Snow tea extract, preparation method and application thereof

Abstract

This application relates to a snow tea extract, a method for preparing the snow tea extract, and its application. The method for preparing the extract includes the following steps: S1. Mixing pulverized snow tea raw materials with water at a mass ratio of 1:7-15 to obtain a first mixture; S2. Culturing Bacillus licheniformis on a culture medium to obtain a seed culture of the Bacillus licheniformis; S3. Using the seed culture as a fermentation broth, adding it to the first mixture at an inoculum of 1%-5%, and obtaining a fermented mixture after fermentation; S4. Filtering the fermented mixture, and concentrating and drying the filtrate to obtain the snow tea extract. The snow tea extract obtained in this application has an excellent promoting effect on the expression of the klotho gene in human fibroblasts and can be applied in products that improve skin health, filling a gap in the prior art.

Description

Technical Field

[0001] This application relates to the field of microbial technology, specifically to a snow tea extract, a method for preparing the snow tea extract, and its application. Background Technology

[0002] The skin is the largest organ in the human body and the first line of defense against environmental damage. Skin aging is a complex and inevitable biological process, driven by both endogenous (such as genetics and hormone levels) and exogenous (such as ultraviolet radiation and pollution) factors. In recent years, scientific research has gradually revealed that aging is closely related to the expression regulation of a series of key genes at the molecular level.

[0003] The Klotho gene, located on human chromosome 13 (13q12), is 50 kb in length and consists of five exons. Through alternative splicing, Klotho can produce two types of proteins: membrane proteins and circulating proteins. Klotho proteins include α-, β-, and γ-types. α-Klotho is a glucuronidase that also exhibits antioxidant and anti-inflammatory properties and has been found to be involved in multiple signaling pathways in aging mechanisms (such as the Wnt, TGF-β, p53 / p21, and IGF-1 pathways). Therefore, targeting and increasing the expression level of endogenous Klotho genes in skin fibroblasts is considered a highly promising intervention strategy, potentially delaying skin aging at its root and promoting skin repair and regeneration.

[0004] Currently, direct supplementation with recombinant Klotho protein or the use of Klotho agonists are potential intervention methods. However, these methods suffer from high production costs, poor protein stability, difficulty in transdermal absorption, and potential immunogenicity, limiting their practical application, especially in the cosmetics field. On the other hand, although some small molecule compounds or biologics have been reported to regulate Klotho expression, most suffer from drawbacks such as low specificity, low efficiency, or unknown safety.

[0005] Finding safe and effective functional extracts from natural products is an important direction in modern anti-aging research and cosmetic development. Extracts from plants, microorganisms, or natural minerals are complex in composition, often possessing advantages such as multi-target and synergistic effects, and typically exhibit good skin compatibility and consumer acceptance. However, to date, very few studies have publicly reported on specific extracts with the core efficacy of "promoting the expression of the klotho gene in human skin fibroblasts" and their precise preparation methods. Summary of the Invention

[0006] Therefore, there is an urgent need to develop a new technical solution that can provide a natural extract with a clear preparation method, rich in active ingredients, and capable of effectively and safely promoting the expression of the klotho gene in human skin fibroblasts, and apply it to products for anti-skin aging and improving skin health, so as to fill the gap in existing technologies.

[0007] To achieve the above objectives, this application provides a method for preparing snow tea extract, comprising the following steps:

[0008] S1. Mix the crushed snow tea raw material with water at a mass ratio of 1:7~15 to obtain the first mixture;

[0009] S2. Culture Bacillus licheniformis on a culture medium to obtain a seed culture of Bacillus licheniformis;

[0010] S3. The seed liquid is used as a fermentation liquid and added to the first mixture at an inoculum amount of 1% to 5%, and a fermentation mixture is obtained after fermentation; wherein, the inoculum amount can be 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, etc.

[0011] S4. The fermented mixture is filtered, and the product obtained by concentrating and drying the filtrate is the snow tea extract.

[0012] Preferably, the Bacillus licheniformis is strain YK-L9, which was deposited on August 21, 2025, at the Guangdong Provincial Microbial Culture Collection Center, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No. 66840. The 16S rDNA sequence of strain YK-L9 is shown in SEQ NO:1 of the sequence listing.

[0013] Preferably, in step S1, the first mixture is further sterilized by steam at 121°C.

[0014] Preferably, in step S2, the revived Bacillus licheniformis YK-L9 strain is inoculated into LB liquid medium and cultured at 25℃~40℃ for 18 h~36 h. After activating for 1~2 generations, the seed culture of Bacillus licheniformis YK-L9 strain is obtained.

[0015] Preferably, in step S3, the fermentation temperature is 25℃~40℃, the fermentation time is 24~48h, and the shaking speed is 150rpm~220rpm. The fermentation temperature can be 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, etc.; the fermentation time can be 24h, 25h, 26h, 27h, 28h, 30h, 35h, 40h, 45h, 46h, 47h, 48h, etc.; and the shaking speed can be 150rpm, 160rpm, 170rpm, 180rpm, 190rpm, 200rpm, 210rpm, 220rpm, etc.

[0016] Preferably, in step S4, the fermentation mixture is filtered using a ceramic membrane with a pore size of 0.1~10μm; after filtration, it is concentrated and dried. The pore size of the ceramic membrane can be 0.1μm, 0.2μm, 0.5μm, 0.8μm, 1μm, 2μm, 3μm, 4μm, 5μm, 6μm, 7μm, 8μm, 9μm, 10μm, etc.

[0017] To achieve the above objectives, this application provides a snow tea extract, wherein the snow tea extract is obtained by fermenting snow tea using Bacillus licheniformis strain YK-L9 with accession number GDMCC No. 66840 and sequence number as shown in sequence listing SEQ NO:1.

[0018] This application also provides the application of snow tea extract in promoting the expression of the klotho gene in human fibroblasts. The snow tea extract is obtained by fermenting snow tea using Bacillus licheniformis strain YK-L9, which has the accession number GDMCC No. 66840 and the sequence number shown in the sequence listing SEQ NO:1.

[0019] This application also provides the application of snow tea extract in the preparation of cosmetics with anti-aging, anti-inflammatory and antioxidant effects. The snow tea extract is obtained by fermenting snow tea with Bacillus licheniformis strain YK-L9, which has accession number GDMCC No. 66840 and serial number as shown in sequence list SEQ NO:1.

[0020] This application also provides a skin care product that promotes the expression of the klotho gene in human fibroblasts. The skin care product contains snow tea extract, which is obtained by fermenting snow tea using Bacillus licheniformis strain YK-L9 with accession number GDMCC No. 66840 and sequence number as shown in sequence listing SEQ NO:1.

[0021] Preferably, the snow tea extract is added to the skin care product at an amount of 0.15-3%. The added amount can be 0.15%, 0.3%, 0.5%, 0.8%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.2%, 2.4%, 2.6%, 3%, etc.

[0022] The technical solution claimed in this application achieves the following beneficial effects:

[0023] 1. A snow tea extract fermented by Bacillus licheniformis strain YK-L9 is provided. This extract has a good promoting effect on the expression of the klotho gene in human fibroblasts and can be used in anti-skin aging products, filling the gap in the existing technology.

[0024] 2. Fermentation using Bacillus licheniformis strain YK-L9 fills the gap in current snow tea fermentation extraction technology that does not involve strains from specific ecological environments. This promotes the release of active ingredients (total polyphenols and total flavonoids) in the snow tea extract, improves extraction efficiency, and enhances the efficacy of snow tea. This process also ensures superior and stable product quality, avoiding the drawbacks of traditional extraction processes such as demanding extraction conditions, low extraction efficiency, complex operation, and high equipment costs.

[0025] 3. The snow tea extract fermented by Bacillus licheniformis strain YK-L9 not only promotes the expression of the klotho gene, but also has excellent in vitro DPPH free radical scavenging ability and anti-inflammatory ability. It has good in vitro antioxidant, anti-inflammatory and anti-aging effects, and is non-allergenic and highly safe, so it can be widely used in the field of health products. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are only embodiments of this application. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0027] Figure 1 The effects of the extracts obtained in Examples 1-9 and Comparative Examples 1-7 on the mRNA expression level of Klotho in human fibroblasts are shown. NC represents the normal group, and D-gal represents the D-galactose-induced senescence group in human fibroblasts.

[0028] Figure 2The results of the anti-inflammatory efficacy test of the extract obtained in Example 1 are shown on the vertical axis, which represents the relative expression levels of TNF-α, IL-7 and SCF mRNA. Detailed Implementation

[0029] To make the objectives, technical solutions, and beneficial effects of the embodiments in this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0030] The source information of the raw materials, materials, and instruments involved in the following embodiments or comparative examples is as follows:

[0031] Bacillus licheniformis strain L9: Self-developed, as described in the examples;

[0032] Bacillus strain L13: Self-developed, as described in the examples;

[0033] Bacillus strain L8: Self-developed, as described in the examples;

[0034] Lactic acid bacteria strain B2: Self-developed, as described in the examples;

[0035] Lactic acid bacteria strain E1: Self-developed, as described in the examples;

[0036] LB medium: purchased from Qingdao Haibo Biotechnology;

[0037] DPPH (1,1-diphenyl-2-trinitrophenylhydrazine), purchased from Aladdin;

[0038] Human fibroblasts (HFF cells) were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences.

[0039] DMEM culture medium, purchased from Eva Cell;

[0040] Fetal bovine serum, Gibco;

[0041] The sample solvent is sterile water.

[0042] <Preparation Example>

[0043] Preparation Example 1

[0044] This preparation example provides Bacillus licheniformis from Usnea yunnanensis (Yunnan Usnea). Bacillus licheniformis The screening method for strain YK-L9 is as follows:

[0045] (1) Select wild Usnea longissima samples growing in Yunnan, wash them and soak them in sterile water at 4℃ for 18h ~ 24h, take an appropriate amount of soaking solution and spread it on LB solid medium, incubate at 25℃ ~ 37℃ for 24h ~ 30h, pick out colonies of different morphologies on LB solid medium, streak them on the surface of LB solid medium for purification, and subculture continuously for no less than 2 times to obtain purified colonies;

[0046] (2) Single colonies on different plates after purification were observed under a microscope. Different strains with Bacillus morphology were selected for screening and identification. After identification, a strain of Bacillus licheniformis was obtained. Bacillus licheniformis The selected strain was named YK-L9 (hereinafter referred to as L9 strain). It was also deposited at the Guangdong Provincial Center for Microbial Culture Collection, located on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou, on August 21, 2025, with accession number GDMCC No. 66840.

[0047] (3) The obtained Bacillus licheniformis YK-L9 strain was sequenced. The 16S rDNA sequence of this L9 strain is shown in the following sequence listing SEQ NO:1:

[0048]

[0049] Preparation Example 2

[0050] This preparation example provides *Bacillus belye* derived from soil from *Meconopsis yunnanensis*. Bacillus velezensis The screening method for strain L13 differs from that in preparation example 1 only in that the strain was not preserved.

[0051] Preparation Example 3

[0052] This preparation example provides Bacillus mycoides derived from wild fungi in Yunnan. Bacillus mycoides The screening method for strain L8 differs from that in preparation example 1 only in that the strain was not preserved.

[0053] Preparation Example 4

[0054] This preparation example provides a fusion of *Lactobacillus weissii* derived from *Cypripedium yunnanensis*. Weissella confuse The screening method for strain B2 differs from that in preparation example 1 only in that MRS medium was used instead of LB medium, and the strain was not preserved.

[0055] Preparation Example 5

[0056] This preparation example provides *Lactobacillus plantarum* derived from *Cypripedium yunnanensis*. Lactiplantibacillus paraplantarum The screening method for strain E1 is the same as that for preparation example 4.

[0057] <Example>

[0058] Example 1

[0059] This embodiment provides a method for preparing snow tea extract, the preparation steps of which are as follows:

[0060] S1. Take snow tea raw materials, dry them in an oven at 60℃ for 4 hours, then crush them and pass them through a 50-mesh sieve. Take 20g of the crushed tea and mix it with water at a mass ratio of 1:10. After sterilizing with steam at 121℃ for 20 minutes, the first mixture is obtained.

[0061] S2. The first mixture was inoculated and fermented using the Bacillus licheniformis L9 strain obtained in Preparation Example 1. The L9 strain was activated by inoculation with LB liquid medium at 37°C for 24 hours. After two generations of activation, the seed culture of Bacillus licheniformis L9 strain was obtained.

[0062] The inoculum was 3% (v / v) of the first mixture, the fermentation temperature was 37℃, the fermentation time was 36h, the shaking speed was 200 rpm, and the second mixture was obtained after fermentation.

[0063] S3. The second mixture was purified by filtration through a 0.22μm ceramic membrane, the filtrate was concentrated under reduced pressure and then freeze-dried at -80℃ to obtain the snow tea extract.

[0064] Example 2

[0065] This embodiment provides a method for preparing snow tea extract, the preparation steps of which are as follows:

[0066] S1. Take snow tea raw materials, dry them in an oven at 60℃ for 4 hours, then crush them and pass them through a 50-mesh sieve. Take 20g of the crushed tea and mix it with water at a mass ratio of 1:7. After sterilizing with steam at 121℃ for 20 minutes, the first mixture is obtained.

[0067] S2. The first mixture was inoculated and fermented using the Bacillus licheniformis L9 strain obtained in Preparation Example 1. The L9 strain was activated by inoculation with LB liquid medium at 37°C for 24 hours. After two generations of activation, the seed culture of Bacillus licheniformis L9 strain was obtained.

[0068] The inoculum amount was 1% (v / v) of the first mixture, the fermentation temperature was 40℃, the fermentation time was 48h, the shaking speed was 150 rpm, and the second mixture was obtained after the fermentation was completed.

[0069] S3. The second mixture was purified by filtration through a 0.1μm ceramic membrane, the filtrate was concentrated under reduced pressure and then freeze-dried at -80℃ to obtain the snow tea extract.

[0070] Example 3

[0071] This embodiment provides a method for preparing snow tea extract, the preparation steps of which are as follows:

[0072] S1. Take snow tea raw materials, dry them in an oven at 60℃ for 4 hours, then crush them and pass them through a 50-mesh sieve. Take 20g of the crushed tea and mix it with water at a mass ratio of 1:15. After sterilizing with steam at 121℃ for 20 minutes, the first mixture is obtained.

[0073] S2. The first mixture was inoculated and fermented using the Bacillus licheniformis L9 strain obtained in Preparation Example 1. The L9 strain was inoculated into LB liquid medium for activation and cultured at 37°C for 24 hours. After two generations of activation, the seed culture of Bacillus licheniformis L9 strain was obtained.

[0074] The inoculum amount was 5% (v / v) of the first mixture, the fermentation temperature was 25℃, the fermentation time was 24h, the shaking speed was 220 rpm, and the second mixture was obtained after the fermentation was completed.

[0075] S3. The second mixture was purified by filtration through a 10μm ceramic membrane, the filtrate was concentrated under reduced pressure and then freeze-dried at -80℃ to obtain the snow tea extract.

[0076] Example 4

[0077] This embodiment provides a method for preparing snow tea extract. The specific preparation steps in this embodiment differ from those in Example 1 in that:

[0078] ①In step S1, the ratio of the tea leaves to the liquid is 1:20;

[0079] ② In step S2, the inoculum amount is 10% (v / v) of the first mixture.

[0080] Other preparation conditions remained the same as in Example 1.

[0081] Example 5

[0082] This embodiment provides a method for preparing snow tea extract. The specific preparation steps in this embodiment differ from those in Example 1 in that:

[0083] ①In step S1, the ratio of the tea leaves to the liquid is 1:5;

[0084] ② In step S2, the inoculum amount is 0.5% (v / v) of the first mixture.

[0085] Other preparation conditions remained the same as in Example 1.

[0086] Example 6

[0087] This embodiment provides a method for preparing snow tea extract. The specific preparation steps of this embodiment differ from those of Embodiment 1 in that: in step S2, the fermentation temperature is 50°C and the shaking speed is 100 rpm.

[0088] Other preparation conditions and maintenance were the same as in Example 1.

[0089] Example 7

[0090] This embodiment provides a method for preparing snow tea extract. The specific preparation steps of this embodiment differ from those of Embodiment 1 in that: in step S2, the fermentation temperature is 20°C and the shaking speed is 280 rpm.

[0091] Other preparation conditions remained the same as in Example 1.

[0092] Example 8

[0093] This embodiment provides a method for preparing snow tea extract. The specific preparation steps in this embodiment differ from those in Example 1 in that:

[0094] ① In step S2, the fermentation time is 12 hours;

[0095] ②The pore size of the ceramic membrane in step S3 is 0.01μm.

[0096] Other preparation conditions remained the same as in Example 1.

[0097] Example 9

[0098] This embodiment provides a method for preparing snow tea extract. The specific preparation steps in this embodiment differ from those in Example 1 in that:

[0099] ①In step S2, the fermentation time is 60 hours;

[0100] ②The pore size of the ceramic membrane in step S3 is 20μm.

[0101] Other preparation conditions remained the same as in Example 1.

[0102] <Comparative Example>

[0103] Comparative Example 1

[0104] This comparative example provides a method for preparing snow tea extract. The specific preparation steps of this comparative example differ from those of Example 1 only in step S2, where the strain used is Bacillus belye L13 obtained in Preparation Example 2.

[0105] Other preparation conditions remained the same as in Example 1.

[0106] Comparative Example 2

[0107] This comparative example provides a method for preparing snow tea extract. The specific preparation steps of this comparative example differ from those of Example 1 only in step S2, where the strain used is Bacillus mycoides L8 strain obtained in Preparation Example 3.

[0108] Other preparation conditions remained the same as in Example 1.

[0109] Comparative Example 3

[0110] This comparative example provides a method for preparing snow tea extract. The specific preparation steps of this comparative example differ from those of Example 1 only in step S2, where the strain used is the fusion Lactobacillus Weissieri B2 strain obtained in Preparation Example 4.

[0111] Other preparation conditions remained the same as in Example 1.

[0112] Comparative Example 4

[0113] This comparative example provides a method for preparing snow tea extract. The specific preparation steps of this comparative example differ from those of Example 1 only in step S2, where the strain used is the *Lactobacillus plantarum* E1 strain obtained in Preparation Example 5.

[0114] Other preparation conditions remained the same as in Example 1.

[0115] Comparative Example 5

[0116] This comparative example provides a method for preparing snow tea extract. The specific preparation steps of this comparative example differ from those of Example 1 only in step S2, where bacterial inoculation and fermentation are not used.

[0117] Other preparation conditions remained the same as in Example 1.

[0118] Comparative Example 6

[0119] This comparative example provides a Bacillus licheniformis extract. The specific preparation steps of this comparative example differ from those of Example 1 only in step S1, where the first mixture is replaced with an equal mass of LB medium. No herbal snow tea was used.

[0120] Other preparation conditions remained the same as in Example 1.

[0121] Comparative Example 7

[0122] This comparative example provides a method for preparing snow tea extract. The specific preparation steps differ from those in Example 1 only in step S3, where the ceramic membrane filtration step is omitted, and the extract is directly concentrated under reduced pressure and freeze-dried.

[0123] Other preparation conditions remained the same as in Example 1.

[0124] <Test Example>

[0125] Test Example 1

[0126] Test Example 1 was used to examine the effect of the extracts from Examples 1-9 and Comparative Examples 1-7 on the cell longevity factor klotho, as detailed below:

[0127] Cell seeding: HFF cells were obtained from the laboratory of Betaine Biotechnology Co., Ltd., and cultured in DMEM + 10% fetal bovine serum medium. HFF cells in the logarithmic growth phase (passages 15-25) were used in the experiment and cultured in 6-well plates at a cell concentration of 1.8 × 10⁶ cells per well. 5 The cells were cultured in 2 mL of medium, and then the culture plate was placed in an incubator with a CO2 concentration of 5% and a temperature of 37°C for 24 hours.

[0128] Cell processing: Remove the culture plate from the incubator, aspirate the original culture medium from the wells, and rinse with PBS. After rinsing, add 500 μL of fresh culture medium to each well (culture medium composition: 1 μL of any extract from Examples 1-9 and Comparative Examples 1-7 (15 mg / mL) + 120 μL D gal + 379 μL DMEM), with a final concentration of snow tea extract of 30 μg / mL. Then, place the culture plate in an incubator with 5% CO2 and a temperature of 37°C for 72 hours. After 72 hours, remove the 6-well plate containing the cultured HFF cells from the incubator, aspirate the original cell culture medium from the wells using a negative pressure aspirator, and rinse each well with PBS.

[0129] PCR experiments: Total RNA was extracted from cells in each treatment group using Trizol reagent, and the purity of the RNA was determined. cDNA was synthesized using reverse transcriptase (MLV). Using an appropriate amount of cDNA as a template, a specific fragment of the klotho gene was amplified in PCR using Taq DNA polymerase. The RT-PCR reaction conditions were set as follows: pre-denaturation at 94℃ for 5 minutes; followed by 40 cycles, each consisting of 94℃ for 30 seconds, 57℃ for 35 seconds, and 72℃ for 35 seconds; and a final extension at 72℃ for 10 minutes.

[0130] like Figure 1 As shown, the expression level of the klotho gene decreased significantly after D-gal induction, and the addition of the extract in the examples significantly improved the decrease in klotho gene expression induced by D-gal. This result indicates that the snow tea extract fermented by Bacillus licheniformis strain YK-L9 can be used in skin anti-aging products that promote klotho gene expression in human skin fibroblasts, filling a gap in the existing technology.

[0131] Test Example 2

[0132] This test case examines the antioxidant effects of the extracts from Examples 1-9 and Comparative Examples 1-7.

[0133] Prepare a 0.1 mg / mL DPPH solution with anhydrous ethanol and store it protected from light. Set up a sample group, a sample blank control group, a control group, and a control blank control group, with two replicates for each group.

[0134] In the sample group, 100 μL of the fermentation product filtrate of Examples 1-9 and Comparative Examples 1-7 were taken into 96-well plates as the test samples. Then, 100 μL of DPPH solution was added in equal volume, shaken and mixed, and reacted at room temperature in the dark for 30 min. The absorbance was measured at a wavelength of 517 nm.

[0135] The blank control group was treated with an equal volume of anhydrous ethanol instead of DPPH solution, and all other conditions were the same as those for the sample group.

[0136] The control group used an equal volume of ultrapure water instead of the fermentation product filtrate, and all other conditions were the same as the sample group.

[0137] The control group used an equal volume of ultrapure water instead of the fermentation product filtrate and anhydrous ethanol instead of the DPPH solution, with other conditions being the same as the sample group.

[0138] The specific composition of each sample to be tested is as follows:

[0139] Sample blank group: 100 μL sample solution + 100 μL DPPH alcohol solution;

[0140] Sample group: 100 μL DPPH solution + 100 μL sample solution;

[0141] Control group: 100 μL DPPH solution + 100 μL sample solvent;

[0142] Control blank group: 100 μL of anhydrous ethanol + 100 μL of sample solvent.

[0143] The DPPH clearance rate was calculated using the following formula, and the results are shown in Table 1.

[0144]

[0145] Table 1

[0146]

[0147] As shown in Table 1, the extracts obtained in Examples 1-9 exhibit good antioxidant effects. Among them, the DPPH inhibition rate of the fermentation product filtrate obtained in Examples 1-3 is the highest. Furthermore, the ratio of tea leaves to liquid, the inoculum size, the fermentation temperature, the fermentation time, the shaking speed, and the ceramic membrane pore size all significantly affect the DPPH free radical inhibition rate of the extracts.

[0148] Test Example 3

[0149] This test example measures the flavonoid and polyphenol content of the extracts prepared in Examples 1-9 and Comparative Examples 1-7.

[0150] Determination of total polyphenol content:

[0151] 1. Reagent preparation

[0152] 10% Folin-Ciocalteu reagent (freshly prepared): Transfer 20 mL of Folin-Ciocalteu reagent (1 mol / L) to a 200 mL volumetric flask, dilute to volume with water and shake well.

[0153] 7.5% sodium carbonate solution: Weigh 37.50g ± 0.01g sodium carbonate (Na2CO3), add an appropriate amount of water to dissolve, transfer to a 500mL volumetric flask, dilute to the mark, and shake well (can be stored at room temperature for 1 month).

[0154] 2. Sample Preparation

[0155] Test samples: extracts from Examples 1-9 and extracts from Comparative Examples 1-7.

[0156] Mother liquor for test sample: Accurately weigh 0.1 g of the concentrated and dried test sample into a 10 mL centrifuge tube, add 5 mL of 70% methanol aqueous solution preheated at 70℃, stir thoroughly with a glass rod to moisten, immediately transfer to a 70℃ water bath, and extract for 10 min (stirring every 5 minutes). After extraction, cool to room temperature. Transfer to a centrifuge at 3500 r / min for 10 min, transfer the supernatant to a 10 mL volumetric flask, and extract the residue again with 5 mL of 70% methanol aqueous solution. Repeat the above operation, combine the extracts and dilute to 10 mL, shake well. Filter through a 0.45 μm membrane and set aside for use (this extract can be stored at 4℃ for up to 24 h).

[0157] Test solution: Transfer 1 mL of the above stock solution into a 100 mL volumetric flask, dilute to the mark with water, shake well, and prepare for testing.

[0158] Preparation of standard solutions:

[0159] Gallic acid standard stock solution (1000 μg / mL): Weigh 0.110 g ± 0.001 g gallic acid (CAS: 149917) standard, dissolve it in a 100 mL volumetric flask and dilute to the mark, then shake well (prepare fresh).

[0160] Gallic acid working solution: Transfer 1.0 mL, 2.0 mL, 3.0 mL, 4.0 mL, and 5.0 mL of gallic acid standard stock solution into 100 mL volumetric flasks, respectively, and dilute to the mark with water. Shake well. The concentrations are 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL, respectively.

[0161] 3. Testing

[0162] Transfer 1.0 mL each of gallic acid working solution, water (for blank control), and test solution to graduated test tubes. Add 5 mL of 10% Folin-Ciocalteu reagent to each tube and mix well. React for 5 min, then add 4 mL of 7.5% sodium carbonate solution to each tube, dilute to the mark with water, and mix well. Incubate at room temperature for 60 min, and measure the absorbance at 765 nm, using the reagent blank solution as a reference. Plot a standard curve using the absorbance measured for the working curve solution and the corresponding gallic acid concentration. Read the concentration of total polyphenols in the test solution from the standard curve. Calculate the total polyphenol content in the sample using the following formula. The results are shown in Table 2.

[0163]

[0164] The method for detecting total flavonoid content was the sodium nitrite-aluminum nitrate method.

[0165] Specifically: Rutin reference standard was prepared into a 0.5 mg / mL standard solution, which was then diluted using a 2:1 dilution method to obtain rutin standard solutions of different concentrations. The extracts obtained in Examples 1-9 and Comparative Examples 1-7 were prepared into 0.5 mg / mL solutions as test samples. 1.0 mL of each standard solution and test sample was placed in a 10 mL volumetric flask, and 0.4 mL of 5% sodium nitrite solution was added to each. After mixing and standing for 6 min, 0.4 mL of 1% aluminum nitrate solution was added to each, and the mixture was mixed and stood for 6 min. Then, 2.0 mL of 4% sodium hydroxide solution was added to each, mixed, and the mixture was brought to volume. The flasks were then incubated at room temperature for 15 min, and the absorbance was measured at 500 nm. A standard curve was plotted based on the absorbance values ​​and concentrations, yielding the standard curve y = 0.4252x + 0.0527, R0. 2 =0.99;

[0166] The absorbance values ​​of the extracts obtained in Examples 1-9 and Comparative Examples 1-7 were substituted into the standard curve to calculate the total flavonoid content in the extracts.

[0167] The results are shown in Table 2.

[0168] Table 2

[0169]

[0170] As can be seen from the data in Table 2, the extracts prepared by the methods described in Examples 1-3 of this invention have high contents of total flavonoids and total polyphenols. Furthermore, the content of total flavonoids and total polyphenols will be significantly affected if the ratio of tea leaves to liquid, the amount of inoculum, the fermentation temperature, the fermentation time, the shaking speed, and the pore size of the ceramic membrane are outside the specified range.

[0171] Test Example 4

[0172] This test example demonstrates the anti-inflammatory properties of the extract prepared in Example 1.

[0173] The test cases used UVB-induced HaCaT cells (Chinese Academy of Sciences Cell Bank) as a model for testing the inhibitory effect on inflammatory factors.

[0174] UVB-induced inflammation and drug administration: Morphologically healthy HaCaT cells in the logarithmic growth phase were seeded into 12-well plates at a density of 1.0 × 10⁶ cells per well. 5 Cells were incubated in an incubator for 24 hours. A blank control group, a UVB-induced inflammation group, and a sample group were set up (the extract concentrations from Example 1 were 0.3%, 0.6%, and 1.2%, respectively), with three replicates for each treatment. Before UVB irradiation, the sample group cells were pretreated with the target concentration of the test substance for 2 hours. After 2 hours, the culture medium was replaced with PBS, and the UVB-induced inflammation group and the sample group were irradiated at an energy of 30 mJ / cm². 2 The samples were treated with UVB irradiation. After irradiation, the target concentration of the test substance was added to the sample group and incubated in an incubator at 37°C and 5% CO2 for 24 hours.

[0175] Gene expression level detection: RNA was extracted from each group of cells according to the RNA extraction kit instructions. After determining the RNA concentration, the mRNA was reverse transcribed into cDNA according to the reverse transcription kit instructions. The amount of RNA in each group was standardized based on the RNA concentration. The expression levels of TNF-α, IL-7, and SCF genes were detected using real-time quantitative PCR.

[0176] Results statistics: The change in expression level of the target gene in the treatment group relative to the negative control group was calculated according to the formula 2^-ΔΔct. The specific formula is: relative gene expression level = 2^- [Δct(tg) - Δct(nc)], where Δct(tg): ct (target gene in the treatment group) - ct (internal reference gene in the treatment group), and Δct(nc): ct (target gene in the negative control group) - ct (internal reference gene in the negative control group). If the ratio is greater than 1, it indicates that the gene expression is upregulated after treatment. If the ratio is less than 1, it indicates that the gene expression is downregulated after treatment.

[0177] Data Analysis: Experimental data were analyzed using GraphPad Prism 8 software. A two-tailed Student's t-test was used to compare the UVB-induced inflammation group with each other, with at least three replicates per group. Data are expressed as mean ± SEM. *P < 0.05 was considered statistically significant, **P < 0.01, and ***P < 0.001. Results are as follows: Figure 2 As shown.

[0178] according to Figure 2The results showed that the snow tea extract obtained in Example 1 had a significant inhibitory effect on the upregulation of TNF-α, IL-7 and SCF gene expression induced by UVB at concentrations of 0.3%, 0.6% and 1.2%, and was considered to have a good anti-UV-induced inflammation effect.

[0179] Test Example 5

[0180] This test example describes the sensitization testing of the extracts obtained in Examples 1-3. The specific procedures are as follows:

[0181] The sensitization potential of the obtained snow tea extract was tested using the human acute monocytic leukemia cell line THF-1 via the H-CLAT cell assay. CD86 refers to the CD86 protein, belonging to the immunoglobulin superfamily, and is a molecule expressed on antigen-presenting cells; CD54 refers to intercellular adhesion molecule 1. A "-" indicates negative CD86 / CD54 expression. The H-CLAT assay was considered negative if the expression levels of the surface markers CD86 and CD54 in the snow tea extract did not exceed the threshold at a concentration of 0.1 mg / mL; otherwise, it was considered positive. "Negative" indicates no sensitization risk, while "positive" indicates a potential sensitization risk. The results are shown in Table 3.

[0182] Table 3

[0183]

[0184] As shown in Table 3, the test results for Examples 1-3 of this invention were all negative, indicating that the extracts prepared by the methods provided in Examples 1-3 of this invention are safe and non-irritating, pose no risk of sensitization, and can be safely applied in the field of skincare products. Furthermore, they fully comply with all indicators in the "Cosmetic Safety Technical Specifications (2015 Edition)" promulgated by the state, are non-irritating to the skin, and are safe and reliable.

[0185] The results above indicate that, on the one hand, the snow tea extract fermented by Bacillus licheniformis strain YK-L9 promoted the expression of the klotho gene in human skin fibroblasts. On the other hand, fermentation by Bacillus licheniformis strain YK-L9 also promoted the release of the active ingredients (total polyphenol content and total flavonoid content) in the snow tea extract, and it exhibits excellent in vitro DPPH free radical scavenging ability and anti-inflammatory ability, demonstrating good in vitro antioxidant, anti-inflammatory, and anti-aging efficacy. Furthermore, it is non-sensitizing, highly safe, and can be widely used in the cosmetics field.

[0186] The embodiments and application examples described above are merely illustrative descriptions of this application and are not intended to limit the scope of this application. Any modifications and improvements made to the technical solutions of this application by those skilled in the art without departing from the spirit of this application should fall within the protection scope defined by this application.

Claims

1. A method for preparing a snow tea extract, characterized in that: The preparation method includes the following steps: S1. Mix the crushed snow tea raw material with water at a mass ratio of 1:7~15 to obtain the first mixture; S2. Culture Bacillus licheniformis on a culture medium to obtain a seed culture of Bacillus licheniformis; S3. The seed liquid is used as a fermentation liquid and added to the first mixture at an inoculation rate of 1% to 5%, and the fermentation mixture is obtained after fermentation. S4. The fermentation mixture is filtered, and the product obtained by concentrating and drying the filtrate is the snow tea extract; The Bacillus licheniformis strain mentioned is Bacillus licheniformis YK-L9, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 21, 2025, with the accession number GDMCC No. 66840.

2. The preparation method according to claim 1, characterized in that, In step S1, the first mixture is further sterilized by steam at 121°C.

3. The preparation method according to claim 1, characterized in that, In step S2, the revived Bacillus licheniformis YK-L9 strain is inoculated into LB liquid medium and cultured at 25℃~40℃ for 18 h~36 h. After activating for 1~2 generations, the seed culture of Bacillus licheniformis YK-L9 strain is obtained.

4. The preparation method according to claim 1, characterized in that, In step S3, the fermentation temperature is 25℃~40℃, the fermentation time is 24~48h, and the shaking speed is 150 rpm~220 rpm.

5. The preparation method according to claim 1, characterized in that, In step S4, the fermentation mixture is filtered using a ceramic membrane with a pore size of 0.1~10μm.

6. A snow tea extract, characterized in that, The snow tea extract is obtained by fermenting snow tea using Bacillus licheniformis as the fermentation bacteria. The Bacillus licheniformis strain mentioned is Bacillus licheniformis YK-L9, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 21, 2025, with the accession number GDMCC No. 66840.

7. The use of the snow tea extract according to claim 6 in the preparation of cosmetics with anti-aging, anti-inflammatory and antioxidant effects.

8. A skincare product that promotes the expression of the klotho gene in human fibroblasts, characterized in that, The skincare product contains the snow tea extract as described in claim 6.

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