Cordyceps militaris fermentation product with effects of resisting aging, enhancing mitochondrial function, resisting oxidation and promoting autophagy as well as preparation method and application of cordyceps militaris fermentation product

Abstract

The invention provides a preparation method of a cordyceps militaris fermentation product with anti-aging, mitochondrial function enhancing, anti-oxidation and autophagy promoting effects, and the preparation method comprises the following steps: inoculating cordyceps militaris into a fermentation substrate for liquid fermentation culture to obtain a fermentation mycelium; the fermented mycelium is subjected to wall breaking or centrifugal separation, concentration and freeze-drying, and the cordyceps militaris fermented product is prepared; wherein the fermentation substrate comprises oat bran and / or lily. The cordyceps militaris fermentation product prepared by the method shows remarkable anti-aging, anti-oxidation, mitochondrial function enhancing and autophagy promoting effects in in-vitro cell experiments and in-vivo animal models, the process period is short, the preparation process is simple and easy, liquid fermentation in the whole process is suitable for large-scale production, and the preparation cost is low.

Description

Technical Field

[0001] The present application belongs to the technical field of beauty and skin care, and specifically relates to a Cordyceps militaris fermentation product having anti-aging, mitochondrial function enhancement, anti-oxidation, and autophagy promotion effects, as well as a preparation method and application thereof. Background Art

[0002] Cordyceps militaris (L.ex Fr.) Link.) is an edible and medicinal fungus belonging to the family of ergot and the genus Cordyceps. It contains a variety of active ingredients and has multiple edible and medicinal values ​​such as anti-tumor, anti-radiation, immunity regulation, lowering blood sugar and lipoprotein, relieving cough, resolving phlegm, and moistening the lungs. People usually eat Cordyceps militaris fruiting bodies directly, or use the extraction method to obtain Cordyceps militaris extracts for consumption. It is mainly used in the fields of food and medicine, and relatively little research has been done in the field of cosmetics.

[0003] Although it has been discovered that the active ingredients of Cordyceps militaris have certain antibacterial and anti-inflammatory effects and can be used as raw materials for cosmetics, their use in the cosmetics field has problems such as long growth cycle, large differences in fruiting body morphology, unstable active ingredient content, high cost, and less than ideal beauty and skin care effects.

[0004] Therefore, there is an urgent need to develop a method for extracting active ingredients with excellent beauty and skin care effects from Cordyceps militaris with a short production cycle and stable active ingredient content, so as to expand the application of Cordyceps militaris in the field of cosmetics. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defects in the prior art and provide a Cordyceps militaris fermented product and a preparation method and application thereof. The Cordyceps militaris fermented product prepared by the present invention exhibits significant anti-aging, anti-oxidation, mitochondrial function enhancement and autophagy promotion effects in both in vitro cell experiments and in vivo animal models. In addition, the process cycle of the present invention is short, the preparation process is simple, the whole process liquid fermentation is suitable for large-scale production, and the preparation cost is low.

[0006] The present invention adopts the following technical solutions to solve the above technical problems:

[0007] The first aspect of the present invention provides a Cordyceps militaris strain YCC127, whose classification name is Cordyceps militaris, deposited in the China Microorganism Culture Collection Committee on November 19, 2024, with a deposit number of CGMCC No. 41663, and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101.

[0008] The Cordyceps militaris strain YCC127 provided by the present invention is suitable for producing a fermented product with skin care effects of anti-aging, anti-oxidation, enhancing mitochondrial function and promoting autophagy.

[0009] The second aspect of the present invention provides a method for preparing a Cordyceps militaris fermented product having anti-aging, mitochondrial function enhancement, anti-oxidation, and autophagy promotion effects, comprising: inoculating Cordyceps militaris into a fermentation substrate for liquid fermentation culture to obtain fermentation mycelium; breaking the fermentation mycelium through wall breaking or centrifugal separation, concentrating, and freeze-drying to obtain the Cordyceps militaris fermented product; wherein the fermentation substrate comprises oat bran and / or lily.

[0010] In one embodiment, the Cordyceps militaris is inoculated in the form of Cordyceps militaris seed solution, wherein the volume of the Cordyceps militaris mycelium in the Cordyceps militaris seed solution accounts for at least 50% of the volume of the culture medium, preferably 60% to 75%, and more preferably 65% ​​to 70%.

[0011] In one embodiment, the Cordyceps militaris seed solution is obtained by primary culture and secondary culture in sequence.

[0012] In one embodiment, the preliminary culturing step comprises: culturing Cordyceps militaris in a first culture medium to obtain a fermentation liquid with bacterial balls, and homogenizing the bacterial balls to break them into fine first-generation mycelia to obtain a homogenized bacterial liquid;

[0013] And / or, the secondary culturing step includes: inoculating the homogenized bacterial liquid into a second culture medium for culturing to obtain second-generation mycelium, until the volume of the second-generation mycelium accounts for at least 50% of the volume of the second culture medium, preferably 60% to 75%, and more preferably 65% ​​to 70%.

[0014] In the present invention, the tiny first-generation mycelium may be a mycelium the size of a rice grain; the volume of the second-generation mycelium refers to the volume of the second-generation mycelium after being left to stand.

[0015] In one embodiment, the culture temperature of the preliminary culture is 23-29° C., preferably 25-28° C.; the culture time is 4-8 days, preferably 5-7 days; the rotation speed is 140-190 rpm, preferably 150-180 rpm; preferably, the rotation speed is the shaking table speed;

[0016] And / or, the culture temperature of the secondary culture is 23-29° C., preferably 25-28° C.; the culture time is 4-8 days, preferably 5-7 days; the rotation speed is 140-190 rpm, preferably 150-180 rpm; preferably, the rotation speed is the shaking table speed.

[0017] In one embodiment, the homogenized bacterial liquid is inoculated into the second culture medium at an inoculation volume of 1% (v / v) to 18% (v / v), preferably 5% to 12% (v / v). In the present invention, the inoculation volume v / v of the homogenized bacterial liquid refers to the volume ratio of the homogenized bacterial liquid to the second culture medium, for example, 6% (v / v) means that 6 mL of the homogenized bacterial liquid is inoculated into 100 mL of the second culture medium.

[0018] In one embodiment, the homogenization treatment can be carried out by a commonly used homogenization method in the art, such as mechanical homogenization, ultrasonic homogenization, bead milling homogenization, etc. Mechanical homogenization is preferred, and manual homogenization among mechanical homogenizations is further preferred. Specifically, a sterilized glass homogenizer can be manually rotated or moved up and down in a clean bench to break the mycelium. The manual homogenization method can be used to manually control the strength and speed to avoid excessive breaking.

[0019] And / or, the first culture medium and the second culture medium can be culture medium commonly used in the art, such as potato dextrose water culture medium. The first culture medium and the second culture medium can be the same or different, preferably the first culture medium and the second culture medium are the same.

[0020] Specifically, the components of the first culture medium and the second culture medium may include: potatoes, glucose and water.

[0021] In one embodiment, the oat bran is oat bran powder or fermented oat bran freeze-dried powder, preferably fermented oat bran freeze-dried powder; the lily is lily powder or fermented lily freeze-dried powder, preferably fermented lily freeze-dried powder.

[0022] In the present invention, the fermented oat bran freeze-dried powder is obtained by fermenting oat bran powder; and the fermented lily freeze-dried powder is obtained by fermenting lily powder.

[0023] In one embodiment, the fermentation substrate further comprises fermentation medium or water, preferably water. For example, the fermentation substrate can be oat bran powder and water, oat bran powder and fermentation medium, fermented oat bran freeze-dried powder and water, fermented oat bran freeze-dried powder and fermentation medium, lily powder and water, lily powder and fermentation medium, fermented lily freeze-dried powder and water, fermented lily freeze-dried powder and fermentation medium, preferably fermented oat bran freeze-dried powder and water or fermented lily freeze-dried powder and water.

[0024] In one embodiment, based on the mass of the fermentation medium or the water, the oat bran or lily accounts for 0.5% to 3%, preferably 1% to 2%, of the mass of the fermentation medium or the water.

[0025] Specifically, the fermentation medium can be selected from potato glucose water medium. The components of the fermentation medium can include potatoes and glucose.

[0026] And / or, the water is any one or more of distilled water, mineral water and tap water.

[0027] In one embodiment, the fermentation substrate further includes a sterilization operation before use; and the sterilization operation further includes an operation of cooling to room temperature.

[0028] In one embodiment, the inoculum amount of the Cordyceps militaris seed solution is 1% (v / v) to 18% (v / v), preferably 5% to 12% (v / v) in the fermentation substrate. In the present invention, the inoculum amount v / v of the Cordyceps militaris seed solution refers to the volume ratio of the Cordyceps militaris seed solution to the volume of the fermentation substrate, for example, 10% (v / v) refers to accessing 10 mL of Cordyceps militaris seed solution to 100 mL of the fermentation substrate.

[0029] In one embodiment, the Cordyceps militaris includes a Cordyceps militaris strain YCC127 deposited in the General Microbiological Center of the China Microorganism Culture Collection Administration Committee, with a deposit number of CGMCC No. 41663. The Cordyceps militaris strain YCC127 is classified and named Cordyceps militaris, with a deposit date of November 19, 2024, and the China Microorganism Culture Collection Administration Committee General Microbiological Center is abbreviated as CGMCC, with an address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and a zip code of 100101.

[0030] In one embodiment, the liquid fermentation culture conditions are: the fermentation temperature is 25-30°C, preferably 25-28°C; and / or the fermentation time is 3-10 days, preferably 5-8 days; and / or the rotation speed is 150-200rpm, preferably 180-200rpm, and further, the rotation speed is the shaking table speed.

[0031] In one embodiment, the method of obtaining the fermented mycelium may be centrifugal filtration. The conditions of the centrifugal filtration are: a centrifugal filtration speed of 3000-6500 rpm, preferably 4500-5000 rpm; and a centrifugal filtration time of 15-40 min, preferably 20-30 min.

[0032] Furthermore, after the fermentation of the fermentation substrate is completed, the Cordyceps militaris seed liquid also includes the operations of sterilization and cooling to room temperature before the centrifugal filtration.

[0033] In one embodiment, the cell wall breaking treatment method can be conventional in the art. For example, the cell wall breaking method can be at least one of hot water extraction, ultrasonic crushing, surfactant treatment, organic solvent treatment, acid-base treatment, etc., preferably hot water extraction. The centrifugal separation method can be gauze filtration, centrifugal filtration, etc., preferably centrifugal filtration.

[0034] In one embodiment, the hot water extraction treatment includes: adding distilled water to the fermented mycelium, the solid-liquid ratio of the fermented mycelium to the distilled water is 1:10-1:30, preferably 1:15-1:25; the extraction temperature is 70-90°C, preferably 75-85°C, the number of extractions is 2-5 times, preferably 2-3 times; the extraction time is 0.5h / time to 1.5h / time, preferably 0.8h / time to 1h / time.

[0035] The material-liquid ratio refers to the mass-to-volume ratio between the fermented mycelium and distilled water. For example, the material-to-liquid ratio is 1:20, that is, 20 ml of distilled water is added to 1 g of fermented mycelium.

[0036] In one embodiment, after the hot water extraction is completed, the extract and the residue are separated, collected, concentrated, and freeze-dried.

[0037] The concentration and freeze-drying methods may be conventional in the art. For example, the concentration may be concentrated by vacuum rotary evaporation.

[0038] Furthermore, the freeze-drying further includes the operation of crushing into powder.

[0039] In one embodiment, the preparation method of the fermented oat bran freeze-dried powder or the fermented lily freeze-dried powder comprises: inoculating yeast liquid into a fermentation system, fermenting, and freeze-drying, wherein the fermentation system comprises oat bran powder and / or lily powder and water.

[0040] In one embodiment, the oat bran or lily accounts for 0.5% to 5% of the water, preferably 1% to 2% by mass.

[0041] In one embodiment, the fermentation system further includes a sterilization operation before use; and the sterilization operation further includes an operation of cooling to room temperature.

[0042] In one embodiment, the concentration of the yeast in the yeast solution is 10 5 ~10 9 CFU / mL, preferably 10 6 ~10 8 CFU / mL.

[0043] In one embodiment, the yeast solution is inoculated into the fermentation system at an inoculation volume of 1% to 15% (v / v), preferably 5% to 10% (v / v). In the present invention, the inoculation volume v / v of the yeast solution refers to the volume ratio of the yeast solution to the volume of the fermentation system, for example, 5% (v / v) means inoculating 5 mL of yeast solution into 100 mL of the fermentation system.

[0044] In one embodiment, the yeast liquid can be prepared according to conventional methods in the art.

[0045] In one embodiment, the yeast is inoculated into the fermentation system and the fermentation conditions are as follows: the fermentation temperature is 25-32°C, preferably 28-30°C; the fermentation time is 20-48h, preferably 25-32h; the fermentation speed is 100-300rpm, preferably 100-220rpm.

[0046] In one embodiment, the yeast is inoculated into the fermentation system for fermentation. After the fermentation is completed, the freeze-drying step further includes a sterilization operation. After the freeze-drying step, the freeze-drying step further includes a step of crushing to powder. The freeze-drying method can be conventional in the art.

[0047] In one embodiment, the yeast may be Saccharomyces cerevisiae. Further, the Saccharomyces cerevisiae may be Saccharomyces cerevisiae strain YWY-1, which has been disclosed in CN202010372740.3, and the depository is the General Microbiological Center of the China Microbiological Culture Collection Administration (CGMCC for short), with an address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a postal code of 100101, a deposit date of March 27, 2019, and a deposit number of CGMCC No. 17452.

[0048] In the present invention, any conventional sterilization method in the art may be used for the sterilization method involved; for example, the sterilization temperature is 115-125°C, preferably 118-121°C; the sterilization time is 15-35 min, preferably 28-32 min, and more preferably 30 min.

[0049] The third aspect of the present invention provides a Cordyceps militaris fermented product having anti-aging, mitochondrial function enhancement, anti-oxidation, and autophagy promotion effects. The Cordyceps militaris fermented product is prepared by the preparation method of the Cordyceps militaris fermented product described in the second aspect.

[0050] The fourth aspect of the present invention provides the use of the Cordyceps militaris fermented product with anti-aging, mitochondrial function enhancement, anti-oxidation and autophagy promotion effects in the preparation of skin topical preparations, hair preparations or foods as described in the third aspect.

[0051] In one embodiment, the Cordyceps militaris fermented product is directly used as a product or as an additive in the skin topical preparation, hair preparation or food.

[0052] In one embodiment, the Cordyceps militaris fermented product can be used as at least one of the antioxidant active ingredient, skin repair active ingredient and anti-aging active ingredient in the skin external preparation.

[0053] In one embodiment, the skin topical agent may be skin care lotion, lotion, cream, facial mask, etc.; and / or, the food may be a health food.

[0054] The fifth aspect of the present invention provides a skin topical preparation, which includes the Cordyceps militaris fermented product prepared by the preparation method of the Cordyceps militaris fermented product with anti-aging, mitochondrial function enhancement, antioxidant and autophagy promotion effects described in the second aspect, or the Cordyceps militaris fermented product with anti-aging, mitochondrial function enhancement, antioxidant and autophagy promotion effects described in the third aspect.

[0055] 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.

[0056] The positive effects of this application are:

[0057] (1) Optimization of the fermentation process. During the strain culture stage, a two-step culture of Cordyceps militaris strain liquid was adopted: the Cordyceps militaris strain was first cultured in a potato glucose water culture medium to allow it to gradually grow and form mycelium, and then a secondary culture was performed to ensure that the Cordyceps militaris strain grew fully in the culture medium, providing more active bacteria for subsequent fermentation.

[0058] (2) The present invention utilizes oat bran or lily as a substrate for combined fermentation with Cordyceps militaris to prepare a fermented product with multiple biological activities. Different from the fermentation of Cordyceps militaris alone, oat bran itself is rich in nutritional sources such as dietary fiber, B vitamins, minerals (such as calcium, iron, magnesium), and antioxidants (such as polyphenols), providing a better growth environment for probiotics and microorganisms. These ingredients interact with the ingredients of Cordyceps militaris during the fermentation process, which helps the reproduction and metabolic activities of microorganisms during the fermentation process, improves the release of the effective ingredients of Cordyceps militaris, improves the overall nutritional value of the fermented product, improves the biological activity and bioavailability of Cordyceps militaris, and thus enhances its anti-aging, antioxidant and other effects.

[0059] (3) The present application does not select conventional microbial culture medium, such as salts such as glucose, yeast extract, magnesium sulfate, potassium dihydrogen phosphate, and potassium hydrogen phosphate, but selects medicinal edible grains, especially medicinal and edible substances processed by microbial fermentation, such as fermented oat bran, assisted by conventional potato glucose water culture medium, and cultured Cordyceps militaris at conventional culture temperature. After a certain number of mycelium are grown, the culture process is terminated (without the need to cultivate into fruiting bodies), and the post-processing stage is entered. Mycelium is obtained by centrifugation, cell wall is broken to obtain lysate or fermentation product filtrate is obtained by centrifugation, and the obtained fermentation product is found to have anti-aging, mitochondrial enhancement, anti-oxidation, and autophagy promotion effects through functional experiments. This preparation process greatly shortens the utilization cycle of Cordyceps militaris, and the production process reduces multiple variable factors, improves controllability, and contributes to large-scale production of fermentation products with stable performance. In addition, oat bran is rich in nutrients such as β-glucan and polypeptides, which can not only provide Cordyceps militaris with nutrients necessary for growth, but also be decomposed and release more active ingredients into the fermentation product. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] The present application may be better understood by referring to the following description in conjunction with the accompanying drawings. The accompanying drawings, together with the following detailed description, are included in 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. Among them:

[0061] Figure 1 This is a diagram showing the cytotoxicity test results of the product prepared in Example 2 at different concentrations;

[0062] Figure 2 Figure 2 shows the established UVA-induced HFF cell senescence model and hydrogen peroxide-induced HFF cell senescence model;

[0063] Figure 3 This is a comparison result diagram of the senescent cell ratio of the product prepared in Example 2;

[0064] Figure 4 This is a graph showing the antioxidant test results of the product obtained in Example 2;

[0065] Figure 5 This is a graph showing the effect of the product prepared in Example 2 on COL-I, COL-III, MMP-1, MMP-3 and TIMP-1;

[0066] Figure 6 This is a test result diagram of the product prepared in Example 2 on the MTC of model zebrafish;

[0067] Figure 7 This is a microscope picture of the effect of the product prepared in Example 2 on cell aging in zebrafish;

[0068] Figure 8 This is a microscope photo of the fluorescence intensity of zebrafish body muscle mitochondria by the product prepared in Example 2;

[0069] Fig. 9 The fluorescence intensity analysis results of the product prepared in Example 2 on zebrafish body muscle mitochondria;

[0070] Fig.10 This is a microscope photo of the fluorescence intensity of the product prepared in Example 2 on zebrafish yolk sac;

[0071] Fig.11 This is a graph showing the results of the fluorescence intensity analysis of the product prepared in Example 2 on zebrafish yolk sac;

[0072] Fig.12 This is a photo of the product prepared in Example 2 on zebrafish heart autophagosomes;

[0073] Fig.13 The results of the detection of cell cycle regulatory proteins p53, p21, and p16 by Cordyceps militaris fermentation products;

[0074] Fig.14 This is a diagram showing the relationship between Cordyceps militaris fermentation products and the mutual regulation between Nrf2 pathway and autophagy. DETAILED DESCRIPTION

[0075] The present application is further described below by way of examples, but the present application is not limited to the scope of the examples described. The experimental methods in the following examples that do not specify specific conditions are selected according to conventional methods and conditions, or according to the product specifications. The reagents and raw materials used in this application are commercially available unless otherwise specified. The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0076] In the following examples, PDA medium (potato dextrose agar medium) was purchased from Haibo Biotechnology Co., Ltd., model HB0233; potato dextrose water medium was purchased from Haibo Biotechnology Co., Ltd., model HB0233-4. Oat bran was purchased from Yili flagship store; dried lily was purchased from Jinliang Food Store, product number 12153370668; Chinese yam was purchased from Yonghuaitang flagship store; seabuckthorn was purchased from Shaanxi Guben Biotechnology Co., Ltd., number GBSJF20200524; rice was purchased from Shiyue Rice Field flagship store, product number: 6971599052029-3; Panax notoginseng was purchased from Wenshan Dongzhi Health Hall; Angelica dahurica was purchased from Songcaotang flagship store.

[0077] In the following embodiments, Cordyceps militaris YCC127 is Cordyceps militaris YCC127, the deposit number is CGMCC No.41663, the deposit date is November 19, 2024, the depository is the General Microbiological Center of China Microorganism Culture Collection Administration (referred to as CGMCC), the address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the zip code is 100101.

[0078] In the following examples, the yeast is Saccharomyces cerevisiae YWY-1, with a deposit number of CGMCC No. 17452 and a deposit date of March 27, 2019. The depository is the General Microbiological Center of China National Microbiological Culture Collection Administration (CGMCC for short), with an address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a zip code of 100101. In the following examples, the preparation method of YWY-1 yeast culture liquid is as follows: (1) YWY-1 yeast is inoculated into yeast extract peptone glucose agar medium (YPD, Haibo Biotechnology Co., Ltd., batch number HB5193), cultured at 30°C for 3-5 days, and set aside. (2) The single colony obtained in step (1) is inoculated into 300 mL of YPD liquid culture medium, cultured at 30°C and 200 rpm for 30 hours, and a YWY-1 culture liquid with a concentration of 10 6 -10 7 of CFU / mL.

[0079] Example 1

[0080] 1. Isolation of Cordyceps militaris YCC127

[0081] In this study, wild fruiting bodies of Cordyceps militaris were collected from Kunming, Yunnan Province (25°04'41.3"N 102°50'11.5"E). First, the collected fruiting bodies of Cordyceps militaris were thoroughly washed with running water to remove dirt and impurities, and then soaked in 75% alcohol for 1-2 minutes. After disinfecting the surface, they were rinsed with sterile water three times to remove residual alcohol. Then, the base of the fruiting body was cut with a sterile scalpel, and its healthy, disease-free tissue was taken for inoculation.

[0082] Place the treated tissue in a culture dish containing PDA (potato dextrose agar medium). After inoculation, place the culture dish in a 25°C incubator and culture it in a dark environment for 7-10 days to observe whether mycelium grows. When mycelium begins to grow, pick up the mycelium part with a sterile inoculation loop and transfer it to a new culture medium to ensure the purity of the strain.

[0083] In order to further purify the strain, two or more purification cultures were performed. During each purification, a healthy and uncontaminated part of the mycelium was selected and transferred to a new PDA medium, and cultured for 5-7 days in a dark environment at 25°C to finally obtain a pure Cordyceps militaris strain YCC127.

[0084] 2. Morphological identification of Cordyceps militaris YCC127

[0085] The purified mycelium of Cordyceps militaris was inoculated on PDA medium. After the mycelium was fully grown, samples were taken for microscopic observation. The mycelium of Cordyceps militaris was white or light beige, with relatively thick mycelium, about 3-5μm in diameter, densely distributed mycelium, and radial or radial growth direction. The mycelium cell wall was relatively strong and uniform, straight or slightly curved, and the internode distance was relatively short. The mycelium was undivided or occasionally had short transverse septa. The surface of the mycelium was relatively smooth, and it was common to see obvious connections or interweaving between the mycelium, showing a typical group growth pattern.

[0086] 3. Storage of Cordyceps militaris YCC127

[0087] The YCC127 strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, zip code 100101) on November 19, 2024, with a deposit number of CGMCC No. 41663 and a classification name of Cordyceps militaris. The full name of YCC127 is Cordyceps militaris YCC127 CGMCC No. 41663.

[0088] Example 2 Preparation of fermented oat bran-Cordyceps militaris fermentation product

[0089] (1) Preparation of Cordyceps militaris YCC127 seed solution: Two culture pieces (circular bacterial pieces with a diameter of 0.5 cm) were cut from the solid plate Cordyceps militaris YCC127 strain and transferred to 300 mL potato glucose water culture medium for preliminary culture at a temperature of 25° C. and a shaking speed of 180 rpm. The preliminary culture was carried out for 7 days to obtain a fermentation liquid with bacterial balls (at this time, it can be observed that the Cordyceps militaris strain gradually grows in the culture medium and forms bacterial balls of different sizes and large morphological differences). Then, the fermentation liquid with the bacterial balls is manually homogenized in a clean bench with a sterilized glass homogenizer to break the homogenized bacterial balls into fine (rice-sized) first-generation mycelia to obtain a homogenized bacterial seed liquid. 20 mL of the homogenized bacterial seed liquid is inoculated again into a new 300 mL potato glucose water culture medium for secondary activation culture at a temperature of 28° C. and a shaking speed of 180 rpm. The culture is continued for 7 days to obtain a second-generation mycelia until the volume of the second-generation mycelia fills two-thirds of the volume of the entire culture medium to obtain a Cordyceps militaris seed liquid.

[0090] (2) Preparation of fermented oat bran freeze-dried powder: Oat bran is dried and crushed with a pulverizer to obtain oat bran powder for later use. 3 g of oat bran powder is added to 300 mL of distilled water to obtain a fermentation system, sterilized at 121° C. for 30 min, cooled to room temperature, and then YWY-1 yeast liquid is inoculated into the above fermentation system at an inoculation amount of 10% (volume ratio v / v), and fermented and cultured for 30 h at a temperature of 30° C. and a rotation speed of 200 rpm. After the fermentation and culture is completed, the fermentation product is obtained by sterilization at 121° C. for 30 min, and the fermentation product is freeze-dried and crushed into powder with a pulverizer to obtain fermented oat bran freeze-dried powder.

[0091] (3) Preparation of fermented oat bran-Cordyceps militaris fermentation product: 3 g of the fermented oat bran freeze-dried powder prepared in step (2) was added to 300 mL of distilled water, sterilized at 121° C. for 30 min, cooled to room temperature, and then the Cordyceps militaris seed solution prepared in step (1) was inoculated at an inoculum amount of 10% (volume ratio v / v) into the sterilized fermented oat bran freeze-dried powder aqueous solution, and fermented and cultured at a temperature of 28° C. and a shaking speed of 180 rpm for 5 days. After the fermentation and culture was completed, the mixture was sterilized at 121° C. 30 minutes, cooled to room temperature, centrifuged and filtered at a speed of 4800 rpm for 20 minutes to obtain fermented mycelium; distilled water was added to the obtained fermented mycelium at a solid-liquid ratio of 1:20 (i.e., 20 ml of distilled water was added to 1 g of fermented mycelium), stirred at 80°C, and water extracted 3 times, each extraction time was 1 hour. After the extraction was completed, the extract and the residue were separated, the extract was collected, and the extract was concentrated by vacuum rotary evaporation, freeze-dried, and crushed into powder to obtain fermented oat bran-Cordyceps militaris fermentation product.

[0092] Example 3 Preparation of oat bran-Cordyceps militaris fermentation product

[0093] (1) Preparation of Cordyceps militaris YCC127 seed solution: same as step (1) of Example 2.

[0094] (2) Dry the oat bran, and grind it with a grinder to obtain oat bran powder for later use.

[0095] (3) Preparation of oat-Cordyceps militaris fermented product: 3 g of oat bran powder was added to 300 mL of distilled water, sterilized at 121° C. for 30 min, and cooled to room temperature; then the Cordyceps militaris seed solution prepared in step (1) was inoculated into the sterilized oat bran aqueous solution at an inoculum amount of 10% (volume ratio v / v); fermentation was carried out at 28° C. and a shaking speed of 180 rpm for 5 days; after the fermentation was completed, the solution was sterilized at 121° C. for 30 min, cooled to room temperature, and centrifuged at a speed of 4800 rpm for 20 min to obtain fermented mycelium; distilled water was added to the obtained fermented mycelium at a solid-liquid ratio of 1:20 (i.e., 20 ml of distilled water was added to 1 g of fermented mycelium); the solution was stirred at 80° C. and extracted with water for 3 times, each extraction time being 1 h; after the extraction was completed, the extract and the residue were separated, the extract was collected, the extract was concentrated by vacuum rotary evaporation, freeze-dried, and pulverized into powder to obtain oat bran-Cordyceps militaris fermented product.

[0096] Example 4 Preparation of fermented lily-Cordyceps militaris fermentation product

[0097] (1) Preparation of Cordyceps militaris YCC127 seed solution: same as step (1) of Example 2.

[0098] (2) Preparation of lyophilized fermented lily powder: Dry the dried lily and crush it with a pulverizer to obtain lily powder for later use. Add 3 g of lily powder to 300 mL of distilled water to obtain a fermentation system, sterilize it at 121° C. for 30 min, cool it to room temperature, and then inoculate YWY-1 yeast liquid in the fermentation system at an inoculation rate of 10% (volume ratio v / v), and ferment and culture it at 30° C. and a rotation speed of 200 rpm for 30 h. After the fermentation and culture is completed, sterilize it at 121° C. for 30 min to obtain a fermentation product, freeze-dry the fermentation product, and crush it into powder with a pulverizer to obtain fermented lily lyophilized powder.

[0099] (3) Preparation of Cordyceps militaris fermented product: 3 g of the fermented lily freeze-dried powder obtained in step (2) was added to 300 mL of distilled water, sterilized at 121° C. for 30 min, and cooled to room temperature. Then, the Cordyceps militaris seed solution prepared in step (1) was inoculated into the sterilized fermented lily freeze-dried powder aqueous solution at a volume ratio of 10% (v / v), and fermented and cultured at 28° C. with a shaking speed of 180 rpm for 5 days. After the fermentation and culture was completed, the solution was sterilized at 121° C. for 30 min, cooled to room temperature, and centrifuged at a speed of 4800 rpm for 20 min to obtain fermented mycelium; distilled water was added to the obtained fermented mycelium at a solid-liquid ratio of 1:20 (i.e., 20 ml of distilled water was added to 1 g of fermented mycelium), stirred at 80° C., and extracted with water for 3 times, each extraction time being 1 h. After the extraction was completed, the extract and the residue were separated, the extract was collected, and the extract was concentrated by vacuum rotary evaporation, freeze-dried, and crushed into powder to obtain the fermented lily-Cordyceps militaris fermented product.

[0100] Example 5 Lily-Cordyceps militaris fermentation product

[0101] (1) Preparation of Cordyceps militaris YCC127 seed solution: same as step (1) of Example 2.

[0102] (2) Dry the dried lily bulbs, and grind them into powder using a grinder. Set aside.

[0103] (3) Preparation of Cordyceps militaris fermented product: 3 g of lily powder prepared in step (2) was added to 300 mL of distilled water, sterilized at 121° C. for 30 min, and cooled to room temperature. Then, the Cordyceps militaris seed solution prepared in step (1) was inoculated into the sterilized lily powder aqueous solution at a volume ratio of 10% (volume ratio v / v), and fermented and cultured at 28° C. and a shaking speed of 180 rpm for 5 days. After the fermentation and culture was completed, the product was sterilized at 121° C. for 30 min, and centrifuged and filtered at a speed of 4800 rpm for 20 min to obtain fermented mycelium. Distilled water was added to the obtained fermented mycelium at a solid-liquid ratio of 1:20 (i.e., 20 ml of distilled water was added to 1 g of fermented mycelium), and the product was stirred at 80° C. and extracted with water for 3 times, each extraction time being 1 h. After the extraction was completed, the extract and the residue were separated, the extract was collected, and the extract was vacuum rotary evaporated to obtain lily-Cordyceps militaris fermented product.

[0104] Example 6 Preparation of oat bran PD-Cordyceps militaris fermentation product

[0105] (1) Preparation of Cordyceps militaris YCC127 seed solution: the same as step (1) of Example 2.

[0106] (2) Preparation of oat bran PD-Cordyceps militaris fermentation product: 3 g of oat bran powder (obtained by drying and crushing oat bran) was added to 300 mL of potato dextrose water medium (PD), sterilized at 121° C. for 30 min, cooled to room temperature, and then the Cordyceps militaris seed solution prepared in step (1) was inoculated into the sterilized medium at an inoculum amount of 10% (volume ratio v / v), and fermented and cultured for 5 days at a temperature of 28° C. and a shaking speed of 180 rpm. After the fermentation and culture was completed, 121° C. was added to the medium. ℃ sterilization for 30 minutes, cooled to room temperature, centrifuged and filtered at a speed of 4800 rpm for 20 minutes to obtain fermented mycelium; distilled water was added to the obtained fermented mycelium at a solid-liquid ratio of 1:20 (i.e., 20 ml of distilled water was added to 1 g of fermented mycelium), stirred at 80℃, and water extracted 3 times, each extraction time was 1 hour. After the extraction was completed, the extract and the residue were separated, the extract was collected, the extract was concentrated by vacuum rotary evaporation, freeze-dried, and crushed into powder to obtain oat bran PD-Cordyceps militaris fermentation product.

[0107] Example 7 Preparation of Lily PD-Cordyceps militaris Fermentation

[0108] (1) Preparation of Cordyceps militaris YCC127 seed solution: the same as step (1) of Example 6.

[0109] (2) Preparation of Lily PD-Cordyceps militaris fermentation product: The same as step (2) of Example 6, except that the oat bran powder is replaced by lily powder (obtained by drying and crushing lily).

[0110] Comparative Example 1 Preparation of Cordyceps militaris PD fermentation product

[0111] (1) Preparation of Cordyceps militaris YCC127 seed solution: same as step (1) of Example 2.

[0112] (2) Preparation of Cordyceps militaris PD fermentation product: sterilize a potato glucose water culture medium (PD) at 121° C. for 30 min, cool to room temperature, inoculate the Cordyceps militaris seed solution prepared in step (1) into the sterilized potato glucose water culture medium at an inoculum amount of 10% (volume ratio v / v), and ferment and culture for 5 days at a temperature of 28° C. and a shaking speed of 180 rpm. After the fermentation and culture, sterilize at 121° C. for 30 min, and centrifuge and filter at a speed of 4800 rpm for 20 min to obtain fermentation mycelium; add distilled water to the obtained fermentation mycelium at a solid-liquid ratio of 1:20 (i.e., add 20 ml of distilled water to 1 g of fermentation mycelium), stir at 80° C., and extract three times with water, each extraction time being 1 h. After the extraction is completed, separate the extract and the residue, collect the extract, concentrate the extract by vacuum rotary evaporation, and freeze-dry to obtain Cordyceps militaris PD fermentation product.

[0113] Comparative Example 2 Preparation of Cordyceps militaris fermented products with other different additives

[0114] 2-1 Preparation of Chinese Yam-Cordyceps militaris Fermentation

[0115] (1) Preparation of Cordyceps militaris YCC127 seed solution: the same as step (1) of Example 6.

[0116] (2) Preparation of Chinese yam-Cordyceps militaris fermented product: The same as step (2) of Example 6, except that the oat bran powder is replaced with Chinese yam powder to prepare Chinese yam-Cordyceps militaris fermented product.

[0117] 2-2 Preparation of Seabuckthorn-Cordyceps militaris Fermentation

[0118] (1) Preparation of Cordyceps militaris YCC127 seed solution: the same as step (1) of Example 6.

[0119] (2) Preparation of seabuckthorn-Cordyceps militaris fermentation product: The same as step (2) of Example 6, except that the oat bran powder is replaced by seabuckthorn powder.

[0120] 2-3 Preparation of Rice-Cordyceps militaris Fermentation

[0121] (1) Preparation of Cordyceps militaris YCC127 seed solution: the same as step (1) of Example 2.

[0122] (2) Preparation of rice-Cordyceps militaris fermentation product: The same as step (2) of Example 6, except that the oat bran powder is replaced by rice flour.

[0123] 2-4 Preparation of Panax notoginseng-Cordyceps militaris fermentation product

[0124] (1) Preparation of Cordyceps militaris YCC127 seed solution: the same as step (1) of Example 2.

[0125] (2) The preparation of Panax notoginseng-Cordyceps militaris fermentation product is the same as step (2) of Example 6, except that the oat bran powder is replaced by Panax notoginseng powder.

[0126] 2-5 Preparation of Angelica dahurica-Cordyceps militaris fermentation product

[0127] (1) Preparation of Cordyceps militaris YCC127 seed solution: the same as step (1) of Example 2.

[0128] (2) Preparation of Angelica dahurica-Cordyceps militaris fermentation product: The same as step (2) of Example 6, except that the oat bran powder is replaced by Angelica dahurica powder.

[0129] Effect Example 1 Analyzing the Effect of Different Additives on the Growth of Cordyceps Militaris Mycelium by Evaluating Biomass

[0130] By comparing the biomass, the adaptability of Cordyceps militaris in the fermentation system was confirmed. The fermentation mycelium was re-prepared according to the methods of Examples 2-7, Comparative Examples 1 and 2, and the mycelium obtained according to the methods of Examples 2-7, Comparative Examples 1 and 2 was rinsed with distilled water, dried at 60°C to constant weight, and weighed. Three parallel experiments were set up to record the biomass. The results are shown in Table 1.

[0131] Table 1 Mycelium weight (g) test results of the embodiments and comparative examples

[0132]

[0133] From the results, it can be seen that compared with Comparative Example 1, the addition of other substrates in Comparative Examples 2-1 to 2-5, and the addition of oat bran powder and lily powder in Examples 6-7, all help to increase the mass of Cordyceps militaris mycelium, among which oat bran powder and lily powder have a more significant effect of promoting the growth of Cordyceps militaris. Although the addition of other substances can also appropriately increase the weight of mycelium, none of them is as significant as oat bran powder and lily powder. Compared with Examples 6 and 7, the substrates of Examples 3 and 5 do not have potato glucose water culture medium, and the mass of the mycelium of Examples 3 and 5 is correspondingly lower than that of Examples 6 and 7.

[0134] Effect Example 2 The antioxidant effects of different fermentations were analyzed by biochemical level DPPH free radical scavenging ability test and hydroxyl free radical scavenging ability test.

[0135] 2.1: The products obtained in Examples 2-5 and Comparative Example 1 were prepared with distilled water into test samples with concentrations of 0.25 mg / mL, 0.5 mg / mL, 1.5 mg / mL, 2 mg / mL, and 4 mg / mL, respectively, and the DPPH free radical scavenging ability was determined. The specific test method was as follows: (1) an equal volume (1 mL) of the test sample was mixed with a 0.2 mol / L DPPH solution (A 1 (2) Take an equal volume (1 mL) of anhydrous ethanol (solvent for the test substance) and mix it with 0.2 mol / L DPPH solution (A 2 (3) Take an equal volume (1 mL) of anhydrous ethanol and mix it with the test solution (A 3 (4) After 30 min of reaction in dark, measure A at 517 nm. 1 Tube, A 2 Tube, A 3 The absorbance value of the tube; the calculation formula of DPPH free radical scavenging rate is: DPPH free radical scavenging rate = [(A 2 +A 3 )-A 1 ] / A 2 × 100%. The calculation results of DPPH free radical scavenging rate are shown in Table 2.

[0136] Table 2 DPPH free radical scavenging ability test results

[0137] Sample concentration 0.25mg / mL 0.5mg / mL 1.5mg / mL 2mg / mL 4mg / mL Example 2 43.26％ 48.50％ 57.03％ 60.52％ 77.50％ Example 3 10.38％ 24.93％ 51.11％ 66.05％ 60.23％ Example 4 42.29％ 47.53％ 50.73％ 59.26％ 80.89％ Example 5 15.03％ 37.05％ 48.88％ 59.94％ 73.23％ Comparative Example 1 22.60％ 39.09％ 43.84％ 48.30％ 59.07％

[0138] 2.2: The products obtained in the above-mentioned Examples 2-5 and Comparative Example 1 were prepared with distilled water to prepare test samples of 0.375 mg / mL, 0.75 mg / mL, 1.5 mg / mL, 3 mg / mL, and 6 mg / mL, respectively, and the hydroxyl radical scavenging ability was determined. The specific test method is: using the Fenton reaction to generate hydroxyl radicals (·OH), adding salicylic acid to the reaction system, ·OH reacts with salicylic acid, and the generated colored compound 2,3-dihydroxybenzoic acid has characteristic absorption at 510nm. Using the fixed reaction time method, the absorbance of the reaction solution containing the test substance is measured at 510nm, and compared with the blank solution, the scavenging effect of the tested sample on the ·OH determination is determined.

[0139] The test solution is a test sample prepared from the products prepared in the above embodiments and comparative examples; reagents are added to the test tube according to the formula in Table 3, and 9 mmol / L FeSO is added in sequence. 4 , 9mmol / L salicylic acid ethanol solution, the sample to be tested, appropriate amount of deionized water and 8.8mmol / L H 2 O 2 Shake well, heat in a 37℃ water bath for 15min, take out, and measure the absorbance of the blank control group (marked A) 0 , the absorbance of the sample group to be tested (marked B) is Ax and the absorbance of the sample group without H 2 O 2 The background absorbance of the solution in group (marked C) is Ax 0 . Determination of A 0 The reference solution was a system without hydrogen peroxide. Three parallel experiments were performed in each group. The absorbance values ​​were tested and the average value was taken. The hydroxyl radical scavenging rate of each group was calculated according to the following formula: Hydroxyl radical scavenging rate = [A 0 -(Ax-Ax 0 )] / A 0 × 100%, the results are shown in Table 4 and Figure 2 .

[0140] Table 3 Settings of each group in the hydroxyl radical scavenging experiment

[0141] Group <![CDATA[V(FeSO 4 ) / mL]]> V(salicylic acid) / mL V(sample) / mL V(deionized water) / mL <![CDATA[V(H 2 About 2 ) / mL]]> Blank control 0.5 0.5 0 6 0.5 Sample set 0.5 0.5 2.5 3.5 0.5 Solution background group 0.5 0.5 2.5 4 0

[0142] Table 4 Hydroxyl radical scavenging ability test results

[0143] concentration 0.375mg / mL 0.75mg / mL 1.5 mg / mL 3mg / mL 6mg / mL Example 2 11.45％ 15.51％ 27.45％ 31.91％ 79.08％ Example 3 16.69％ 31.61％ 40.72％ 44.58％ 62.10％ Example 4 12.53％ 26.82％ 47.67％ 51.09％ 53.73％ Example 5 7.05％ 19.18％ 28.53％ 47.17％ 66.75％ Comparative Example 1 8.86％ 14.58％ 15.17％ 43.80％ 69.88％

[0144] According to the relationship between a series of scavenging rates and concentrations commonly used in the art, the concentration of the test sample required when the free radical scavenging rate of Examples 2-5 and Comparative Example 1 is 50%, i.e., the IC50 value, is calculated. The smaller the IC50 value, the better the free radical scavenging ability of the product. The IC50 values ​​of Examples 6 and 7 are measured in the same manner as in Examples 2-5, and the results are shown in Table 5.

[0145] Table 5 Concentration of test samples required for free radical scavenging rate of 50% - IC50 value

[0146] serial number name DPPH free radical IC50 value / mg / mL Hydroxyl free radical IC50 value / mg / mL Example 2 Fermented oat bran-Cordyceps militaris fermented product 0.60 2.75 Example 3 Oat bran-Cordyceps militaris fermentation 1.51 3.01 Example 4 Fermented Lily-Cordyceps militaris fermented product 0.73 2.93 Example 5 Lily-Cordyceps militaris fermentation 1.27 3.17 Example 6 Oat bran PD-Cordyceps militaris fermentation 1.50 3.03 Example 7 Lily PD-Cordyceps militaris fermentation 1.14 3.10 Comparative Example 1 Cordyceps militaris PD fermentation 1.96 3.22

[0147] As shown in Table 5, the IC50 values ​​of DPPH radical scavenging ability of Cordyceps militaris-related fermentations are generally lower than the IC50 values ​​of hydroxyl radical scavenging ability, indicating that the DPPH radical scavenging ability is generally stronger than the hydroxyl radical scavenging ability. In terms of DPPH radical scavenging ability, the fermented oat bran-Cordyceps militaris fermentation has the best scavenging ability, with an IC50 value of only 0.60 mg / mL, followed by the fermented lily-Cordyceps militaris fermentation with an IC50 value of 0.73 mg / mL. The IC50 values ​​of oat bran-Cordyceps militaris fermentation and lily-Cordyceps militaris fermentation are both greater than the corresponding fermented oat bran-Cordyceps militaris fermentation and fermented lily-Cordyceps militaris fermentation. The IC50 value of oat bran PD-Cordyceps militaris fermentation is not much different from the corresponding oat bran-Cordyceps militaris fermentation, indicating that the fermentation with potato glucose water as the substrate has no significant effect on the free radical scavenging ability; the IC50 value of lily PD-Cordyceps militaris fermentation is lower than that of the corresponding lily-Cordyceps militaris fermentation, indicating that the fermentation with potato glucose water as the substrate has an improved effect on the free radical scavenging ability. However, the scavenging ability of both is not as high as that of fermented oat bran-Cordyceps militaris fermentation and fermented lily-Cordyceps militaris fermentation. The control group Cordyceps militaris PD fermentation with no other ingredients added and only potato glucose water is at the end, with an IC50 value of 1.96 mg / mL. This shows that the addition of oat bran and lily powder or their fermentation freeze-dried powder can help improve the DPPH free radical scavenging ability of Cordyceps militaris fermentation.

[0148] In terms of hydroxyl radical scavenging ability, the fermented oat bran-Cordyceps militaris fermentation product has the best scavenging ability, with an IC50 value of 2.75 mg / mL, and the addition of fermented oat bran or fermented lily powder is more conducive to the scavenging of hydroxyl radicals than the direct addition of oat bran or lily powder. The IC50 value of oat bran PD-Cordyceps militaris fermentation product is not much different from the corresponding oat bran-Cordyceps militaris fermentation product, indicating that the fermentation product with potato glucose water as a substrate has no significant effect on the scavenging ability of hydroxyl radicals; the IC50 value of lily PD-Cordyceps militaris fermentation product is lower than that of the corresponding lily-Cordyceps militaris fermentation product, indicating that the fermentation product with potato glucose water as a substrate has an improved effect on the scavenging ability of free radicals. As shown in Table 5, the fermented oat bran-Cordyceps militaris fermentation product has the best free radical scavenging ability.

[0149] In order to facilitate labeling, fermented oat bran-Cordyceps militaris fermentation product will be referred to as YM in the future.

[0150] Effect Example 3 Cytotoxicity Detection

[0151] The HFF cells used in this experiment and subsequent effect experiments were obtained from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, to verify the cytotoxicity of the products prepared in the above examples.

[0152] The manufacturer of CCK-8 kit is Biorigin, model BN15201. The manufacturer of DMEM culture medium is INVITROGEN, model C11995500BT. Fetal bovine serum is purchased from Gibico, model SH30070.03. Streptomycin (double antibody) is purchased from INVITROGEN, model 15140122.

[0153] Preparation of test samples: The product prepared in Example 2 was prepared with serum-free DMEM culture medium into test solutions of the experimental group with concentrations of 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 6 mg / mL, and 8 mg / mL.

[0154] Test method: HFF cells were supplemented with 15% fetal bovine serum and 1% penicillin (1×10 5 U / L) and streptomycin (100 mg / L) in DMEM medium. The HFF cells were cultured at 37°C in an environment with 5% carbon dioxide. When the cell confluence reached 80%, the medium was renewed, and the cells in the logarithmic growth phase were digested with 0.25% (EDTA) trypsin (INVITROGEN, model 25200056). The digestion reaction was terminated with serum-containing DMEM to obtain a cell suspension.

[0155] According to the manufacturer's instructions of the CCK-8 kit, HFF cell suspension was incubated at 37°C at 1 × 10 cells per well. 4 Cells were seeded into 96-well plates at a concentration of 10 cells / mL and incubated in a cell culture incubator at 37°C, 5% CO. 2 Incubate for 12 hours. After incubation, remove the old culture medium and wash the cells twice with phosphate buffered saline.

[0156] In each well of the experimental group, 200 μL of the above-prepared, filtered and sterilized experimental group test solution of different concentrations was added, and 6 replicate wells were made for each test solution. The wells were incubated for 24 hours. The determination method was referred to the CCK-8 kit manual.

[0157] The absorbance value was measured at a wavelength of 450 nm, and the cell survival rate of each group was calculated. The results are shown in Table 2 and Figure 3 The calculation formula of cell survival rate is as follows: Cell survival rate (%) = (A experimental group - A blank control group) / (A control group - A blank control group) × 100%. The results of cytotoxicity experiments at different concentrations are shown in Figure 1 .

[0158] Depend on Figure 1 It can be seen that the fermented oat bran-Cordyceps militaris fermented product of Example 2 has a low cytotoxicity. It has a proliferation-promoting effect on HFF cells under low concentration conditions. The maximum concentration (3 mg / mL) that is non-toxic to cells (cell survival rate of more than 80%) is selected as the action concentration for subsequent cytotoxicity experiments, cell aging marker evaluation, antioxidant capacity test, and dermal collagen content change detection cell experiment.

[0159] Effect Example 4 Establishment of UVA-induced and hydrogen peroxide-induced HFF cell aging models

[0160] The same HFF cell suspension as in Example 1 was added at 1×10 4 The cells were inoculated into each well of a 96-well plate at a density of 10 cells / mL and cultured in a cell culture incubator for 12 h. The cell adhesion status was observed and the cells were washed with 1 mL PBS (pH 7.4, 0.01 M) and aspirated. For the UVA model, 1 mL PBS (pH 7.4, 0.01 M) was added and the concentrations were 0 mJ / cm 2 , 2mJ / cm 2 , 4mJ / cm 2 、6mJ / cm 2 、8mJ / cm 2 、10mJ / cm 2The cells were stimulated with different doses of UVA; the hydrogen peroxide model was stimulated with 250μmol / L, 312μmol / L, 500μmol / L, 525μmol / L, 1000μmol / L, 1250μmol / L, 2000μmol / L, and 2500μmol / L hydrogen peroxide for 2h. The viability of HFF cells was determined by CCK-8 method. The model establishment results are shown in Figure 2 According to the CCK-8 results, 50% survival rate was selected as the standard, and the UVA model establishment condition was determined to be 8mJ / cm 2 , the hydrogen peroxide model was 1250μmol / L, and the treatment was 2h.

[0161] Effect Example 5 Evaluation of Cell Senescence Markers

[0162] β-Galactosidase staining is a classic staining method that is effective in detecting cell senescence. It uses X-Gal as a substrate and catalyzes the stable expression of intracellular β-galactosidase under acidic conditions to generate a dark blue deposition product. Blue-expressing cells are observed under a microscope as the molecular characteristics of positive senescent cells.

[0163] Preparation of the test sample: The product prepared in Example 2 above was prepared into a test solution of the experimental group with a concentration of 3 mg / mL using serum-free DMEM culture medium.

[0164] Test method: The same HFF cell suspension as in Example 1 was incubated at 37°C with 3×10 5 The cells were seeded into six-well plates at a concentration of 10 cells / mL and incubated in a cell culture incubator at 37°C and 5% CO. 2 Incubate for 24 hours. After incubation, remove the old culture medium and wash the cells twice with PBS buffer. In the UVA model group, each well was irradiated with UVA under 1 mL PBS, washed once with PBS after irradiation, and added with 2 mL of serum-free DMEM medium for 24 hours; the experimental group under the UVA model was washed once with PBS after irradiation, and then 2 mL of the above-prepared, filtered and sterilized experimental group test solution with a concentration of 3 mg / mL was added for a certain period of time; the hydrogen peroxide model group was added with 2 mL of 1250 μmol / L hydrogen peroxide solution per well, at 37°C, 5% CO 2Incubate for 2 hours under the environment, then wash once with PBS, add 2mL serum-free DMEM medium and culture for 24 hours; the experimental group under the hydrogen peroxide model was treated with hydrogen peroxide, washed once with PBS, and then added 2mL of the above-prepared filtered and sterilized experimental group test solution with a concentration of 3mg / mL for 24 hours; the positive control used vitamin C (VC), and 2mL of 50μg / mL vitamin C was added to each well of the positive control group for 24 hours. The blank control group was not stimulated by UVA or hydrogen peroxide, and 2mL serum-free DMEM was added for 24 hours.

[0165] The cells were stained with a β-galactosidase staining kit (the kit was purchased from Bio-Tech, model C0602) after stimulation for 0 h, 8 h, 16 h, and 24 h, respectively. The β-galactosidase staining photos were quantitatively analyzed based on the image analysis software Image J. The number of positive cells and the total number of cells were calculated to obtain the senescent cell ratio (i.e., the proportion of positive cells). Figure 3 .from Figure 3 It can be seen that after treating cells with fermented oat bran-Cordyceps militaris fermentation product YM for different time periods (8h, 16h, 24h), the positive proportion of senescent cells decreased, indicating that YM has the effect of delaying the aging of skin cells.

[0166] Effect Example 6 Antioxidant Capacity Test

[0167] When there are too many free radicals in cells and insufficient antioxidant enzymes, free radicals cannot be removed in time, which will cause cell apoptosis and tissue damage. The free radical theory of aging believes that this is also the root cause of aging. In the human body's antioxidant enzyme system, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) work synergistically to effectively remove excess free radicals. The malondialdehyde (MDA) content is an important parameter that reflects the body's antioxidant potential. It can reflect the body's lipid peroxidation rate and intensity, and can also indirectly reflect the degree of peroxidation damage.

[0168] Preparation of the test sample: The product prepared in Example 2 above was prepared into a test solution of the experimental group with a concentration of 3 mg / mL using serum-free DMEM culture medium.

[0169] Test method: The same HFF cell suspension as in Example 1 was incubated at 37°C with 1×10 4 Cells were seeded into six-well plates at a concentration of 10 cells / mL and incubated in a cell culture incubator at 37°C, 5% CO. 2Incubate for 24 hours. After incubation, remove the old culture medium and wash the cells twice with PBS buffer. In the UVA model group, each well was irradiated with UVA under 1 mL PBS, washed once with PBS after irradiation, and added with 2 mL of serum-free DMEM medium for 24 hours; the experimental group under the UVA model was washed once with PBS after irradiation, and then 2 mL of the above-prepared, filtered and sterilized experimental group test solution with a concentration of 3 mg / mL was added for 24 hours; the hydrogen peroxide model group was added with 2 mL of 1250 μmol / L hydrogen peroxide solution per well, at 37°C, 5% CO 2 The cells were incubated for 2 h under the environment, then washed once with PBS, and 2 mL of serum-free DMEM medium was added for 24 h; the experimental group under the hydrogen peroxide model was treated with hydrogen peroxide, washed once with PBS, and then 2 mL of the above-prepared, filtered and sterilized experimental group test solution with a concentration of 3 mg / mL was added for 24 h; the positive control used vitamin C (VC), which was treated with UVA or hydrogen peroxide, and 2 mL of 50 μg / mL vitamin C was added to each well of the positive control group for 24 h; the blank control group was not stimulated with UVA or hydrogen peroxide, and 2 mL of serum-free DMEM was added for 24 h.

[0170] After cultivation, the cell supernatants in the six-well plates were collected and centrifuged at 10000r / min for 10 min. The supernatants were collected for later use. This was the cell culture supernatant. 200 μL of lysis solution was added to each well (the lysis solution was prepared with IP lysis solution and PMSF at a ratio of 100:1). After the cells were completely lysed, the cells were carefully scraped off with a cell scraper and the cell lysate was collected. The plates were centrifuged at 10000r / min for 10 min. The supernatants were collected for later use. This was the cell lysis supernatant.

[0171] The contents of GSH-Px, CAT, SOD and MDA were determined by using the GSH-Px kit (Biyuntian, S0057S), CAT kit (Biyuntian, S0051), SOD kit (Biyuntian, S0101S) and MDA kit (Biyuntian, S0131S) according to their respective test methods. The test results are shown in Figure 4 .Depend on Figure 4 It can be seen that the activities of GSH-Px, CAT and SOD in the cells protected by fermented oat bran-Cordyceps militaris fermentation product YM were significantly restored, and the MDA content was significantly decreased compared with the model group, indicating that fermented oat bran-Cordyceps militaris fermentation product YM has excellent antioxidant effect.

[0172] Effect Example 7 Detection of changes in collagen content in the dermis

[0173] Collagen is the main component of the extracellular matrix and the main component of dermal collagen. There are many types of collagen, including type I collagen (COL-I) and type III collagen (COL-III). Matrix metalloproteinase (MMP) is a type of metalloproteinase that can degrade extracellular matrix molecules of the dermal connective tissue of the skin. When reactive oxygen ROS accumulates, the expression of collagenase (MMP-1) and matrix degrading factor-1 (MMP-3) is induced by activating the expression of the NFκB signaling pathway, resulting in a decrease in the synthesis capacity of collagen and elastin (especially type I and type III collagen) in the extracellular matrix of dermal fibroblasts, which is manifested as thinning of the dermis, decreased skin firmness, and the formation of fine wrinkles. This experiment measured the protective effect of the fermented oat bran-Cordyceps militaris fermented product prepared in Example 2 on collagen at the mRNA level.

[0174] The same HFF cell suspension as in Example 1 was incubated at 37°C at 3×10 5 The cells were seeded into six-well plates at a concentration of 10 cells / mL and incubated in a cell culture incubator at 37°C and 5% CO. 2 Incubate for 24 hours. After incubation, remove the old culture medium and wash the cells twice with PBS buffer. In the UVA model group, each well was irradiated with UVA under 1 mL PBS, washed once with PBS after irradiation, and added with 2 mL of serum-free DMEM medium for 24 hours; the experimental group under the UVA model was washed once with PBS after irradiation, and then 2 mL of the above-prepared, filtered and sterilized experimental group test solution with a concentration of 3 mg / mL was added for 24 hours; the hydrogen peroxide model group was added with 2 mL of 1250 μmol / L hydrogen peroxide solution per well, at 37°C, 5% CO 2 The cells were incubated for 2 h under the environment, then washed once with PBS, and 2 mL of serum-free DMEM medium was added for 24 h; the experimental group under the hydrogen peroxide model was treated with hydrogen peroxide, washed once with PBS, and then 2 mL of the above-prepared, filtered and sterilized experimental group test solution with a concentration of 3 mg / mL was added for 24 h; the positive control used vitamin C (VC), which was treated with UVA or hydrogen peroxide, and 2 mL of 50 μg / mL vitamin C was added to each well of the positive control group for 24 h; the blank control group was not stimulated with UVA or hydrogen peroxide, and 2 mL of serum-free DMEM was added for 24 h.

[0175] Real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to study the expression level of genes. Total RNA extraction and cDNA synthesis were performed using TriQuick reagent (Solarbio, R1100) and UEIris II RT-PCR system. All synthesized cDNAs were purified by Fast Super qPCR Master Mix Kit (BIORIGIN, model BN12008) was used for PCR amplification. The total reaction system was 20 μL. The primer sequences used are shown in Table 6. Primers were designed using PrimerExpress software based on the gene sequences published in NCBI, and β-actin was used as the internal reference gene. The cycle parameters were 94°C pre-denaturation for 30 s, followed by PCR reaction (40 cycles, 94°C for 15 s, 60°C for 15 s, 72°C for 10 s), and fluorescence data was collected at 72°C. The reaction was performed on a QuanStudio 3 fluorescence quantitative PCR instrument.

[0176] Table 6 Real-Time PCR primer sequences

[0177]

[0178] Follow Fast Super The experiment was performed according to the steps specified in the instruction manual of the qPCR Master Mix Kit (BIORIGN, BN12008). The test results are shown in Figure 5 .Depend on Figure 5 It can be seen that H 2 O 2 After stimulation and UVA irradiation, the intracellular COL-I and COL-III contents decreased significantly, while after protection by fermented oat bran-Cordyceps militaris fermentation product YM, the decrease in collagen content was significantly reduced. During skin aging, fibroblasts secrete more MMP-1 and MMP-3. At the same time, ultraviolet rays and free radicals can also stimulate fibroblasts to produce more MMPs. The matrix metalloproteinase inhibitor TIMP-1 is considered to have protective significance for skin aging. Its expression in aging fibroblasts decreases, that is, the Model group of the two models decreases. After YM treatment, it can especially alleviate the decrease in TIMP-1 caused by the UVA model. The gene expression levels of MMP-1 and MMP-3 were measured by H 2 O 2 The expression increased significantly after stimulation and UVA irradiation, and the gene expression level in the model group was significantly reduced after protection by fermented oat bran-Cordyceps militaris fermentation product YM, indicating that fermented oat bran-Cordyceps militaris fermentation product YM alleviates the degradation of skin collagen and reduces the formation of skin wrinkles by reducing the overexpression of MMP-1 and MMP-3 genes.

[0179] In summary, through the changes in the proportion of senescence-positive cells, the activity of antioxidant enzymes, and the content of collagen, it was preliminarily determined that fermented oat bran-Cordyceps militaris fermentation product YM can enhance the activity of antioxidant enzymes and increase the content of collagen to exert anti-wrinkle and firming effects.

[0180] Effect Example 8

[0181] The applicant commissioned Hangzhou Huante Biotechnology Co., Ltd. to conduct zebrafish animal experiments, with the project number HZ9940. In the following examples, the zebrafish used were wild-type AB strain zebrafish, and the zebrafish were all raised in 28°C fish farming water (water quality: 200 mg instant sea salt was added to each 1L of reverse osmosis water, the conductivity was 450-550 μS / cm; pH was 6.5-8.5; hardness was 50-100 mg / L CaCO 3 ), bred and provided by the fish farming center of Hangzhou Huante Biotechnology Co., Ltd. The experimental animal use license number is SYXK (Zhejiang) 2022-0004, and the feeding and management meet the requirements of the international AAALAC certification (certification number: 001458), and the IACUC ethics review number is IACUC-2024-9940-01. Feed three times a day on weekdays, feed brine shrimp in the morning and afternoon, feed powdered flake feed at noon, and feed brine shrimp twice a day on weekends and holidays. The light / dark cycle is 14h / 10h. At about 16:00 one day before the experiment, the male and female broodstock were placed in a spawning tank with a sieve plate in a ratio of 2:1. The plate was removed at about 8:30 on the second day, and the fertilized eggs were collected at 10:00. The collected fish eggs were rinsed with 0.003% (v / v) sodium hypochlorite solution and placed in culture water, placed at 28°C, and continued to incubate in a light incubator.

[0182] 8.1 Zebrafish Animal Model Validation - Zebrafish Maximum Detection Concentration (MTC)

[0183] Preparation of test samples: The product prepared in Example 2 (i.e., fermented oat bran-Cordyceps militaris fermentation product (YM), also referred to as Cordyceps militaris mycelium extract) was prepared into a mother solution with a mass concentration of 1% using standard dilution water, and diluted to test solutions of the experimental group with mass concentrations of 0.01%, 0.025%, 0.05%, 0.1%, 0.25%, and 0.5% as required according to the experimental concentration.

[0184] Zebrafish 6 hours after fertilization (6hpf) were randomly selected and placed in a 6-well plate with a capacity of 3mL per well, with 30 fish per well. Experimental group, model control group, and normal control group (i.e., blank control group, Control group) were set up. In the experimental group, 800μmol / L of hydrogen peroxide was given to each well in water to establish the zebrafish aging model, and different volumes of 1% mother solution were added to each well as needed, so that the final concentration of the experiment was 0.01%, 0.025%, 0.05%, 0.1%, 0.25%, and 0.5%; in the model control group (Model group), 800μmol / L of hydrogen peroxide was given to each well in water to establish the zebrafish aging model, and no other substances were added; in the normal control group (control group), no hydrogen peroxide was given, and no other substances were added.

[0185] The liquid was replaced once a day and incubated at 28°C in the dark for 120 h. The MTC of the sample for the model zebrafish was determined based on the zebrafish mortality and toxicity. Figure 6 .from Figure 6 It can be seen that there was no death in zebrafish when the YM concentration was 0.05%, and the MTC of YM for model zebrafish was 0.05%.

[0186] 8.2 Evaluation of Cellular Senescence Markers in Zebrafish

[0187] β-galactosidase (SA-β-gal) is a marker of cell senescence. In senescent cells, the activity and expression of β-galactosidase increase. Therefore, the average staining intensity of β-galactosidase is detected to evaluate whether the sample has an anti-aging effect.

[0188] Preparation of the sample to be tested: The product prepared in Example 2 above was first prepared into a mother solution with a mass concentration of 1% with standard dilution water, and then diluted with standard dilution water to a 0.1% sub-mother solution, and diluted to a mass concentration of 0.0125%, 0.025%, and 0.05% experimental group test solution as required according to the experimental concentration; resveratrol was prepared into a mother solution with a mass concentration of 1% with DMSO, and diluted to a mass concentration of 0.002% positive control group test solution as required according to the experimental concentration. Zebrafish 6 hours after fertilization (6hpf) were randomly selected in a 6-well plate with a capacity of 3mL per well, 30 tails per well, and set up a sample group, a model control group, a normal control group (blank control group), and a positive control group. The experimental group was given 800 μmol / L hydrogen peroxide in water to establish a zebrafish aging model, and different volumes of 0.1% mother solution were added as needed to make the final concentration of the experimental group test solution 0.0125%, 0.025%, and 0.05%; the model control group was given 800 μmol / L hydrogen peroxide in water to establish a zebrafish aging model, and no other substances were added; the normal control group (blank control group) was not given hydrogen peroxide, and no other substances were added; the positive control group was given 800 μmol / L hydrogen peroxide to establish a zebrafish aging model, and 1% mother solution was added as needed to make the final concentration of the positive control group test solution 0.002%.

[0189] The liquid was changed every day. Incubate at 28°C in the dark for 120 h. The zebrafish were fixed overnight with 4% cell tissue fixative and stained with a β-galactosidase staining kit (Shanghai Biotech Co., Ltd., Cat. No. C0602). After staining overnight, 10 zebrafish were randomly selected from each experimental group and photographed under a dissecting microscope. The data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software. The average zebrafish β-galactosidase staining intensity (S) was analyzed and the following formula was used to calculate and determine whether the sample had an anti-aging effect. The results are shown in Tables 7 and Figure 7 .

[0190]

[0191] Table 7 Results of cell senescence test in zebrafish

[0192] serial number Detection concentration Anti-aging effect% P-value Test results Example 2 0.0125％ 50 <0.01 Significant Example 2 0.025％ 46 <0.05 Significant Example 2 0.05％ 44 >0.05 Not significant Resveratrol group 0.002％ 49 <0.001 Significant

[0193] From Table 7 and Figure 7 It was observed that the average β-galactosidase staining intensity of the sample experimental group was significantly reduced compared with the model control group, and YM had a significant effect in delaying cell aging.

[0194] 8.3 Enhanced mitochondrial function in zebrafish

[0195] Mitochondria are the main site of cellular energy production and the main source of ROS. Damage to mitochondria leads to disordered energy metabolism and the production of a large number of free radicals, which affects the normal growth of cells and causes aging of cells and even the body. Enhancing mitochondrial function can be achieved by increasing mitochondrial biogenesis, improving mitochondrial dynamics (fusion and fission), and activating mitochondrial antioxidant pathways.

[0196] Cobalt chloride is ionized in aqueous solution to produce Co 2+ After acting on cells, it can directly replace Fe in heme 2+ , so that hemoglobin cannot bind to oxygen and remains in a deoxygenated state, thereby simulating hypoxia; on the other hand, Co 2+ Will replace the Fe on the catalytic group 2+ , thereby blocking the activity of proline hydroxylase and aspartate hydroxylase, inhibiting the degradation of HIF-1α, and causing hypoxia damage after HIF-1α aggregation. The fluorescence intensity of mitochondrial fluorescence strain zebrafish is related to the number of mitochondria. Cobalt chloride induces hypoxia and hypoxic damage, affecting the normal function of the mitochondrial respiratory chain, and ultimately leading to the activation of mitochondrial autophagy. The damaged mitochondria are selectively cleared by the lysosomes in the body, thereby affecting the normal number of mitochondria in the zebrafish body and reducing the mitochondrial fluorescence intensity. The fluorescence intensity of mitochondria in the zebrafish body muscle is used to evaluate whether the sample has the effect of enhancing mitochondria.

[0197] Experimental system: Transgenic mitochondrial green fluorescent zebrafish (CZ222), bred and provided by the fish breeding center of Hangzhou Huante Biotechnology Co., Ltd. Zebrafish age: 4 days post fertilization (4dpf).

[0198] Preparation of the sample to be tested: The product prepared in Example 2 above was first prepared into a mother solution with a mass concentration of 1% using standard dilution water, and diluted to a test solution of the experimental group with a mass concentration of 0.05% as required according to the experimental concentration; glutathione was prepared into a mother solution with a mass concentration of 10 mmol / L using ultrapure water, and diluted to a positive control group test solution with a mass concentration of 300 μmol / L as required according to the experimental concentration.

[0199] Zebrafish were randomly selected in a 6-well plate with a capacity of 3 mL per well, with 15 fish per well. Water-soluble samples were given, and experimental group, normal control group (Control group), model control group, and positive control group (glutathione) were set up. Among them, 1% mother solution was added to the experimental group as needed, so that the final concentration of the experimental group test solution was 0.05%; no other substances were added to the normal control group and the model control group; 10mmol / L mother solution was added to the positive control group as needed, so that the final concentration of the experiment was 300μmol / L of the positive control group test solution;

[0200] Incubate at 28°C in the dark for 22 hours. Then, except for the normal control group, all other groups were given 300 μg / mL cobalt chloride hexahydrate in water to establish a zebrafish mitochondrial damage model, and incubated at 28°C in the dark for 4 hours. After the incubation, 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. The data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software, and the fluorescence intensity (S) of zebrafish body muscle mitochondria was analyzed. The formula was used to calculate and determine whether the sample had an enhanced mitochondrial effect.

[0201]

[0202] Fluorescence intensity microscope photos of zebrafish body muscle mitochondria are shown in Figure 8 , the results of the fluorescence intensity analysis of zebrafish body muscle mitochondria are shown in Fig. 9 The calculation results of the enhanced mitochondrial effect in zebrafish are shown in Table 8.

[0203] Table 8 Test results of enhancing mitochondrial function in zebrafish

[0204] serial number Detection concentration Enhance mitochondrial function% P-value Test results Example 2 0.05％ 74 <0.001 Significant Glutathione group 300μM 63 <0.001 Significant

[0205] from Figure 8 It can be observed that the fluorescence intensity of mitochondria in the zebrafish body muscle of the sample fermented oat bran-Cordyceps militaris fermentation product YM group was significantly increased compared with the model control group, indicating that YM has the effect of enhancing mitochondria, as shown in Table 8 and Fig. 9 This effect can also be confirmed.

[0206] 8.4 Evaluation of antioxidant activity in zebrafish

[0207] The reagent is a DNA dye with weak fluorescence itself. It can be oxidized by oxygen free radicals (ROS) in the cell. The oxidation products combine with DNA to produce bright green fluorescence. It is soluble in organic solvents (such as dimethyl sulfoxide), and the main component of zebrafish yolk sac is fat. The permeability of the yolk sac is strong, and the staining is obvious. Menadione can produce active oxygen free radicals. When the amount of free radicals produced is greater than the body's clearance capacity, oxidative stress occurs. The fluorescence intensity of the zebrafish yolk sac is used to evaluate whether the sample has an antioxidant effect.

[0208] Experimental system: melanin allele mutant zebrafish (Albino), bred and provided by the fish breeding center of Hangzhou Huante Biotechnology Co., Ltd. Zebrafish age: 2 days post fertilization (2dpf).

[0209] Preparation of the sample to be tested: The product obtained in Example 2 is first prepared into a mother solution with a mass concentration of 1% with standard dilution water, and then diluted with standard dilution water to a 0.1% sub-mother solution, and diluted to a mass concentration of 0.0125%, 0.025%, and 0.05% as required according to the experimental concentration. N-acetylcysteine ​​(NAC) is prepared into 1.25% (mass concentration) with ultrapure water, and diluted to a positive control group test solution with a mass concentration of 0.00625% as required according to the experimental concentration.

[0210] Zebrafish were randomly selected in a 6-well plate with a capacity of 3 mL per well, with 15 fish per well. Experimental group, normal control group, model control group and positive control group were set up. Except for the normal control group, all other experimental groups were given 1.5 μmol / L menadione in water to establish the zebrafish oxidative stress model. Then the sample was given in water, and different volumes of 0.1% sub-mother-son solution were added as needed to make the final concentration of the experimental group test solution 0.0125%, 0.025%, and 0.05%; no other substances were added to the normal control group; the model control group was given 1.5 μmol / L menadione in water to establish the zebrafish aging model, and no other substances were added.

[0211] The positive control group was added with 1.25% stock solution as needed, so that the final concentration of the positive control group test solution was 0.00625%; incubated at 28°C in the dark for 22 hours. Then, the zebrafish were stained with a specific ROS fluorescent reagent (invitrogen, catalog number C10444). After the staining, 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. The zebrafish intelligent image analysis software 1.31 was used to analyze and collect data, and the fluorescence intensity (S) of the zebrafish yolk sac was analyzed. According to the formula, it was calculated and judged whether the sample had an antioxidant effect.

[0212]

[0213] Fluorescence intensity microscope photos of zebrafish yolk sac are shown in Fig.10 , the results of zebrafish yolk sac fluorescence intensity analysis are shown in Fig.11 The calculation results of the antioxidant effect of zebrafish are shown in Table 9.

[0214] Table 9 Test results of zebrafish antioxidant effect

[0215] serial number Detection concentration Antioxidant effect% P-value Test results Example 2 0.0125％ 18 <0.001 Significant Example 2 0.025％ 21 <0.001 Significant Example 2 0.05％ 40 <0.001 Significant NAC Group 0.00625％ 93 <0.001 Significant

[0216] from Fig.10 It can be seen that the fluorescence intensity of zebrafish yolk sac in the fermented oat bran-Cordyceps militaris YM sample group was significantly reduced compared with the model control group, indicating that the fermented oat bran-Cordyceps militaris YM has an antioxidant effect. Fig.11 Table 9 also confirms this effect.

[0217] 8.5 Promoting autophagy in zebrafish

[0218] Due to its translucent nature, the EGFP-Lc3 transgenic zebrafish embryo can be observed in vivo using a confocal fluorescence microscope to emit specific green fluorescence from the Lc3 protein. Under normal circumstances, the Lc3 protein is distributed throughout the cytoplasm, and the fluorescence signal is diffuse; after autophagy is induced, the Lc3 protein will aggregate on the autophagosome membrane, and the fluorescence signal will become dot-like and bright. By detecting the number of zebrafish heart autophagosomes per unit area, it is possible to evaluate whether the sample has the effect of promoting autophagy.

[0219] Experimental system: Tg (clmc2: EGFP-Lc3) strain zebrafish, bred and provided by the fish breeding center of Nanjing Huante Zhiyu Biotechnology Co., Ltd. Zebrafish age: 4 days after fertilization (4 dpf).

[0220] Preparation of the sample to be tested: The product prepared in Example 2 above was first prepared into a mother solution with a mass concentration of 1% using standard dilution water, and diluted to a test solution of the experimental group with a mass concentration of 0.05% as required according to the experimental concentration. Rapamycin (Sigma, item number 553211) was prepared into 1 mmol / L using DMSO, and diluted to a positive control group test solution with a mass concentration of 1 μmol / L as required according to the experimental concentration.

[0221] Zebrafish were randomly selected in a 6-well plate with a capacity of 3 mL per well, with 15 fish per well. Experimental group, normal control group and positive control group were set up, and water-soluble samples were given. Among them, 1% mother solution was added as needed in the experimental group to make the final concentration of the experimental mass of the experimental group test solution of 0.05%; no other substances were added to the normal control group; the positive control group was diluted as needed according to the experimental concentration to a mass concentration of 1 μmol / L positive control group test solution; incubated at 28°C in the dark for 24 hours. Five zebrafish were randomly selected from each experimental group and placed under a laser confocal microscope (model AX, Nikon, Japan) for imaging. Image processing software was used to analyze and collect data, and the number of zebrafish cardiac autophagosomes per unit area (N) was analyzed, and the formula was used to calculate and determine whether it has the effect of promoting autophagy.

[0222]

[0223] Photo of zebrafish cardiac autophagosomes Fig.12 The results of promoting autophagy in zebrafish are shown in Table 10.

[0224] Table 10 Test results of promoting autophagy in zebrafish

[0225] serial number concentration Promote autophagy% P-value Test results Example 2 0.05％ 94 <0.001 Significant Rapamycin group 1μM 124 <0.001 Significant

[0226] Fig.12 In the figure, the red arrow points to the zebrafish heart, the white dotted line is the quantitative area, and the green fluorescent dots are cardiac autophagosomes. It was observed that the number of cardiac autophagosomes per unit area in the sample group was significantly increased compared with the normal control group, revealing that the fermented oat bran-Cordyceps militaris fermentation product YM has the effect of promoting autophagy. This effect can also be confirmed by Table 10. Cellular autophagy is the process of cell decomposition and recycling of damaged components, which is important for maintaining cell health. Autophagy clears intracellular damage and provides cells with energy and raw materials for repair. After ultraviolet (UV) or oxidative stress, enhancing autophagy may accelerate the repair of skin cell damage and reduce the risk of photoaging. In addition, autophagy can regulate inflammatory responses, reduce the release of reactive oxygen species (ROS) by clearing damaged mitochondria (mitophagy), and thus inhibit inflammatory signaling pathways (such as NLRP3 inflammasomes). Autophagy also indirectly enhances the antioxidant capacity of cells and reduces the effects of free radicals on the skin by clearing oxidatively damaged proteins and organelles.

[0227] In summary, the efficacy of fermented oat bran-Cordyceps militaris ferment YM in the in vivo model was comprehensively evaluated in the zebrafish model through β-galactosidase staining, yolk sac ROS level, mitochondrial enhancement, and promotion of autophagy.

[0228] Effect Example 9

[0229] Through efficacy evaluation, it has been preliminarily determined that YM has anti-wrinkle and firming effects, but human skin aging is a complex and multi-dimensional process. To build a complete scientific evidence chain for anti-wrinkle and firming efficacy evaluation, we must start from the mechanism of action and rationally arrange different categories and dimensions of test methods to ensure that the efficacy evaluation system is scientific and rigorous. Further in-depth basic research will help reveal the mechanism of cosmetics' anti-wrinkle and firming effects, and will help screen out efficient laboratory test methods that are highly correlated with the final efficacy of cosmetics.

[0230] 9.1 Cell cycle regulatory proteins

[0231] Cell cycle arrest is controlled by many regulatory factors in a complex and precise manner. Tumor suppressor genes including p53, p21, and p16 can regulate cell cycle and G1 phase arrest, thereby becoming the central signal for activating the aging signaling pathway. This experiment measured the effect of the fermented oat bran-Cordyceps militaris fermented product prepared in Example 2 on cell cycle regulatory proteins at the mRNA level. The preparation of the sample to be tested and the sample processing method are the same as those in Example 5. The p53, p21, and p16 gene primer sequences and test methods are shown in Example 7.

[0232] Test results see Fig.13 .Depend on Fig.13 It can be seen that UVA irradiation and H 2 O 2 Stimulation can activate the P16 / RB-dependent pathway and the P53 / P21-dependent pathway, which together lead to cell cycle arrest; 2 O 2 In the model, YM significantly downregulated the expression of P53, P21 and P16 mRNA at the 16h node, delaying the premature aging caused by cell cycle arrest; in the UVA photoaging model, UVA irradiation caused the persistent high expression of P53 and P21, and YM alleviated this phenomenon to a certain extent. This shows that YM delays the aging of skin fibroblasts by downregulating the expression of p53, p21, and p16 genes.

[0233] 9.2Keap1-Nrf2-P62

[0234] The signaling pathway Keap1-Nrf2-P62 pathway is one of the important pathways for oxidative stress regulation. After activation, it can accelerate the removal of ROS, maintain the body's redox homeostasis, and reduce oxidative stress damage. Autophagy, as a mechanism of programmed cell death, can remove cells damaged by oxidative stress and maintain physiological function homeostasis. The Keap1-Nrf2-P62 signaling pathway and autophagy interact in series to antagonize a series of skin injuries caused by oxidative stress. By studying the role of YM in the Nrf2 pathway and autophagy, the relationship between the Nrf2 pathway and autophagy was analyzed to regulate each other. This experiment measured the effect of the fermented oat bran-Cordyceps militaris fermented product obtained in Example 2 on Keap1-Nrf2-P62 at the mRNA level. The preparation of the sample to be tested and the sample processing method are the same as those in Example 5. See Example 7 for the primer sequences and test methods of the Nrf2, Keap1, and P62 genes.

[0235] Test results see Fig.14 .Depend on Fig.14 It can be seen that after the cell stress regulation is unbalanced, the intracellular ROS level increases significantly, leading to the disorder of cell autophagy and the increase of p62 content. 2 O 2 Under the model, at different time points after YM treatment (8h, 16h, 24h, 32h, 40h, 48h), Nrf2 expression was increased compared with the Model group (i.e., injury group), and the negative regulatory factor Keap1 was decreased compared with the Model group (i.e., injury group), indicating that the signaling pathway was activated, and P62 expression was decreased compared with the Model group (i.e., injury group). Under the UVA model, at different time points after YM treatment (8h, 16h, 24h, 32h, 40h, 48h), Nrf2 expression was increased compared with the Model group (i.e., injury group), and Keap1 did not show a significant decrease, which may be due to the role of other transcription factors, and the activation of Nrf2 is obvious. P62 expression was decreased compared with the Model group (i.e., injury group).

[0236] In addition, under normal physiological conditions, Nrf2 binds to Keap1 and is degraded. When cells are stressed, ROS increases, Nrf2 separates from Keap1, and antioxidant gene transcription is initiated. When the expression of downstream antioxidant genes regulated by Nrf2 is enhanced, the antioxidant capacity of cells can be improved and ROS damage to cells can be reduced. Although short-term stress activation of the Nrf2 signaling pathway can enhance the antioxidant capacity of cells, stress can cause an imbalance in the regulation of this pathway, resulting in a decrease in antioxidant substances in cells, a weakening of antioxidant capacity, a continuous increase in ROS levels, and an aggravation of oxidative stress, leading to skin aging.

[0237] This study provides new ideas and experience references for the evaluation of the anti-wrinkle and firming effects of cosmetics. Through functional evaluation, molecular mechanism exploration, and zebrafish model verification, a relatively complete discussion of the role of cosmetic samples in anti-wrinkle and firming effects was conducted. This provides direction for the research and development of anti-wrinkle and firming cosmetics and their raw materials, and the multi-dimensional evaluation of the improvement of skin aging.

[0238] 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.

[0239] 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 Cordyceps militaris strain YCC127, characterized in that: The deposit number of the strain is CGMCC No.41663.

2. A method for preparing a Cordyceps militaris fermented product having anti-aging, mitochondrial function enhancement, anti-oxidation, and autophagy promotion effects, characterized in that: include: The Cordyceps militaris is inoculated into a fermentation substrate for liquid fermentation culture to obtain fermentation mycelium; the fermentation mycelium is subjected to cell wall breaking or centrifugal separation, concentration, and freeze-drying to obtain the Cordyceps militaris fermentation product; wherein the fermentation substrate includes oat bran and / or lily.

3. The preparation method according to claim 2, characterized in that: The Cordyceps militaris fungus is inoculated in the form of Cordyceps militaris seed liquid; And / or, the volume of the Cordyceps militaris mycelium in the Cordyceps militaris seed solution accounts for at least 50% of the volume of the culture medium, preferably 60% to 75%, and more preferably 65% ​​to 70%; Preferably, the Cordyceps militaris seed solution is obtained by primary culture and secondary culture in sequence; Further preferably, the preliminary culturing step comprises: culturing Cordyceps militaris in a first culture medium to obtain a fermentation liquid with bacterial balls, and homogenizing the bacterial balls to break them into fine first-generation mycelia to obtain a homogenized bacterial liquid; And / or, the secondary culturing step comprises: inoculating the homogenized bacterial liquid into a second culture medium for culturing to obtain second-generation mycelium, until the volume of the second-generation mycelium accounts for at least 50%, preferably 60% to 75%, and more preferably 65% ​​to 70% of the volume of the second culture medium; And / or, the homogenized bacterial liquid is inoculated into the second culture medium at an inoculation rate of 1% (v / v) to 18% (v / v), preferably 5% to 12% (v / v); And / or, the Cordyceps militaris is the Cordyceps militaris strain YCC127 with a deposit number of CGMCC No.41663.

4. The preparation method according to claim 3, characterized in that: The culture temperature of the preliminary culture is 23-29°C, preferably 25-28°C; the culture time is 4-8 days, preferably 5-7 days; the rotation speed is 140-190 rpm, preferably 150-180 rpm; And / or, the culture temperature of the secondary culture is 23-29° C., preferably 25-28° C.; the culture time is 4-8 days, preferably 5-7 days; the rotation speed is 140-190 rpm, preferably 150-180 rpm.

5. The preparation method according to claim 2, characterized in that: The oat bran is oat bran powder or fermented oat bran freeze-dried powder, preferably fermented oat bran freeze-dried powder; the lily is lily powder or fermented lily freeze-dried powder, preferably fermented lily freeze-dried powder.

6. The preparation method according to claim 2 or 5, characterized in that: The fermentation substrate also includes fermentation medium or water, preferably water; And / or, based on the mass of the fermentation medium or water, the oat bran or lily accounts for 0.5% to 3%, preferably 1% to 2%, of the mass of the fermentation medium or the water; preferably, the fermentation medium is potato dextrose water medium; And / or, the Cordyceps militaris seed solution is inoculated into the fermentation substrate at an inoculation amount of 1% (v / v) to 18% (v / v), preferably 5% to 12% (v / v); and / or, the liquid fermentation culture conditions are: the fermentation temperature is 25-30° C., preferably 25-28° C.; and / or, the rotation speed is 150-200 rpm, preferably 180-200 rpm; preferably, the rotation speed is the shaking table speed; and / or, the fermentation time is 3-10 days, preferably 5-8 days; And / or, the method of obtaining the fermented mycelium may be centrifugal filtration; preferably, the conditions of the centrifugal filtration are: the centrifugal filtration speed is 3000-6500 rpm, preferably 4500-5000 rpm; the centrifugal filtration time is 15-40 min, preferably 20-30 min; and / or, the cell wall breaking treatment method is at least one of hot water extraction, ultrasonic crushing, surfactant treatment, organic solvent treatment, acid-base treatment, preferably hot water extraction; The centrifugal separation method is gauze filtration or centrifugal filtration, preferably centrifugal filtration; Preferably, the hot water extraction treatment includes: adding distilled water to the fermented mycelium, the solid-liquid ratio of the fermented mycelium to the distilled water is 1:10 to 1:30, preferably 1:15 to 1:25; the extraction temperature is 70 to 90°C, preferably 75 to 85°C, the number of extractions is 2 to 5 times, preferably 2 to 3 times; the extraction time is 0.5h / time to 1.5h / time, preferably 0.8h / time to 1h / time.

7. The preparation method according to claim 5, characterized in that: The preparation method of the fermented oat bran freeze-dried powder or the fermented lily freeze-dried powder comprises: inoculating yeast liquid into a fermentation system, fermenting, and freeze-drying, wherein the fermentation system comprises oat bran powder and / or lily powder and water; And / or, in terms of mass fraction, the oat bran or lily accounts for 0.5% to 5%, preferably 1% to 2% of the water; And / or, the concentration of the yeast in the yeast solution is 10 5 ~10 9 CFU / mL, preferably 10 6 ~10 8 CFU / mL; And / or, the yeast liquid is introduced into the fermentation system at an inoculation rate of 1% to 15% (v / v), preferably 5% to 10% (v / v); And / or, the yeast is inoculated into the fermentation system and the fermentation conditions are as follows: the fermentation temperature is 25-32° C., preferably 28-30° C.; the fermentation time is 20-48 hours, preferably 25-32 hours; the fermentation speed is 100-300 rpm, preferably 100-220 rpm; And / or, the yeast is Saccharomyces cerevisiae; preferably, the Saccharomyces cerevisiae is Saccharomyces cerevisiae strain YWY-1, with a deposit number of CGMCC No.17452.

8. A Cordyceps militaris fermented product having anti-aging, mitochondrial function enhancement, anti-oxidation, and autophagy promoting effects, characterized in that: The Cordyceps militaris fermented product is prepared by the method for preparing the Cordyceps militaris fermented product according to any one of claims 2 to 7.

9. Use of the Cordyceps militaris fermented product having anti-aging, mitochondrial function enhancement, anti-oxidation and autophagy promotion effects as claimed in claim 8 in the preparation of external skin preparations, hair preparations or foods; Preferably, the Cordyceps militaris fermented product is directly used as a product or as an additive in the skin topical preparation, hair preparation or food; Preferably, the Cordyceps militaris fermented product is used as at least one of the antioxidant active ingredient, skin repair active ingredient and anti-aging active ingredient in the skin external preparation; Preferably, the skin topical agent is at least one of skin care lotion, lotion, face cream and face mask; and / or the food is a health food.

10. A skin external preparation, characterized in that: The skin topical preparation includes the Cordyceps militaris fermented product with anti-aging, mitochondrial function enhancement, antioxidant, and autophagy promotion effects as described in claims 2 to 7, or the Cordyceps militaris fermented product with anti-aging, mitochondrial function enhancement, antioxidant, and autophagy promotion effects as described in claim 8.

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