2% was added, and the positive control was 50 μg / mL VC. The blank group was added with serum-free medium; placed in a 37°C incubator and incubated for 24 hours; the supernatant was aspirated and discarded; washed twice with PBS, the cells were collected to extract RNA, and the concentration was measured and then reverse transcribed into cDNA, followed by qPCR. The specific process is as follows:

[0164] Primer Information:

[0165]

[0166]

[0167] The qPCR reaction system is as follows: Fast Super qPCR Master Mix Kit (Table 6, Guangzhou Boren 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, calculate the relative expression level. The calculation formula is as follows: Expression ratio = 2^(-△△Ct)

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

[0175] △Ct = Ct value of the gene to be tested - Ct value of the 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 the results of Table 8, after the damaged cells were treated with the products prepared in Examples 1 to 8, the ELN content in the cells increased significantly, which was higher than the ELN content in Comparative Examples 1 to 3. It can be seen that the products prepared in Examples 1 to 8 have good anti-aging and firming effects. Figure 4 In the table, *p<0.05 indicates that there is a statistical difference compared with the model group; **p<0.01 indicates that there is a significant statistical difference compared with the model group; ***p<0.001 indicates that there is an extremely significant statistical difference compared with the model group; ### p<0.001, indicating that there is an extremely significant statistical difference compared with the blank control group.)

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

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

Claims

1. A method for preparing a cicada fungus fermentation lysate, characterized in that: The method comprises the following steps: inoculating cicada fungus into a fermentation substrate, fermenting and culturing for 4 to 8 days, breaking the cell wall and sterilizing; Wherein, the fermentation substrate includes edible fungi and water.

2. The method for preparing the cicada fungus fermentation lysate according to claim 1, characterized in that: The method for preparing the cicada fungus fermentation lysate satisfies at least one of the following conditions: The edible fungi include one or more of morels, matsutake mushrooms, white fungus, and red mushrooms; The mass ratio of the usable fungus to the water is 1:(200-500), preferably 1:500; The water may include purified water and / or deionized water; The method for preparing the fermentation substrate comprises: mixing the components in the fermentation substrate until the components are completely dissolved; The fermentation substrate further includes a sterilization operation before use; preferably, when the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization temperature is 110°C to 125°C, more preferably 110°C to 121°C, for example 121°C; when the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization time is 20 to 45 minutes, more preferably 25 to 40 minutes, for example 30 minutes.

3. The method for preparing the cicada fungus fermentation lysate according to claim 1, characterized in that: The cicada fungus is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit date of August 9, 2024, a deposit number of CGMCC No.41430, a classification name of cicada fungus (Cordyceps cicadae), a depository name of China Microorganism Culture Collection Administration General Microbiology Center (referred to as CGMCC), and an address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a zip code of 100101; Preferably, the cicada fungus is added in the form of cicada fungus liquid.

4. The method for preparing the cicada fungus fermentation lysate according to claim 3, characterized in that: The method for preparing the cicada fungus fermentation lysate satisfies at least one of the following conditions: The preparation method of the cicada fungus liquid comprises: inoculating cicada fungus into a PDA liquid culture medium, then culturing at 28°C and 180 rpm for 5 days, then coating on a PDA solid plate, placing in a 28°C constant temperature incubator for 7 days, then picking a single colony into 300 ml of PDA liquid culture medium, culturing at 28°C and 180 rpm for 7 days, and using the cicada fungus liquid as standby; The concentration of live bacteria of Cicadae in the Cicadae fungus liquid is 10 7 CFU / mL; preferably, based on the water in the fermentation substrate, the number of cicada fungi inoculated per unit mass of the water is 10 5 ~10 6 CFU / mL, preferably 10 6 CFU / mL.

5. The method for preparing the cicada fungus fermentation lysate according to claim 1, characterized in that: The method for preparing the cicada fungus fermentation lysate satisfies at least one of the following conditions: The fermentation culture time is 5 to 7 hours, preferably 5 days; The fermentation culture is aerobic fermentation; preferably, the aerobic fermentation is carried out in a constant temperature oscillating box, and the rotation speed of the constant temperature oscillating box is 150-200rpm, more preferably 180rpm; The temperature of the wall breaking is high temperature and high pressure wall breaking; preferably, the temperature of the wall breaking is 110-121°C, more preferably 110°C; preferably, the pressure of the wall breaking is 0.10-0.15Mpa, more preferably 0.1Mpa; The sterilization method is high-temperature sterilization; preferably, when the high-temperature sterilization method is used for sterilization, the sterilization temperature is 100°C to 110°C, more preferably 110°C; when the high-temperature sterilization method is used for sterilization, the sterilization time is 20 to 45 minutes, more preferably 25 to 40 minutes, for example 30 minutes.

6. The method for preparing the cicada fungus fermentation lysate according to claim 1, characterized in that: The cell wall breaking further includes the operations of centrifugation, filtration and collection of supernatant.

7. The method for preparing the cicada fungus fermentation lysate according to claim 6, characterized in that: The method for preparing the cicada fungus fermentation lysate satisfies at least one of the following conditions: The centrifugal speed is 3000-7000 rpm, preferably 3500-6000 rpm, more preferably 5000 rpm; The centrifugation time is 20 to 45 minutes, preferably 25 to 40 minutes, and more preferably 30 minutes; The filter membrane used for the filtration is 10 to 15 μm.

8. A cicada fungus fermentation lysate, which is obtained by the preparation method of the cicada fungus fermentation lysate according to any one of claims 1 to 7.

9. A use of the cicada fungus fermentation lysate as claimed in claim 8 directly as a product, as an additive or as a base in the preparation of a skin external preparation; Preferably, the Cicadae Concolor fermentation lysate is used as at least one of the anti-aging active ingredient, moisturizing and water-locking active ingredient, and soothing active ingredient of the skin external preparation; More preferably, the anti-aging active ingredient is an anti-aging active ingredient having the ability to increase lysosomal activity or a firming anti-aging active ingredient having the ability to increase the intracellular ELN1 mRNA content; More preferably, the moisturizing and water-locking active ingredient is a moisturizing and water-locking active ingredient that increases the intracellular HAS content; More preferably, the soothing active ingredient is a soothing active ingredient that has the effect of reducing the intracellular TRPV1 mRNA content.

10. A skin external preparation comprising the Cordyceps sinensis fermentation lysate according to claim 8; Preferably, the skin topical preparation further comprises at least one of a moisturizing active ingredient, a whitening active ingredient, an anti-inflammatory active ingredient, an anti-allergic active ingredient and an antioxidant active ingredient; Preferably, the skin topical preparation includes but is not limited to a facial mask, an essence or a toner; Preferably, the mass percentage of the Cordyceps sinensis fermentation lysate in the skin external preparation is 5% to 99%, preferably 60% to 99%.

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