Leuconostoc citreum and application thereof

Abstract

The application discloses a Leuconostoc strain and application thereof, and the Leuconostoc strain HX-236 is screened by the inventors. The Leuconostoc strain HX-236 uses cordyceps militaris powder as the only nitrogen source, and after fermentation culture, a fermentation filtrate can be obtained. The fermentation filtrate has high contents of cordycepin and cordycepic acid, is easily soluble in water, is convenient to use, has good stability, has good compatibility, can significantly improve the expression of hypoxia inducible factor (HIF-1 alpha) in cells, thereby activating downstream signal pathways for regulating inflammatory reactions, cell autophagy and tissue neogenesis, and then regulating cell hypoxia pressure, improving problems of skin aging such as cell activity reduction, collagen loss, pigmentation and skin elasticity reduction caused by hypoxia, and having wide application prospects in the fields of medicines, cosmetics and the like.

Description

Technical Field

[0001] This invention relates to a Leuconostoc sp. strain, its application in the fermentation of Cordyceps militaris, and the application of fermented products obtained from the fermentation of Cordyceps militaris by Leuconostoc sp. in the fields of pharmaceuticals and cosmetics. Background Technology

[0002] Skin is the first tissue to show signs of aging. Skin aging is characterized by increased wrinkles, sagging skin, decreased luster, smoothness, tension, and elasticity, coarser skin texture, irregular skin grooves, and increased pigmentation—all major enemies of beauty. Therefore, people often use anti-aging skincare products to reduce the signs of aging on their faces. Skin aging is mainly divided into natural aging and extrinsic aging. Natural aging refers to the phenomenon of decreased cell vitality, reduced cell migration and proliferation rates caused by factors such as uncontrollable forces (e.g., changes in the body's endocrine and immune functions) and genetics. During this process, cellular oxygen consumption increases, leading to insufficient oxygen in the skin to maintain cell vitality, resulting in decreased cell vitality, collagen loss, pigmentation, skin sagging, and the appearance of wrinkles. Exogenous aging refers to the skin's aging process caused by continuous exposure to environmental factors such as work stress, lack of sleep, smoking, alcohol consumption, ultraviolet radiation, and contact with chemicals. This leads to an increased metabolic burden on the body, increased oxygen demand in other organs, and reduced oxygen allocation to the skin, resulting in signs such as deeper and thicker wrinkles, sagging skin, dilated blood vessels, rough skin, and accumulation of degradation products. With increasing consumer awareness and health consciousness, the selection of anti-aging products is undergoing a fundamental shift. However, most commercially available anti-aging cosmetics primarily alleviate skin aging by providing sufficient skin hydration, lacking a comprehensive approach to improving cellular oxygenation and addressing overall aging issues.

[0003] Cordyceps militaris, the type species of the genus Cordyceps in the family Clavicipitaceae, is a fungus belonging to the phylum Ascomycota, order Hypocreales, and family Clavicipitaceae. It is also known as North Cordyceps or simply Cordyceps militaris. Generally, Cordyceps militaris cultivated from live insect pupae is called Cordyceps militaris. The main active ingredients in Cordyceps militaris include cordycepin and cordycepic acid. Cordycepic acid has antioxidant and free radical scavenging effects; cordycepin is the first nucleoside antibiotic isolated from fungi, possessing unique antibacterial and antiviral effects and significantly inhibiting various inflammatory factors. Therefore, Cordyceps militaris is a promising raw material for skin care.

[0004] Reports indicate that lactic acid bacteria fermentation can further increase the content of active ingredients in Cordyceps militaris. Furthermore, the peptides and extracellular polysaccharides in the fermentation products have been proven to be natural antioxidants, showing significant effects in scavenging free radicals and reducing oxidative damage. The enzymes and other active ingredients in the lactic acid bacteria extract can promote skin wound healing, while the lysate of lactic acid bacteria can improve skin hydration, enhance the skin barrier, and combat photoaging. Therefore, as a novel natural fermented product, Cordyceps militaris lactic acid bacteria fermentation extract has broad application prospects in the pharmaceutical, cosmetic, and other fields. Summary of the Invention

[0005] For skin inflammation, decreased cell vitality, collagen loss, and pigmentation caused by skin hypoxia, resisting cell damage and aging, strengthening the skin's physical barrier function, and maintaining the skin's internal environment homeostasis are good solutions. This invention provides a Leuconostoc mesenteroides strain, which has been verified to have a good effect on the release of active ingredients in Cordyceps militaris. The resulting Cordyceps militaris fermented product has the effects of regulating skin inflammatory response, soothing, anti-oxidation, improving cell autophagy activity, promoting tissue regeneration, and promoting collagen endogenous growth. It has good application prospects in the fields of pharmaceuticals and cosmetics.

[0006] The specific technical solution of this invention is as follows:

[0007] A strain of Leuconostoc sp. HX-236 was screened from waste sugarcane bagasse. This strain was deposited on September 8, 2022, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Bayi Road, Wuchang District, Wuhan, with accession number CCTCC NO: M20221389.

[0008] The present invention also provides a microbial agent comprising the aforementioned Leuconostoc sp. HX-236. This microbial agent can be a solid or liquid formulation.

[0009] This invention also provides the application of *Leuconostoc sp.* HX-236 or the above-mentioned *Leuconostoc sp.* HX-236-containing inoculum in the preparation of fermented products. In this application, *Leuconostoc sp.* HX-236 or the above-mentioned inoculum is generally added to a substrate for fermentation to obtain the fermented product. According to prior art reports, the *Leuconostoc sp.* HX-236 of this invention can replace other *Leuconostoc* strains in the prior art for product fermentation; specific fermentation methods can be referred to in the prior art.

[0010] Preferably, the Leuconostoc sp. HX-236 or the above-mentioned Leuconostoc sp. HX-236-containing inoculum is used to ferment Cordyceps militaris to obtain a fermented Cordyceps militaris product.

[0011] This invention also provides a method for preparing a Cordyceps militaris fermented product. The method includes a step of fermenting Cordyceps militaris with the aforementioned Leuconostoc sp. HX-236, and the fermented product is the Cordyceps militaris fermented product. In addition to the fermentation step, the method may also include subsequent processing steps for the fermentation broth, including but not limited to pH adjustment, filtration, sterilization, protein removal, concentration, and drying.

[0012] Furthermore, in the above preparation method, the culture medium used for fermentation is composed of carbon source, nitrogen source, inorganic salt, water and other components. The carbon source can be sugars such as glucose and fructose, the nitrogen source can be Cordyceps militaris powder, and the inorganic salt can be sodium acetate, magnesium sulfate and other components.

[0013] Preferably, in the above preparation method, Cordyceps militaris is selected as the sole nitrogen source for the fermentation culture medium.

[0014] In a specific embodiment of the present invention, the fermentation medium formula adopts the medium formula disclosed in CN111808901A.

[0015] Furthermore, in the above preparation method, the optimal culture temperature for Leuconostoc mesenteroides HX-236 is 35-38℃.

[0016] Furthermore, in the above preparation method, the fermentation process includes: inoculating the *Leuconostoc mesenteroides* HX-236 seed culture into a fermentation medium, culturing with aeration and stirring at 35-38℃, without controlling the pH value during fermentation, and ending fermentation when no residual sugar remains, thereby obtaining the fermentation broth. The *Leuconostoc mesenteroides* HX-236 seed culture can be obtained by culturing *Leuconostoc mesenteroides* HX-236 in liquid MRS medium at a temperature of 35-38℃ and a pH of 6.2-6.6.

[0017] This invention also provides a Cordyceps militaris fermented product, which is obtained by fermenting Cordyceps militaris with Leuconostoc sp. HX-236, preferably prepared by the above-described method for preparing Cordyceps militaris fermented products. This fermented product can be in a liquid or solid state; it can be a fermentation broth obtained by fermenting Cordyceps militaris with Leuconostoc sp. HX-236, or a liquid or solid product obtained by further processing the fermentation broth. Further processing includes, but is not limited to, adjusting the pH of the fermentation broth, filtration, sterilization, protein removal, concentration, and drying.

[0018] Preferably, the Cordyceps militaris fermented product contains 80-200 mg / L of cordycepin and 5-30 g / L of cordycepic acid.

[0019] Furthermore, the fermented product is rich in a variety of natural active ingredients and has multiple functions, showing great application prospects in the fields of pharmaceuticals and cosmetics.

[0020] The present invention also provides the application of the above-mentioned Leuconostoc sp. HX-236, the above-mentioned microbial agent, or the above-mentioned Cordyceps militaris fermentation product in the preparation of pharmaceuticals and cosmetics.

[0021] The present invention also provides a composition comprising the above-mentioned Leuconostoc sp. HX-236, or the above-mentioned microbial agent, or the above-mentioned Cordyceps militaris fermentation product.

[0022] The present invention also provides the use of the above-mentioned Leuconostoc sp. HX-236, the above-mentioned microbial agent, the above-mentioned Cordyceps militaris fermentation product, or the above-mentioned composition in anti-aging, or promoting collagen production, or promoting cell repair and regeneration, or anti-inflammatory, or whitening, or increasing skin elasticity.

[0023] This invention has independently screened a strain of Leuconostoc mesenteroides HX-236, which can ferment Cordyceps militaris to fully release its active ingredients. During the fermentation process, Cordyceps militaris is the sole nitrogen source, and the resulting fermentation broth is rich in various natural active ingredients, with high levels of cordycepic acid and cordycepin. It is easily soluble in water, convenient to use, and exhibits good stability and compatibility, making it widely applicable in various skincare products.

[0024] Verification has shown that the Cordyceps militaris fermentation product obtained by this invention can increase the expression level of hypoxia-inducible factor HIF-1α (increasing the expression level of HIF-1α in the skin by 2%-8%), thereby activating downstream signaling pathways that regulate inflammatory responses, autophagy, and tissue regeneration, restoring cell vitality. It can also increase the expression levels of skin type I collagen and the autophagy marker LC3B, thereby improving skin aging problems such as decreased cell vitality, collagen loss, pigmentation, and reduced skin elasticity caused by hypoxia by regulating cellular hypoxia pressure. It plays a role in regulating skin inflammatory responses, promoting cell autophagy activity, promoting tissue regeneration, soothing and anti-oxidation, and promoting collagen endogenous growth. It can be used in pharmaceuticals, cosmetics, and other fields.

[0025] Preservation Information

[0026] The present invention, Leuconostoc sp. HX-236, was deposited at the China Center for Type Culture Collection (CCTCC) on September 8, 2022. The deposit address is: China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, China. The deposit number is CCTCC NO: M20221389. Attached Figure Description

[0027] Figure 1 This is a microscope image of the anti-aging efficacy test in Experiment Example 3. Figure 1 A is a fluorescence micrograph of hypoxia-inducible factor HIF-1α in a human immortalized epidermal cell (HaCaT) model without the addition of Cordyceps militaris fermentation filtrate. Figure 1 B is a fluorescence micrograph of hypoxia-inducible factor HIF-1α in a human immortalized epidermal cell (HaCaT) model containing 0.01% Cordyceps militaris fermentation filtrate. Figure 1 Fluorescence micrograph of hypoxia-inducible factor HIF-1α in a human immortalized epidermal cell (HaCaT) model of 0.1% Cordyceps militaris fermentation filtrate at C concentration.

[0028] Figure 2 This is a microscope image of Experiment 4, which demonstrates the effects of promoting collagen endogenous regeneration and repairing damage. Figure 2 A represents a human primary dermal fibroblast (FB) model without the addition of Cordyceps militaris fermentation filtrate. Type II Collagen Fluorescence micrographs, Figure 2 B represents a human primary dermal fibroblast (FB) model containing 0.01% Cordyceps militaris fermentation filtrate. Type II Collagen Fluorescence micrographs, Figure 2 C represents a human primary dermal fibroblast (FB) model containing 0.1% Cordyceps militaris fermentation filtrate. Type II Collagen Fluorescence micrograph.

[0029] Figure 3 Microscopic image of the experiment to enhance autophagy activity in Example 5. Figure 3 A shows a fluorescence micrograph of the autophagy marker LC3B in a human immortalized epidermal cell (HaCaT) model without the addition of Cordyceps militaris. Figure 3 B is a fluorescence micrograph of LC3B, an autophagy marker in a human immortalized epidermal cell (HaCaT) model, obtained from 0.01% Cordyceps militaris fermentation filtrate. Figure 3 C is a fluorescence micrograph of LC3B, an autophagy marker, in a human immortalized epidermal cell (HaCaT) model, obtained from 0.1% Cordyceps militaris fermentation filtrate.

[0030] Figure 4 The graph shows the results of the inflammatory factor inhibition rate in the anti-inflammatory test of Experiment 6. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0032] Unless otherwise specified, all concentrations below are mass percentages.

[0033] Example 1: Screening and identification of Leuconostoc mesenteroides HX-236

[0034] Using waste sugarcane bagasse as raw material, the bagasse was serially diluted and dispersed with sterile water. Each solution was evenly spread onto MRS solid agar plates using a spreader and incubated statically at 30°C for 1-2 days. Single colonies with smooth, rounded morphology were selected and inoculated onto MRS solid agar containing calcium carbonate. These were then incubated statically at 30°C for 1-2 days. Single colonies producing calcium dissolution zones were repeatedly streaked for purification. Pure cultures were preserved on slant agar and stored at 4°C. The composition of the MRS solid agar was: 10 g / L peptone, 5 g / L beef extract, 5 g / L yeast extract, 2 g / L dipotassium hydrogen phosphate, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 2 g / L magnesium sulfate, 2 g / L manganese sulfate, and 20 g / L agar powder. The composition of the MRS solid medium containing calcium carbonate is as follows: peptone 10 g / L, beef extract 5 g / L, yeast extract 5 g / L, dipotassium hydrogen phosphate 2 g / L, diammonium citrate 2 g / L, sodium acetate 5 g / L, glucose 20 g / L, Tween 80 1 mL / L, magnesium sulfate 2 g / L, manganese sulfate 2 g / L, agar powder 20 g / L, and calcium carbonate 5 g / L.

[0035] The preserved strains were subjected to Cordyceps militaris fermentation. The strain with the highest content of cordycepic acid and cordycepin in the fermentation broth was selected as the target strain. The fermentation method was as follows: aeration and stirring were carried out at 35~38℃, the rotation speed was 200 r / min, the aeration rate was 0.25 vvm, the pH value was not controlled during the fermentation process, and the fermentation was stopped when no residual sugar was obtained to obtain the fermentation broth.

[0036] The selected target bacterial strain was named HX-236 and sent to Shanghai Sangon Biotech (Shanghai) Co., Ltd. for genome sequencing identification. The results showed that the strain was *Leuconostoc mesenteroides*. The 16S rRNA gene sequence of this strain is shown in SEQ ID NO: 1. The morphological characteristics of *Leuconostoc mesenteroides* HX-236 are as follows: the colonies of *Leuconostoc mesenteroides* are round or bean-shaped, with a diameter of less than 1.0 mm, a smooth surface, milky white, and do not produce any pigment; the cells are spherical, bean-shaped, or short stem-shaped, some in pairs or arranged in short chains, non-motile, and non-spore-forming; Gram staining is positive.

[0037] Example 2

[0038] Seed culture preparation: Leuconostoc mesenteroides HX-236 was inoculated into liquid MRS medium at pH 6.2-6.6 and cultured in a shake flask at 37°C for 2 days to obtain the seed culture. The liquid MRS medium consisted of: 10 g / L peptone, 5 g / L beef extract, 5 g / L yeast extract, 2 g / L dipotassium hydrogen phosphate, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 2 g / L magnesium sulfate, and 2 g / L manganese sulfate.

[0039] Fermentation culture: The seed culture was inoculated into the fermentation medium at an inoculum rate of 5 wt%. The fermentation medium formula was: Cordyceps militaris powder 8 g / L, glucose 5 g / L, fructose 18 g / L, sodium acetate 5 g / L, magnesium sulfate 2 g / L, water balance, and pH 6.2–6.6. The culture was carried out at 37℃ with aeration and stirring at a rotation speed of 200 r / min and an aeration rate of 0.25 vvm. The pH was not controlled during fermentation. Fermentation was stopped when no residual sugar was found, yielding the fermentation broth. The fermentation broth was heated in a 65℃ water bath for 1 h, and then the pH was adjusted to 5.0–6.0. ​​The broth was then filtered through a 0.45 μm filter membrane to obtain Cordyceps militaris fermentation filtrate S1.

[0040] Comparative Example 1

[0041] Cordyceps militaris fermentation filtrate D1 was prepared according to the method of Example 2, except that Leuconostoc mesenteroides HX-236 was replaced with Leuconostoc mesenteroides dextran subsp. SHBCC D14110.

[0042] Comparative Example 2

[0043] Cordyceps militaris fermentation filtrate D2 was prepared according to the method in Example 2, except that Leuconostoc mesenteroides HX-236 was replaced with Leuconostoc mesenteroides dextran subsp. SHBCC D16368.

[0044] Comparative Example 3

[0045] Without inoculating the culture medium of Example 2, the culture medium was heated in a water bath at 65°C for 1 hour, and then the pH was adjusted to 5.0~6.0. The medium was then filtered through a 0.45 μm filter membrane to obtain Cordyceps militaris extract D3.

[0046] Experimental Example 1: Cordycepin Content

[0047] The cordycepin content in the samples prepared in each example and comparative example was determined, and the specific steps are as follows:

[0048] 1.1 Sample solution preparation: The samples of each example and comparative example were diluted 10 times to obtain sample solutions.

[0049] 1.2 Preparation of standard sample solution: Accurately weigh 1 mg of standard sample cordycepin, add 50 mL of distilled water to dissolve, transfer to a 100 mL volumetric flask, add water to the mark, and dilute 10 times before use to obtain the standard sample solution.

[0050] 1.3 Chromatographic conditions: Detection was performed using a high-performance liquid chromatograph (HPLC). The column was a CAPCELL PAK-C18 column. The mobile phase was an aqueous solution containing 0.04 M potassium dihydrogen phosphate and 5 wt% acetonitrile. The flow rate was 1.0 ml / min, and the column temperature was 35℃. The detection wavelength was 260 nm, and the injection volume was 10 μl.

[0051] 1.4 Determination: The cordycepin content was calculated based on the peak area, and the results are shown in Table 1 below.

[0052]

[0053] Experimental Example 2: Cordycepic Acid Content

[0054] 2.1. Reagent preparation:

[0055] Nash reagent: Must be freshly prepared. Accurately weigh 150 g of ammonium acetate, dissolve in distilled water, add 2 mL of glacial acetic acid and 2 mL of acetylacetone, and bring the volume to 1000 mL.

[0056] 0.015 mol / L sodium periodate solution: Accurately weigh 3.2 g of sodium periodate and dissolve it in 0.12 mol / L hydrochloric acid.

[0057] 0.1% L-rhamnose solution: Accurately weigh 0.1g of L-rhamnose, dissolve in distilled water, and bring the volume to 100ml.

[0058] Preparation (storage) of cordycepic acid standard solution: Weigh 100 mg of cordycepic acid standard, dissolve in distilled water, and dilute to 100 ml to obtain a 1 mg / ml cordycepic acid standard solution. Dilute tenfold before use.

[0059] Test solution: Samples prepared in each example and comparative example, diluted 400 times.

[0060] 2.2. Measurement Method:

[0061] (1) Preparation of Cordycepic Acid Standard Curve

[0062]

[0063] Mix the cordycepic acid standard solution with water according to Table 2 above, then add 1 ml of sodium periodate solution to each test tube, mix well, and let stand at room temperature for 10 min. Add 2 ml of 0.1% rhamnose solution to each test tube to remove excess sodium periodate, shake well, add 4 ml of freshly prepared Nash reagent, and incubate in a 53℃ water bath for 15 min, then rapidly cool to room temperature. Measure the absorbance at 412 nm and plot a standard curve of cordycepic acid content (μg / ml) versus A412.

[0064] (2) Sample testing

[0065] Take 50 μl of each test solution and dilute to 100 ml in a volumetric flask. Then, take 1 ml of each solution and place it in a stoppered test tube. Add 1 ml of water to the blank control, then add 1 ml of sodium periodate solution, mix well, and let stand at room temperature for 10 min. Add 2 ml of 0.1% rhamnose to each test tube to remove excess sodium periodate, shake well, add 4 ml of freshly prepared Nash reagent, and incubate in a 53℃ water bath for 15 min, then rapidly cool to room temperature. Measure the absorbance at 412 nm. Substitute the absorbance values ​​into the standard curve and multiply by the dilution factor to obtain the cordycepic acid content. The results are shown in Table 3 below.

[0066]

[0067] Experimental Example 3: Anti-aging Efficacy Test

[0068] Human immortalized epidermal cell model establishment experiment

[0069] 1. Group settings

[0070] Control group: DMEM medium containing 10% FBS. Experimental group: DMEM medium containing 10% FBS, including 0.01% and 0.1% Cordyceps militaris fermentation filtrate S1.

[0071] 2. Experimental Procedure

[0072] (1) Cell seeding: HaCaT cells were cultured in T75 culture flasks with DMEM medium until the cell density was about 80%. They were then seeded into 24-well plates with coverslips and cultured at 37°C and 5% CO2.

[0073] (2) Sample treatment: After 24 h, the supernatant was removed. The experimental group was given a complete culture medium containing 0.01% and 0.1% Cordyceps militaris fermentation filtrate S1. At the same time, a control group was set up and incubated at 37℃ and 5% CO2 for 72 h.

[0074] (3) Immunofluorescence detection: Remove the supernatant, wash the cells twice, fix the cells with ice-cold methanol at -20℃, then add HIF-1α primary antibody and incubate overnight at 4℃. The next day, wash three times with PBS, add the corresponding secondary antibody and incubate at room temperature in the dark for 1.5 hours, stain the nuclei with DAPI and mount the slides, then observe and take pictures under a light microscope.

[0075] Immunofluorescence assay is based on the principle of antigen-antibody reaction. First, a known antigen or antibody is labeled with a fluorescein to create a fluorescent label. Then, this fluorescent antibody (or antigen) is used as a molecular probe to examine the corresponding antigen (or antibody) within cells or tissues. The antigen-antibody complex formed in the cells or tissues contains fluorescein. When the specimen is observed using a fluorescence microscope, the fluorescein emits bright fluorescence (yellow-green or orange-red) when exposed to excitation light, making the cells or tissues containing the fluorescence visible. This allows for the determination of the nature and location of the antigen or antibody, as well as the quantitative determination of its content using quantitative techniques.

[0076] 2. HIF-1α content detection

[0077] The fluorescence data from the photographs were semi-quantitatively analyzed using ImageJ software. After background removal, the readings were normalized and expressed as Means ± SEM, compared to a control. Statistical analysis was performed using Graphpad Prism software. The results are shown in Table 4 below.

[0078]

[0079] Microscopic images of the test results are shown below. Figure 1 As shown in A-1C, the denser the distribution of green fluorescent regions, the higher the brightness.

[0080] The higher the expression level of HIF-1α, the better. Figure 1 As can be seen from A-1C, the expression level of HIF-1α was increased in both the control group and the group that added Cordyceps militaris fermentation filtrate. The expression level of HIF-1α increased most significantly with the addition of 0.1% Cordyceps militaris fermentation filtrate, which significantly upregulated the expression level of HIF-1α factor by 8%, thereby improving the skin's hypoxia and enhancing cell vitality.

[0081] Experiment Example 4: Trial on Promoting Collagen Endogenous Growth and Repairing Damage

[0082] 1. In human primary dermal fibroblasts (FB) Type II Collagen Immunohistochemical detection

[0083] 1.1 Group Settings

[0084] Control group: DMEM medium containing 10% FBS. Experimental group: DMEM medium containing 10% FBS, including 0.01% and 0.1% Cordyceps militaris fermentation filtrate S1.

[0085] 1.2 Experimental Procedure

[0086] 1) Cell seeding: Take FB cells and culture them in DMEM medium in T75 culture flasks. When the cell density is about 80%, seed them into 24-well plates with coverslips and culture them at 37°C and 5% CO2.

[0087] 2) Sample processing: After 24 hours, the supernatant was removed. The experimental group was given complete culture medium containing 0.01% and 0.1% of the sample, while the control group was incubated at 37℃ in a 5% CO2 incubator for 72 hours.

[0088] 3) Immunofluorescence detection: The supernatant was aspirated, the cells were washed twice, and then fixed with ice-cold methanol at -20°C. Collagen I primary antibody was added and incubated overnight at 4°C. The next day, the cells were washed three times with PBS, and the corresponding secondary antibody was added and incubated at room temperature in the dark for 1.5 hours. The nuclei were stained with DAPI and the slides were mounted. The slides were then observed and photographed under a fluorescence microscope. Finally, the fluorescence data of the slides were semi-quantitatively analyzed using ImageJ software.

[0089] 2. Human primary dermal fibroblasts Type II Collagen Immunoassay Quantitative Analysis

[0090] Fluorescence data from the photographs were analyzed semi-quantitatively using ImageJ software. After background removal, the readings were normalized and expressed as Means ± SEM, compared to a control. Statistical analysis was performed using Graphpad Prism software. The results are shown in Table 5 below.

[0091]

[0092] The microscopic images of the test results are shown in 2A-2C. The denser the distribution of green fluorescent areas and the higher the brightness, the stronger the Collagen. The higher the expression level, the better. Figure 2 As shown in A-2C, compared with the control group, the addition of different concentrations of Cordyceps militaris fermentation filtrate resulted in Collagen... The expression levels of all components were significantly increased, especially in the Collagen model with the addition of 0.1% Cordyceps militaris fermentation filtrate. The expression level of the most significant effect was increased, indicating that the fermentation filtrate of worm pupae can significantly upregulate the synthesis of type I collagen. Compared with the control group, 0.01% and 0.1% fermentation filtrate of worm pupae increased the expression level of type I collagen by 17% and 23%, respectively, thereby making the skin elastic and plump.

[0093] Experiment 5: Enhancing Cellular Autophagy Activity

[0094] 1. Group settings

[0095] Control group: DMEM medium containing 10% FBS.

[0096] Experimental group: DMEM medium containing 10% FBS, which contains 0.01% and 0.1% Cordyceps militaris fermentation filtrate S1.

[0097] 2. Experimental Procedure

[0098] (1) Cell seeding: HaCaT cells were cultured in DMEM medium in T75 culture flasks. When the cell density was about 80%, they were seeded into 24-well plates with coverslips and cultured at 37°C and 5% CO2.

[0099] (2) Sample treatment: After 24 hours, the supernatant was removed. The experimental group was given a complete culture medium containing 0.01% and 0.1% Cordyceps militaris fermentation filtrate S1. The control group was incubated at 37°C and 5% CO2 for 72 hours.

[0100] (3) Immunofluorescence detection: The supernatant was aspirated, the cells were washed twice, and then fixed with ice-cold methanol at -20°C. LC3B primary antibody was added and incubated overnight at 4°C. The next day, the cells were washed three times with PBS, and the corresponding secondary antibody was added and incubated at room temperature in the dark for 1.5 hours. The nuclei were stained with DAPI and the slides were mounted. The slides were then observed and photographed under a fluorescence microscope. Finally, the fluorescence data of the photographs were semi-quantitatively analyzed using ImageJ software. The results are shown in Table 6 below.

[0101]

[0102] Microscopic images of the immunohistochemical detection results of the autophagy marker LC3B are shown in 3A-3C. The denser the distribution and the higher the brightness of the green fluorescent areas, the higher the expression level of the autophagy marker LC3B. Figure 3As shown in A-3C, the expression levels of the autophagy marker LC3B were significantly increased in the addition of Cordyceps militaris fermentation filtrate compared to the control group. Specifically, the addition of 0.01% Cordyceps militaris fermentation filtrate upregulated the expression level of LC3B in cells by 12%, indicating that Cordyceps militaris fermentation filtrate can significantly improve the autophagy capacity of cells, promote cell repair and regeneration, and thus help the skin resist damage and aging.

[0103] Experiment 6: Soothing and Anti-inflammatory Trial

[0104] Using Cordyceps militaris fermentation filtrate S1 as the test sample, it was diluted with water to a concentration of 1.5% (v / v). The inhibitory effect of the test sample on three pro-inflammatory factors at this concentration was determined, as follows:

[0105] A mouse macrophage model was used, and LPS was used to stimulate the production of inflammatory factors. The study investigated whether the samples could inhibit the secretion of these factors. A stock solution of LPS at a concentration of 500,000 units / mL was prepared using serum-free 1640 medium, sterilized by filtration through a 0.22 µm filter, and stored at -20°C. The stock solution was diluted to 10,000 units / mL before use. Samples that came into contact with the cells were prepared using the LPS reaction solution.

[0106] Raw264.7 cells in the logarithmic growth phase were harvested at a dose of 1×10⁻⁶. 5 Inoculated at / mL in 24-well plates and cultured at 37℃ and 5% CO2 for 24 h. Experimental groups were treated with 1 mL of test sample per well, the model group was treated with LPS-free reaction medium (without sample), and the negative control group was treated with serum-free 1640 medium (without LPS). Cultured for another 24 h, the levels of inflammatory factors in the culture supernatant were measured according to the instructions of the ELISA kit for mouse IL-6, TNF-α, and IL-1β, and the relative decrease in inflammatory factors was calculated.

[0107]

[0108] The results are as follows Figure 4 As shown, under LPS stimulation, the secretion of pro-inflammatory factors IL-6, IL-1β, and TNF-α by mouse macrophages was significantly reduced, with the reduction in IL-1β being the most significant. Using these three inflammatory factors as the main indicators, the sample showed a certain inhibitory effect on the three inflammatory factors IL-6, TNF-α, and IL-1β in mice. The 1.5% Cordyceps militaris fermentation filtrate S1 reduced the relative values ​​of the three inflammatory factors by 11.5%, 100%, and 8.7%, respectively. Therefore, Cordyceps militaris fermentation filtrate can effectively reduce skin problems caused by inflammation.

[0109] Experimental Example 7: Melanin Inhibition Test

[0110] 1. Effects of Cordyceps militaris fermentation filtrate on the proliferation of melanoma cells in B16 mice

[0111] Melanoma cells from B16 mice in the logarithmic growth phase were used at a concentration of 2 × 10⁻⁶. 4 Cells were seeded at a density of 100 μL / mL in 96-well cell culture plates and incubated overnight at 37°C with 5% CO2. The S1 samples from Example 1 were prepared into 1.5% and 3% serum-containing complete culture media, respectively, and sterilized by filtration through a 0.22µm filter. The old culture medium was discarded, and the experimental groups were treated with 1.5% and 3% S1 sample solutions, respectively. The normal control group was treated with an equal volume of serum-containing cell culture medium. After culturing for 24 hours, the relative cell proliferation rate was detected using the WST-1 method. The relative proliferation rate (RGR) was the ratio of the absorbance of the experimental group to that of the normal control group.

[0112] 2. Melanin content detection

[0113] Take B16 cells in logarithmic growth phase, and use 2×10 4 Inoculate 3 mL of Cordyceps militaris fermentation filtrate into 6-well plates at a density of 1 / mL and incubate at 37°C with 5% CO2 for 24 h. Discard the old culture medium. Add 3 mL of serum-containing medium to the normal control and model groups, and add 3 mL of sample solution to the experimental groups. Add 60 μL of 2 mM linolenic acid solution to each well of the model and experimental groups to stimulate melanin production. Continue incubation for another 72 h. The sample solution is 1.5% and 3.0% Cordyceps militaris fermentation filtrate S1.

[0114] The method for determining intracellular melanin content is as follows: trypsin digestion, centrifugation to remove supernatant, addition of 500 L of 1 mol / L (containing 10% DMSO) NaOH solution to lyse cells, heating at 80℃ for 30 min, centrifugation at 3000 rpm for 10 min, collection of supernatant added to 96-well plates, 100 μL / well, measurement of absorbance at 450 nm to obtain total melanin. With the melanin content of the model group as 100%, the relative value of melanin content in the experimental group is obtained. If the sample has a significant effect on the proliferation of B16 cells, the ratio of the relative value of melanin content to the corresponding B16 cell proliferation rate is the melanin production rate per unit cell.

[0115] 3. Tyrosinase activity detection

[0116] Within melanocytes, tyrosine is activated by tyrosinase to form dopa. Dopa then undergoes dehydrogenation to form dopaquinone, which rearranges to form 5,6-indole. This 5,6-indole polymerizes and binds to structural proteins within the melanocyte to form melanin. Tyrosinase is a pluripotent enzyme in this process; inhibiting its activity can suppress melanin formation. The experimental principle involves determining the relative activity of tyrosinase in a sample by measuring its ability to oxidize the substrate dopa to dopaquinone.

[0117] Take B16 cells in logarithmic growth phase, and use 2×10 4 Inoculate 3 mL of Cordyceps militaris fermentation filtrate into 6-well plates at a density of 1 / mL and incubate at 37°C with 5% CO2 for 24 h. Discard the old culture medium. Add 3 mL of serum-containing medium to the normal control and model groups, and add 3 mL of sample solution to the experimental groups. Add 60 μL of 2 mM linolenic acid solution to each well of the model and experimental groups to stimulate melanin production. Continue incubation for 72 h. The sample solution is 1.5% and 3.0% Cordyceps militaris fermentation filtrate S1.

[0118] Discard the old culture medium, wash the cells twice with PBS, then lyse the cells with Tirs-HCl (0.02 mol / L, pH 6.8) containing 0.1% Triton-100, sonicate for 10 min, add 0.1% L-DOPA (0.01 mol / L, pH 8.0) and incubate at 37℃ for 60 min. After centrifugation, collect the supernatant and add 100 μl / well of a 96-well plate. Measure the absorbance at 450 nm using a microplate reader. The ratio of absorbance between the experimental group and the model group is used as an indicator to evaluate the activity of tyrosinase.

[0119]

[0120] The results in the table above show that different concentrations of Cordyceps militaris fermentation filtrate can significantly reduce intracellular tyrosinase activity and melanin production rate. The tyrosinase activity of 1.5% and 3.0% concentrations of Cordyceps militaris fermentation filtrate decreased by 23.3% and 38.0%, respectively, and the melanin production rate decreased by 18.4% and 36.7%, respectively. Therefore, Cordyceps militaris fermentation filtrate can alleviate pigmentation and reduce age-related pigmentation.

[0121] Experimental Example 8: Human Skin Elasticity Test

[0122] 8.1 Recruit 30 healthy volunteers, regardless of gender, aged 20-65 years. Before the trial, each participant signed an informed consent form.

[0123] 8.2 Fifteen minutes after the subjects cleansed their faces, the initial R2 values ​​of skin elasticity on both sides of the face were measured using an instrument and recorded.

[0124] 8.3 Subjects used the sample twice a day, morning and evening. The method of use was as follows: placebo (essence without Cordyceps militaris ferment filtrate) and test sample (essence containing 1% Cordyceps militaris ferment filtrate S1) were applied to the left and right sides of the face, respectively. The skin elasticity R2 value was measured again at 1 week, 2 weeks and 4 weeks after the initial use.

[0125] 8.4 Calculate the relative growth rate of skin elasticity for each subject according to the following calculations, and then take the average value.

[0126]

[0127] 8.5 Test Results:

[0128]

[0129] As can be seen from the data in the table above, after using the essence containing 1% Cordyceps militaris ferment filtrate for 2 weeks, the relative growth rate of skin elasticity in volunteers increased significantly by 20%, and by 27.87% after 4 weeks. Therefore, Cordyceps militaris ferment filtrate can tighten the skin and effectively improve the problem of reduced elasticity in aging skin.

Claims

1. A type of Leuconostoc sp. HX-236, characterized by: The accession number is CCTCC NO: M20221389.

2. A microbial agent, characterized in that: Includes Leuconostoc sp. HX-236 as described in claim 1.

3. The application of Leuconostoc sp. HX-236 as described in claim 1 or the microbial agent as described in claim 2 in the preparation of Cordyceps militaris fermented products.

4. A method for preparing a Cordyceps militaris fermented product, characterized in that: The method includes the step of fermenting Cordyceps militaris using Leuconostoc sp. HX-236 as described in claim 1.

5. The preparation method according to claim 4, characterized in that: Cordyceps militaris was used as the sole nitrogen source in the fermentation medium.

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