Bud extract, preparation method and application thereof

Abstract

This application relates to a plum blossom bud extract, its preparation method, and its application, belonging to the field of cosmetic raw material technology. The plum blossom bud extract is obtained by fermenting plum blossom buds using *Bacillus belye* as the fermentation bacteria; wherein the *Bacillus belye* strain is *Bacillus belye* BTN-BL13, classified and named... Bacillus from Velez This application was deposited on November 3, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 67198. The fermentation process used in this application is efficient and low-cost, improving the utilization efficiency of plum blossom bud raw materials and enhancing the skin safety of plum blossom bud extract, thus ensuring the quality and efficacy of the product.

Description

Technical Field

[0001] This disclosure relates to the field of cosmetic raw material technology, specifically to a plum blossom bud extract, its preparation method, and its application. Background Technology

[0002] Plum blossom buds are the buds of the plum tree (Prunus mume), a plant in the Rosaceae family. Prunus mume Siebold & Zucc., commonly known as green plum, Chinese plum, dried plum, sour plum, black plum, white plum, wild plum, western plum, and spring plum, contains flower buds. Plum buds contain components with excellent antioxidant, anti-aging, skin-brightening, and anti-inflammatory effects. Currently, research on plum bud extracts (… Prunus mume The preparation of bud extract mainly focuses on methods such as direct pressing and ethanol extraction.

[0003] For example, CN109303723A discloses a plum blossom embryo extract, its preparation method, and its application. The method involves harvesting the flower embryos of green plum blossoms at a highly active stage, i.e., in the bud stage. Within 24 hours of harvesting, the embryos are rapidly frozen to -28 degrees Celsius, and then frozen-pressed at -196 degrees Celsius to obtain fresh whole fruit pulp, which is the plum blossom embryo extract. The obtained extract has the function of promoting the expression of hyaluronic acid, type I collagen, and elastin in the skin. It can be used as an active ingredient in the preparation of topical skin agents, serving as an active ingredient for moisturizing, anti-aging, or skin repair.

[0004] CN101536967A discloses a method for extracting plum blossoms, a new use for plum blossoms, and an anti-aging skin cream made from plum blossoms. The method uses supercritical CO2 to extract the non-polar components of dried plum blossoms and refluxes 30% ethanol to extract the polar components. The obtained plum blossom extract is further compounded with other skincare ingredients to prepare an anti-aging skin cream with antioxidant properties and the ability to promote skin cell metabolism and delay skin aging.

[0005] For example, CN112386543A discloses a method for preparing and applying a total flavonoid extract from white plum blossoms. This method involves extracting white plum blossoms with 50% ethanol under reflux for 2-4 times, combining the filtrates, and concentrating to obtain an extract. After removing impurities by water precipitation, the extract is purified by macroporous resin column separation. The concentrated eluent is then dried to obtain the total flavonoid extract from white plum blossoms. The prepared total flavonoid extract has a purity of over 70%, and its main components are a mixture of hyperoside, isoquercitrin, quercetin-3-O-neohesperidin, and rutin, all possessing flavonoid structural cores. It exhibits activities such as inhibiting tyrosinase activity, preventing pigmentation, reducing melanin, scavenging free radicals, and clearing heat, detoxifying, and reducing inflammation. It can be used to prepare whitening, spot-removing, anti-aging, and acne-removing cosmetics.

[0006] However, the above-mentioned and similar preparation methods have drawbacks such as low extraction efficiency and poor environmental friendliness. Furthermore, the residual organic solvents in the obtained extracts can easily cause skin sensitivity in users. With the increasing number of people with sensitive skin globally, higher requirements are being placed on the safety of cosmetic ingredients. Moreover, current extraction methods may result in the loss of plum blossom bud raw materials, leading to low utilization efficiency and impacting the quality and efficacy of the final product.

[0007] Therefore, current methods for preparing plum blossom bud extract are insufficient to meet the modern cosmetics industry's demand for efficient, safe, and natural raw materials. Summary of the Invention

[0008] Therefore, the purpose of this application is to improve the efficiency and convenience of preparing plum blossom bud extract, as well as to improve the safety of plum blossom bud extract for skin use.

[0009] To achieve the above objectives, this application provides a plum blossom bud extract, which is obtained by fermenting plum blossom buds using Bacillus belye as the fermenting bacteria.

[0010] Among them, the *Bacillus belyssus* strain is *Bacillus belyssus* BTN-BL13, classified and named as follows: Bacillus velezensis It was obtained from Meconopsis plants from Haba Snow Mountain in Yunnan Province and was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 3, 2025, with accession number GDMCC No: 67198.

[0011] To achieve the above objectives, this application provides a method for preparing plum blossom bud extract, comprising the following steps:

[0012] S1. Mix dried plum blossom buds with water at a mass ratio of 1:30 to 1:10 to obtain the first mixture;

[0013] S2. Culture Bacillus belye to obtain a seed culture of Bacillus belye;

[0014] S3. Using the seed liquid as a fermentation liquid, add it to the first mixture at an inoculum amount of 3% to 8% to obtain a second mixture; wherein the inoculum amount can be 3%, 4%, 5%, 6%, 7%, 8%, etc.

[0015] S4. The second mixture is centrifuged and filtered, and the filtrate is dried to obtain the plum blossom bud extract;

[0016] Among them, the *Bacillus belyssus* strain is *Bacillus belyssus* BTN-BL13, classified and named as follows: Bacillus velezensis It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 3, 2025, with accession number GDMCC No: 67198.

[0017] Preferably, in step S1, the first mixture is sterilized by steam at 121°C.

[0018] Preferably, step S2 is as follows: the revived Bacillus belye BTN-BL13 strain is inoculated into LB medium and cultured at 25℃~40℃ for 18 h~48 h, and the seed liquid is obtained after 1~2 generations of activation.

[0019] More preferably, the LB medium is LB liquid medium or LB solid medium; the LB liquid medium includes tryptone, yeast extract, NaCl and ultrapure water; the LB solid medium includes tryptone, yeast extract, NaCl, ultrapure water and agar powder.

[0020] Preferably, in step S3, the fermentation temperature is 25~40℃, the fermentation time is 16~36h, and the shaking speed is 100 rpm~220 rpm. Specifically, 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 16h, 18h, 20h, 22h, 24h, 25h, 26h, 27h, 28h, 30h, 35h, 36h, etc.; and the shaking speed can be 100rpm, 110rpm, 120rpm, 130rpm, 140rpm, 150rpm, 160rpm, 170rpm, 180rpm, 190rpm, 200rpm, 210rpm, 220rpm, etc.

[0021] Preferably, in step S4, the second mixture is taken, centrifuged, and the supernatant is collected. The supernatant is then filtered through a ceramic membrane with a pore size of 0.1~10μm to obtain the filtrate. 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.

[0022] This application also provides the application of plum blossom bud extract in skin care products, which have soothing, emotional beauty, barrier repair, anti-glycation, anti-inflammatory, antioxidant, anti-aging and photoprotective effects. The plum blossom bud extract is obtained by fermenting plum blossom buds with Bacillus vesiculosus as the fermentation bacteria.

[0023] Among them, the *Bacillus belyssus* strain is *Bacillus belyssus* BTN-BL13, classified and named as follows: Bacillus velezensis It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 3, 2025, with accession number GDMCC No: 67198.

[0024] This application further provides a skin care product containing plum blossom bud extract, which has soothing, emotional beauty, barrier repair, anti-glycation, anti-inflammatory, antioxidant, anti-aging and photoprotective effects. The plum blossom bud extract is obtained by fermenting plum blossom buds with Bacillus vesiculosus as the fermentation bacteria.

[0025] Among them, the *Bacillus belyssus* strain is *Bacillus belyssus* BTN-BL13, classified and named as follows: Bacillus velezensis It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 3, 2025, with accession number GDMCC No: 67198.

[0026] Preferably, the plum blossom bud extract is added to the skin care product at a rate of 0.75% to 3% by weight; wherein the added amount may be 0.75%, 0.85%, 0.9%, 0.95%, 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.

[0027] The solution claimed in this application achieves the following beneficial effects:

[0028] 1. For the first time, *Bacillus belye* strain BTN-BL13 was used as the fermentation agent to extract plum blossom bud extract through fermentation. This process is highly efficient and low-cost, suitable for industrial-scale production. Furthermore, this method avoids the drawbacks of traditional extraction processes, such as harsh extraction conditions, residual organic solvents, low extraction efficiency, complex operation, and high equipment costs. It improves the utilization efficiency of plum blossom bud raw materials and enhances the skin safety of the extract, ensuring product quality and efficacy.

[0029] 2. The extraction method using Bacillus vesiculosus strain BTN-BL13 as the fermentation agent promotes the release of active ingredients in plum blossom bud extract, resulting in increased yield, total polyphenol content (>25%), and total polysaccharide content (>20%). This also enhances the soothing, emotional beauty, barrier repair, anti-glycation, antioxidant, and photodamage-resistant effects of the plum blossom bud extract. Furthermore, the obtained plum blossom bud extract is safe and non-irritating, with no risk of sensitization, and can be safely used as an active ingredient in skincare products, meeting the modern cosmetics industry's demand for highly effective, safe, and natural raw materials. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in this embodiment, 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.

[0031] Figure 1 This is a 3D skin-soothing activity test of the plum blossom bud extract obtained in Example 1. In this test, Control represents the normal group, SLS represents the sensitive skin model group, and dexamethasone is the positive control group.

[0032] Figure 2 This image shows skin HE staining / immunofluorescence images for the 3D skin anti-aging activity test of the plum blossom bud extract obtained in Example 1. In the images, Control represents the normal group, UVA represents the aging model group, and VC+VE represents the positive control group. The top row of the image shows the skin HE staining image for the 3D skin anti-aging activity test of the obtained plum blossom bud extract, and the bottom row shows the fluorescence staining image with COL4 (type IV collagen) marked as green fluorescence.

[0033] Figure 3 This is a qRT-PCR image of the 3D skin anti-aging activity test of the plum blossom bud extract obtained in Example 1. In the image, Control represents the normal group, UVA represents the aging model group, and VC+VE represents the positive control group.

[0034] Figure 4 The results of the antioxidant activity test of the plum blossom bud extract obtained in Example 1 are shown.

[0035] Figure 5 This test examines the photodamage resistance activity of the plum blossom bud extract obtained in Example 1. BC represents the normal control group, and UVB represents the photodamage model group. Detailed Implementation

[0036] 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 disclosure, not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0037] <Materials and Reagents>

[0038] Unless otherwise specified, all raw materials and reagents used in this section were purchased through general commercial channels.

[0039] The source information of the raw materials, materials, and instruments involved in the following examples, preparation examples, and comparative examples is as follows:

[0040] Bacillus belyssus BTN-BL13: Self-developed, as described in the examples;

[0041] Lactobacillus plantarum BTN-E1: Self-developed, as described in the examples;

[0042] Bacillus BTN-D16: Self-developed, as described in the examples;

[0043] Bacillus subtilis BTN-P43: Self-developed, as described in the examples;

[0044] Candida bacillus BTN-M19: Self-developed, as described in the examples;

[0045] LB medium: Qingdao Haibo Biotechnology;

[0046] DPPH (1,1-Diphenyl-1-picrylhydrazyl), Aladdin;

[0047] Skin fibroblasts (HFF cells) were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences.

[0048] DMEM culture medium, Eva cells;

[0049] Fetal bovine serum, Gibco;

[0050] The sample solvent is sterile water.

[0051] <Culture medium>

[0052] LB liquid medium: 10 g tryptone, 5 g yeast extract, 10 g NaCl and 1 L ultrapure water;

[0053] LB solid medium: Add 2% agar powder to LB liquid medium;

[0054] MRS liquid culture medium: 10 g peptone, 8 g beef meal, 4 g yeast powder, 20 g glucose, 2 g dipotassium hydrogen phosphate, 2 g diammonium hydrogen citrate, 5 g sodium acetate, 0.2 g magnesium sulfate, 0.04 g manganese sulfate, 1 g Tween and 1 L ultrapure water;

[0055] MRS solid medium: Add 2% agar powder to MRS liquid medium;

[0056] YPD liquid culture medium: 10 g peptone, 5 g yeast extract, 20 g glucose and 1 L ultrapure water;

[0057] YPD solid medium: Add 2% agar powder to YPD liquid medium;

[0058] The above culture medium was sterilized at 121°C for 15 min before use.

[0059] <Preparation Example 1>

[0060] This preparation example provides Bacillus belyssus isolated from the symbiotic bacteria of Meconopsis plants from Haba Snow Mountain in Yunnan. Bacillus velezensis The screening method for BTN-BL13, and the specific steps are as follows:

[0061] 1) Select wild Meconopsis 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℃ ~ 40℃ for 18h ~ 48h. Pick out colonies of different morphologies on LB solid medium, streak them on the surface of LB solid medium for purification, and subculture them at least twice to obtain purified colonies.

[0062] 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 belye was obtained and named BTN-BL13. At the same time, the selected strain was deposited at the Guangdong Provincial Microbial Culture Collection Center, located on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou, on November 3, 2025, with accession number GDMCC No: 67198.

[0063] Sequencing of the selected Bacillus belyssus BTN-BL13 revealed that its ITS sequence is shown in the following sequence listing SEQ NO:1:

[0064]

[0065] <Preparation Example 2>

[0066] This preparation example provides *Lactobacillus plantarum* derived from *Cypripedium yunnanensis*. Lactiplantibacillus paraplantarum The screening method for BTN-E1 differs from that in Preparation Example 1 only in that the medium used is MRS solid medium instead of LB solid medium, and the strain was not preserved.

[0067] <Preparation Example 3>

[0068] This preparation example provides Bacillus subtilis derived from Rhodiola rosea (Yunnan Rhodiola) ( Bacillus subtilis The screening method for BTN-P43 differs from that in Preparation Example 1 only in that the strain was not preserved.

[0069] <Preparation Example 4>

[0070] This preparation example provides Yunnan green-head mushroom (i.e., green-head mushroom that grows in Yunnan (scientific name: *Pleurotus ostreatus*)). Russula virescens (Schaeff.) Fr ))) from the source of Candida glabrata ( Candida parapsilosis The screening method for BTN-M19 differs from that in Preparation Example 1 only in that YPD solid medium is used instead of LB solid medium, and the strain is not preserved.

[0071] <Preparation Example 5>

[0072] This preparation example provides *Candida santalis* from the Yunnan sea cucumber (*Scutellaria baicalensis*). Candida santamariae The screening method for BTN-P49 is the same as that in preparation example 4.

[0073] <Example 1>

[0074] This embodiment provides a method for preparing plum blossom bud extract, the preparation steps of which are as follows:

[0075] S1. Take 10g of plum blossom bud raw material and mix it with water at a material-to-liquid ratio of 1:20 by weight; after steam sterilization at 121℃ for 20 min, the first mixture is obtained;

[0076] S2. The first mixture is inoculated and fermented using Bacillus berreatus BTN-BL13 obtained in Preparation Example 1:

[0077] Bacillus belysin BTN-BL13 strain was inoculated into LB liquid medium for activation. During activation, the culture was carried out at 37°C for 24 hours. After two generations of activation, seed culture of Bacillus belysin BTN-BL13 was obtained.

[0078] The above seed liquid was inoculated into the first mixture at an inoculum amount of 5% (v / v) of the first mixture for fermentation. The fermentation temperature was 37°C, the fermentation time was 24 hours, and the shaking speed was 200 rpm. After the fermentation was completed, the second mixture was obtained.

[0079] S3. Centrifuge the second mixture at 5000 rpm for 15 min, take the supernatant and filter it through a 0.22 μm ceramic membrane for purification, concentrate the filtrate under reduced pressure and freeze dry at -80℃ to obtain plum blossom bud extract.

[0080] <Example 2>

[0081] This embodiment provides a method for preparing plum blossom bud extract, the preparation steps of which are as follows:

[0082] S1. Take 10g of plum blossom bud raw material and mix it with water at a ratio of 1:10 by weight; after steam sterilization at 121℃ for 20 min, the first mixture is obtained;

[0083] S2. The first mixture is inoculated and fermented using Bacillus berreatus BTN-BL13 obtained in Preparation Example 1:

[0084] Bacillus belysin BTN-BL13 strain was inoculated into LB liquid medium for activation. During activation, the culture was carried out at 25°C for 36 hours. After two generations of activation, the seed culture of Bacillus belysin BTN-BL13 was obtained.

[0085] The above seed liquid was inoculated into the first mixture at an inoculum amount of 3% (v / v) of the first mixture for fermentation. The fermentation temperature was 40℃, the fermentation time was 36h, and the shaking speed was 150 rpm. After the fermentation was completed, the second mixture was obtained.

[0086] S3. Centrifuge the second mixture at 5000 rpm for 15 min, take the supernatant and filter it through a 0.10 μm ceramic membrane for purification, concentrate the filtrate under reduced pressure and freeze dry at -80℃ to obtain plum blossom bud extract.

[0087] <Example 3>

[0088] This embodiment provides a method for preparing plum blossom bud extract, the preparation steps of which are as follows:

[0089] S1. Take 10g of plum blossom bud raw material and mix it with water at a ratio of 1:30 by weight; after steam sterilization at 121℃ for 20 min, the first mixture is obtained;

[0090] S2. The first mixture is inoculated and fermented using Bacillus berreatus BTN-BL13 obtained in Preparation Example 1:

[0091] Bacillus belyssus strain BTN-BL13 was inoculated into LB liquid medium for activation. During activation, the culture was carried out at 40℃ for 36 h. After two generations of activation, seed culture of Bacillus belyssus strain BTN-BL13 was obtained.

[0092] The above seed liquid was inoculated into the first mixture at an inoculum amount of 8% (v / v) of the first mixture for fermentation. The fermentation temperature was 25℃, the fermentation time was 16h, and the shaking speed was 220 rpm. After the fermentation was completed, the second mixture was obtained.

[0093] S3. Centrifuge the second mixture at 5000 rpm for 15 min, take the supernatant and filter it through a 10 μm ceramic membrane for purification, concentrate the filtrate under reduced pressure and freeze dry at -80℃ to obtain plum blossom bud extract.

[0094] <Example 4>

[0095] This embodiment provides a method for preparing plum blossom bud extract, the specific steps of which differ from those in Example 1 only in that:

[0096] In step S1, the ratio of plum blossom buds to water is 1:50;

[0097] In step S2, the inoculum amount is 12% (v / v) of the first mixture.

[0098] <Example 5>

[0099] This embodiment provides a method for preparing plum blossom bud extract, the specific steps of which differ from those in Example 1 only in that:

[0100] In step S1, the ratio of plum blossom buds to water is 1:5;

[0101] In step S2, the inoculum amount is 1% (v / v) of the first mixture.

[0102] <Example 6>

[0103] This embodiment provides a method for preparing plum blossom bud extract, the specific steps of which differ from those in Example 1 only in that:

[0104] In step S2, the fermentation temperature is 50°C and the shaking speed is 100 rpm.

[0105] <Example 7>

[0106] This embodiment provides a method for preparing plum blossom bud extract, the specific steps of which differ from those in Example 1 only in that:

[0107] In step S2, the fermentation temperature is 20°C and the shaking speed is 280 rpm.

[0108] <Example 8>

[0109] This embodiment provides a method for preparing plum blossom bud extract, the specific steps of which differ from those in Example 1 only in that:

[0110] In step S2, the fermentation time is 12 hours;

[0111] In step S3, the pore size of the ceramic membrane is 0.01 μm.

[0112] <Example 9>

[0113] This embodiment provides a method for preparing plum blossom bud extract, the specific steps of which differ from those in Example 1 only in that:

[0114] In step S2, the fermentation time is 48 hours;

[0115] In step S3, the pore size of the ceramic membrane is 20 μm.

[0116] <Comparative Example 1>

[0117] This comparative example provides a method for preparing plum blossom bud extract, the specific steps of which differ from those in Example 1 only in that:

[0118] In step S2, the strain used is *Lactobacillus plantarum* BTN-E1 obtained in Preparation Example 2.

[0119] <Comparative Example 2>

[0120] This comparative example provides a method for preparing plum blossom bud extract, the specific steps of which differ from those in Example 1 only in that:

[0121] In step S2, the strain used is Bacillus subtilis BTN-P43 obtained in Preparation Example 3.

[0122] <Comparative Example 3>

[0123] This comparative example provides a method for preparing plum blossom bud extract, the specific steps of which differ from those in Example 1 only in that:

[0124] In step S2, the strain used is *Candida parapsilosis* BTN-M19 obtained in Preparation Example 4.

[0125] <Comparative Example 4>

[0126] This comparative example provides a method for preparing plum blossom bud extract, the specific steps of which differ from those in Example 1 only in that:

[0127] In step S2, the strain used is Candida Santamarie BTN-P49 obtained in Preparation Example 5.

[0128] <Comparative Example 5>

[0129] This comparative example provides a method for preparing plum blossom bud extract, the specific steps of which differ from those in Example 1 only in that:

[0130] In step S2, fermentation is not carried out by inoculating with a bacterial strain.

[0131] <Comparative Example 6>

[0132] This comparative example provides a method for preparing plum blossom bud extract, the specific steps of which differ from those in Example 1 only in that:

[0133] In step S1, the first mixture is replaced with an equal mass of LB medium.

[0134] <Comparative Example 7>

[0135] This comparative example provides a method for preparing plum blossom bud extract, the specific steps of which differ from those in Example 1 only in that:

[0136] In step S3, the step of filtration with a ceramic membrane is omitted, and the product is directly concentrated under reduced pressure after centrifugation.

[0137] <Test Example 1>

[0138] This test example measures the yield, polyphenol and polysaccharide content of the plum blossom bud extracts prepared in Examples 1-9 and Comparative Examples 1-7.

[0139] (1) Yield

[0140] The yield was calculated using the formula: Yield (%) = (M1 / M2) × 100%, where M1 is the weight of the plum blossom bud extract and M2 is the amount of plum blossom bud extract fed into the feed. The results are shown in Table 1.

[0141] (2) Determination of total polyphenol content

[0142] 1. Reagent preparation

[0143] 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.

[0144] 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).

[0145] 2. Sample Preparation

[0146] Test samples: Plum blossom bud extracts obtained in Examples 1-9 and Comparative Examples 1-7.

[0147] Sample stock solution: Accurately weigh 0.2 g of the 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, and immediately transfer to a 70℃ water bath for extraction for 10 min (stirring every 5 minutes). After extraction, cool to room temperature. Transfer to a centrifuge and 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, and 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).

[0148] 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.

[0149] Preparation of standard solution (gallic acid standard stock solution (1000 μg / mL)): Weigh 0.100 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).

[0150] 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.

[0151] 3. Testing

[0152] 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, and calculate the total polyphenol content in the sample. The results are shown in Table 1. The total polyphenol content is calculated using the following formula:

[0153]

[0154] (3) Determination of total polysaccharide content

[0155] The total polysaccharide content in the test samples was determined using the sulfuric acid-phenol method.

[0156] Preparation of standard solution: Prepare a 0.1 mg / mL glucose standard solution by successively pipetting 0, 0.1, 0.2, 0.4, 0.8, 1.6, and 2 mL into test tubes, adding ddH2O to each test tube to make up to 2 mL, and set aside for use.

[0157] Preparation of sample solution: Accurately weigh the test sample powder, add ddH2O to prepare a sample solution of 0.1 mg / mL, ready for testing.

[0158] Preparation of 5% phenol solution: Accurately weigh 2.5g of phenol solid powder, add 47.5mL of ddH2O and dissolve at 75℃, then store in the dark.

[0159] Reaction of standard and sample solutions: 1 mL of each of the prepared standard and sample solutions was added sequentially to a test tube. 0.5 mL of 5% phenol solution and 2.5 mL of concentrated sulfuric acid solution were added, and the mixture was gently shaken. ddH₂O was used as a blank control. The absorbance was measured at 490 nm using an ELISA reader. A standard curve was plotted with concentration on the x-axis and absorbance on the y-axis. The absorbance of the sample solution was substituted into the standard curve for calculation. The polysaccharide content determination results are shown in Table 1.

[0160] Table 1. Results of yield, total polyphenol content and total polysaccharide content of plum blossom bud extract.

[0161] Group Yield (%) Polyphenols (%) Polysaccharides (%) Example 1 19.62 30.19 25.45 Example 2 17.98 27.69 22.78 Example 3 20.96 25.63 23.17 Example 4 17.26 20.29 17.48 Example 5 10.41 13.65 10.19 Example 6 12.68 19.78 16.83 Example 7 12.35 18.25 15.22 Example 8 8.01 12.32 13.75 Example 9 15.16 18.48 15.71 Comparative Example 1 13.25 18.02 16.65 Comparative Example 2 10.13 19.65 12.96 Comparative Example 3 12.02 14.39 14.61 Comparative Example 4 11.29 16.47 6.21 Comparative Example 5 11.83 17.62 15.27 Comparative Example 6 3.95 0 1.25 Comparative Example 7 25.91 16.31 14.28

[0162] As shown in Table 1, the extracts prepared by the methods in Examples 1-3 have high yields and high contents of total polyphenols and total polysaccharides. The ratio of plum blossom buds to water, the amount of inoculum, the fermentation temperature, the fermentation time, the shaking speed, and the pore size of the ceramic membrane will have a significant impact on the yield and the contents of total polyphenols and polysaccharides if they exceed the specified range.

[0163] <Test Example 2>

[0164] This test example performs a soothing test on the plum blossom bud extracts prepared in Examples 1-9 and Comparative Examples 1-7, specifically testing the inhibitory activity of the soothing target TRPV1 (transient receptor potential vanillic acid subtype 1).

[0165] TRPV1 is a key ion channel. TRPV1 is easily activated by various physicochemical factors, including heat (>42℃), low pH (<5.9), capsaicin, and nerve growth factor. When these stimuli bind to TRPV1 receptors, they induce calcium influx, thereby triggering action potentials in cells and sending signals of skin discomfort to the brain. This activation not only leads to sensations such as stinging, itching, and burning, but also affects the integrity of the skin barrier, delaying barrier recovery and further exacerbating the symptoms of sensitive skin. Sensitive skin is related to damage to the epidermal barrier function, and TRPV1 plays an important role in the pathogenesis of sensitive skin. TRPV1 activation also affects barrier integrity, making the skin more susceptible to damage from external stimuli. Therefore, TRPV1 activation is a detrimental factor for sensitive skin.

[0166] The testing method used in this test case is as follows:

[0167] A keratinocyte-based soothing efficacy testing model was used to evaluate the soothing effects of the plum blossom bud extracts prepared in Examples 1-9 and Comparative Examples 1-7 by immunofluorescence (IF) detection of their TRPV1 protein inhibition effect. Four groups were set up: a blank control group, a model group, a positive control group, and a sample group. The blank control group was a normal group without any treatment. The model group was an inflammatory state group with abnormally elevated TRPV1 expression after stimulation with 15 μM CAP reagent. 15.6 μg / mL of trans-4-tert-butylcyclohexanol was used as a positive control. The sample group consisted of plum blossom bud extracts prepared in Examples 1-9 and Comparative Examples 1-7 at a mass concentration of 15 μg / mL. Keratinocytes were used as the detection model.

[0168] The results of the TRPV1 protein content detection model are shown in Table 2.

[0169] Table 2. TRPV1 inhibition results of plum blossom bud extract

[0170] Group TRPV1 inhibition rate (%) Example 1 83.15 Example 2 72.39 Example 3 74.52 Example 4 36.26 Example 5 35.72 Example 6 45.43 Example 7 33.65 Example 8 43.51 Example 9 40.93 Comparative Example 1 38.46 Comparative Example 2 23.96 Comparative Example 3 18.89 Comparative Example 4 15.34 Comparative Example 5 30.24 Comparative Example 6 0 Comparative Example 7 38.28 trans-4-tert-butylcyclohexanol 60.98

[0171] <Test Example 3>

[0172] This test case examines the plum blossom bud extracts prepared in Examples 1-3 and Comparative Examples 1-7 for testing genes related to emotional beauty and barrier repair (Piezo2 and ZO-1).

[0173] Piezo2 is a mechanosensitive ion channel protein that dominates the skin's perception of mechanical stimuli such as light touch and mechanical itch. Piezo2 activation can enhance the perception of gentle touch (sensing light touch and caresses, promoting pleasure and social bonding), barrier repair (mild mechanical stimulation may trigger barrier repair signals through Piezo2), and hair growth promotion (mild traction on hair follicles may promote growth through Piezo2).

[0174] ZO-1 is a cytoplasmic scaffold protein, and its specific mechanism of action is as follows:

[0175] 1) Constructing a physical barrier: In the granular and spinous layers, ZO-1 binds to transmembrane proteins such as Claudin-1, Claudin-4, and Occludin, forming a continuous tight junction chain. These structures act like a "fence," selectively preventing the passive diffusion of water, ions, and macromolecules through intercellular spaces, which is crucial for maintaining moisture within the skin and blocking external substances.

[0176] 2) Maintaining cell polarity: Tight junctions separate the membrane proteins on the apical and basal sides of the cell, maintaining the normal polarity of keratinocytes, which is crucial for their orderly differentiation.

[0177] 3) Regulation of barrier plasticity: Tight junctions require dynamic reconstruction during wound healing, cell differentiation, and other processes. The phosphorylation / dephosphorylation state of ZO-1 can sensitively regulate its binding ability with other proteins, thereby dynamically controlling the "opening and closing" state of tight junctions to adapt to physiological needs.

[0178] 4) Synergistic effects with other junctions: ZO-1 also interacts with adhesion junctions, desmosomes, and other structures to jointly maintain the overall structure and mechanical strength of the epidermis. When ZO-1 expression is downregulated, its distribution is abnormal, or its function is impaired, the tight junctions of the skin become loose and broken, leading to severe damage to the barrier function. This may trigger atopic dermatitis, skin sensitivity, etc.

[0179] The testing process for this test case is as follows:

[0180] Cell seeding: HaCaT cells were obtained from the laboratory of Betaine Biotechnology Co., Ltd., and cultured in DMEM + 10% fetal bovine serum medium. HaCaT cells in the logarithmic growth phase at passages 15-25 were selected for the experiment and cultured in 6-well plates with a cell concentration of 1.8 × 10⁶ cells per well. 5 The cells were cultured in 2.5 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.

[0181] Cell treatment: Remove the culture plate from the incubator, aspirate the original culture medium from the wells, and rinse with PBS. Set up a blank control group (NC reagent group) and a sample group (plum blossom bud extract from Examples 1-3 and Comparative Examples 1-7, all prepared at a concentration of 120 μg / mL), with 3 replicates for each treatment. Then, place the culture plate in an incubator with a CO2 concentration of 5% and a temperature of 37°C for 24 hours. After 24 hours, remove the 6-well plate containing the cultured HaCaT cells from the incubator, aspirate the original cell culture medium from the wells using a negative pressure aspirator, and rinse each well with PBS.

[0182] Gene expression level detection: RNA was extracted from each group of cells according to the RNA extraction kit instructions. After the RNA concentration was measured, the mRNA was reverse transcribed into cDNA according to the reverse transcription kit instructions. The amount of RNA in each group was standardized according to the RNA concentration. The expression levels of piezo2 and ZO-1 genes in HaCaT cells were detected by real-time quantitative PCR.

[0183] Results statistics: The 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)], Δct(tg) = ct (target gene in the treatment group) - ct (internal reference gene in the treatment group), Δct(nc) = ct (target gene in the negative control group) - ct (internal reference gene in the negative control group).

[0184] Data Analysis: The experimental data were analyzed using the software GraphPad Prism 8, and the results are shown in Table 3.

[0185] Table 3. Effects of plum blossom bud extract on Piezo2 and ZO-1 gene expression

[0186]

[0187] According to the results in Table 3, the plum blossom bud extracts obtained in Examples 1-3, compared with those obtained in Comparative Examples 1-7, significantly upregulated the expression of the emotion-beauty-related gene piezo2 and the barrier repair-related gene ZO-1 at a concentration of 120 μg / mL. Therefore, it is believed that they have good emotion-beauty and barrier repair effects.

[0188] <Test Example 4>

[0189] In this test example, the plum blossom bud extracts prepared in Examples 1-3 and Comparative Examples 1-7 were used as test samples for anti-glycation testing.

[0190] Glycation is a fundamental mechanism in the human aging process. It is a spontaneous, non-enzymatic reaction between sugars and proteins, leading to the formation of advanced glycation end products (AGEs). AGEs bind to collagen or elastin, reducing skin elasticity and causing sagging and fine lines. The accumulation of glycated proteins can darken and yellow the skin. In the epidermis, AGEs disrupt keratinization, slow skin repair, and damage the skin barrier. Glycation begins around age 35 and accelerates with endogenous aging.

[0191] The testing method for this test case is as follows:

[0192] A 0.05 mg / mL sample solution was prepared using phosphate buffer (50 mmol / L, pH 7.4). 1 mL of the sample, 0.8 mg / mL bovine serum albumin (BSA) solution, 200 mM glucose solution, and phosphate buffer (PBS) were added to separate test tubes. The tubes were heated at 60 °C for 24 h. Aminoguanidine hydrochloride (AG) (250 μg / mL) was used as a positive control (the setup for each experimental group is shown in Table 4). Fluorescent AGEs were measured using a fluorescence microplate reader with an excitation wavelength of 370 nm and an emission wavelength of 420 nm. The content of fluorescent AGEs was expressed as fluorescence intensity (AU).

[0193] Table 4. Setup of each experimental group in the anti-glycation test

[0194]

[0195] The inhibition rate of fluorescent AGEs formation by the test sample is calculated as follows:

[0196]

[0197] Each group was measured an average of 3 times, and the average value was taken to calculate the AGEs formation inhibition rate. The results are shown in Table 5.

[0198] Table 5. Results of inhibition of AGEs formation by plum blossom bud extract

[0199] Group AGEs (%) Example 1 91.93 Example 2 88.07 Example 3 85.21 Comparative Example 1 50.88 Comparative Example 2 52.93 Comparative Example 3 49.72 Comparative Example 4 51.21 Comparative Example 5 50.48 Comparative Example 6 6.21 Comparative Example 7 46.25 aminoguanidine 38.69

[0200] As shown in Table 5, the plum blossom bud extracts obtained in Examples 1-3 have a better inhibitory effect on AGEs compared with those obtained in Comparative Examples 1-7, indicating that they have a good anti-glycation aging effect.

[0201] <Test Example 5>

[0202] This test example examines the allergenicity of the plum blossom bud extracts prepared in Examples 1-3.

[0203] The testing method for this test case is as follows:

[0204] The sensitization potential of plum blossom bud extract was tested using the human acute monocytic leukemia cell line THF-1 via the H-CLAT cell assay. The results are shown in Table 6. 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 expression levels of the surface markers CD86 and CD54 in the plum blossom bud extract did not exceed the threshold at a concentration of 0.1 mg / mL, and the H-CLAT assay was considered negative; otherwise, it was considered positive. "Negative" indicates no sensitization risk, while "positive" indicates a potential sensitization risk.

[0205] Table 6. Results of sensitization test of plum blossom bud extract

[0206]

[0207] As shown in Table 6, the test results for the plum blossom bud extracts obtained in Examples 1-3 were all negative, indicating that the extracts prepared by the methods provided in Examples 1-3 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.

[0208] <Test Example 6>

[0209] This test case uses a 3D skin model to detect the soothing and anti-inflammatory activity of the plum blossom bud extract prepared in Example 1.

[0210] The experimental materials and equipment used in this test case are as follows:

[0211] 3D Skin Model: The 3D skin model used in this test was the Episkin large-pore skin model, purchased from Shanghai Sianfuno Biotechnology Co., Ltd.

[0212] Reagents: 3D skin model kit maintenance medium, test medium, IL-1α ELISA kit, IL-8 ELISA kit, DPBS, SLS and dexamethasone.

[0213] Experimental conditions: The incubator temperature was set to 37°C and the relative humidity was set to 95%.

[0214] Equipment: Microplate reader (Thermo), biosafety cabinet (Thermo), etc.

[0215] Plum blossom bud extract concentration: 100 μg / mL.

[0216] The experimental method for this test case is as follows:

[0217] A skin model sensitive to SLS was constructed using an in vitro reconstructed 3D epidermal model. The model was then incubated with a solution of plum blossom bud extract, and the skin culture fluid was collected for the detection of inflammatory factors IL-1α and IL-8. The results were then evaluated. The control group consisted of untreated normal skin, the sensitive skin model group consisted of 0.25% SLS (sodium lauryl sulfate), and the positive control group consisted of 10 μM dexamethasone.

[0218] The evaluation criteria for this test case are as follows:

[0219] If the relative expression levels of IL-1α and IL-8 in 3D skin treated with the test sample are lower than those in the negative control, it indicates that the test sample has a soothing effect in the SLS-sensitive model. If the relative expression levels of IL-1α and IL-8 in 3D skin treated with the test sample are higher than or equal to those in the negative control, it indicates that the test sample does not have a soothing effect in the SLS-sensitive model.

[0220] The results are as follows Figure 1 As shown, the expression of IL-1α and IL-8 in 3D skin treated with the plum blossom bud extract obtained in Example 1 was downregulated, indicating that the plum blossom bud extract has a soothing and anti-inflammatory effect.

[0221] <Test Example 7>

[0222] This test case uses a 3D skin model to detect the anti-aging activity of the plum blossom bud extract prepared in Example 1.

[0223] 1. Experimental Materials

[0224] 1.1 3D Skin Model

[0225] The 3D skin model used in this test case is the T-skin full-thickness skin model, purchased from Shanghai Sianfuno Biotechnology Co., Ltd.

[0226] 1.2 Reagents

[0227] The 3D skin model kit includes maintenance culture medium, test culture medium, isopropanol, PBS, Triton X-100, sodium citrate, citric acid, xylene, DPBS, SLS, anhydrous ethanol, 4% paraformaldehyde, paraffin, BSA, antibodies (primary / secondary antibodies; the type and quantity of immunofluorescence hybridization proteins can be selected according to sample requirements, which will extend the lead time accordingly), DAPI staining solution, SYBR, Trizol, and primers (the type and quantity of genes to be detected can be selected according to sample requirements, which will extend the lead time accordingly).

[0228] 1.3 Experimental Conditions

[0229] Incubator temperature: 37°C

[0230] Relative humidity: 95%.

[0231] 1.4 Equipment

[0232] Microplate reader (Thermo), paraffin microtome (Leica), biosafety cabinet (Thermo), upright fluorescence microscope (ZESS), real-time quantitative PCR instrument (Roche), etc.

[0233] 2. Sample information: Plum blossom bud extract obtained in Example 1 at a concentration of 100 μg / mL.

[0234] 3. Experimental Methods

[0235] Using an in vitro reconstructed normal 3D whole-skin model, an aging 3D skin model was constructed through UVA irradiation. Plum blossom bud extract was added for incubation, and skin samples were collected for HE staining / immunofluorescence staining. Skin RNA was extracted for gene detection, and the overall experimental results were evaluated. The normal group without any treatment served as the Control group, the group irradiated only with UVA served as the aging model group, and the group supplemented with 110 μg / mL VC and 10 μg / mL VE and irradiated with UVA served as the positive control group.

[0236] 4. Evaluation Criteria

[0237] The intensity of immunofluorescence of related proteins; the higher the intensity, the better the anti-aging effect.

[0238] The higher the expression of the relevant collagen genes, the more collagen is synthesized, and the better the anti-aging effect.

[0239] The thicker the dermis, the better the anti-aging effect.

[0240] Anti-aging activity results as follows Figure 2 and Figure 3 As shown, Figure 2 The top row shows skin HE staining images of the obtained plum blossom bud extract for 3D skin anti-aging activity testing. From the top row of images, it can be seen that compared with the Control group, the dermis of the UVA-induced aging model group is significantly thinner, while the plum blossom bud extract obtained in Example 1 significantly thickens the dermis. Figure 2 The bottom row shows the fluorescence staining diagram of COL4 (type IV collagen) marked with green fluorescence. It can be seen from the bottom row that, compared with the Control group, the green fluorescence (i.e., COL4) in the dermis of the UVA-induced aging model group was significantly reduced, while the plum blossom bud extract obtained in Example 1 significantly increased the green fluorescence (i.e., COL4), indicating that it has the effect of promoting the increase of COL4. Figure 3 This indicates that the plum blossom bud extract obtained in Example 1 can enhance COL4 expression. In summary, the above results suggest that the plum blossom bud extract has anti-aging effects.

[0241] <Test Example 8>

[0242] This test example examines the antioxidant activity of the plum blossom bud extract prepared in Example 1. The specific procedure is as follows:

[0243] DPPH solution was prepared with anhydrous ethanol to a concentration of 0.1 mg / mL. Simultaneously, vitamin C (positive control) and sample (plum blossom bud extract) solutions were prepared (concentration: 1–0.00391 mg / mL, diluted downwards using a 2:1 dilution method). 150 μL of DPPH ethanol solution and 150 μL of sample solution were mixed thoroughly. A blank control group was included. The mixture was reacted at room temperature in the dark for 30 min, shaken well, and the absorbance was measured at 517 nm. The DPPH free radical scavenging rate of the sample was calculated using the following formula:

[0244]

[0245] The test groups are configured as follows:

[0246] Sample blank group: 150 μL sample solution + 150 μL DPPH alcohol solution

[0247] Sample group: 150 μL DPPH solution + 150 μL sample solvent

[0248] Control group: 150 μL sample solution + 150 μL anhydrous ethanol

[0249] Control blank group: 150 μL of anhydrous ethanol + 150 μL of sample solvent.

[0250] A scatter plot with smoothed lines and data markers was created, with the sample concentration as the X-axis and absorbance as the Y-axis. Three points (upper, middle, and lower) representing approximately 50% scavenging rates were selected to plot a straight line, and the equation of the line was derived. The IC50 value and scavenging rate of the DPPH radical scavenging of the sample were then calculated.

[0251] The results are as follows Figure 4 As shown, the plum blossom bud extract obtained in Example 1 has strong antioxidant activity and can effectively scavenge free radicals.

[0252] <Test Example 9>

[0253] Lactate dehydrogenase (LDH) is a marker of cell damage; increased LDH release indicates cell membrane damage. This test case examines the photodamage resistance of the plum blossom bud extract prepared in Example 1. The test was conducted as follows:

[0254] 1. Material Preparation

[0255] Cell line: HaCaT cells (human immortalized keratinocyte cell line), owned by the laboratory.

[0256] Reagents: LDH detection kit (commercially available, such as Roche or Sigma brand), cell culture medium (DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin), PBS buffer (pH 7.4), UVB light source (such as ultraviolet lamp, wavelength 280~315nm).

[0257] Instruments and equipment: Incubator, UV radiometer, ELISA reader, cell culture plate (96-well plate), sterile workbench.

[0258] 2. HaCaT cell culture

[0259] HaCaT cells were cultured in DMEM complete medium at 37°C and 5%... Subculture the cells in a humid environment. When the cells reach 80-90% confluence, digest them with 0.25% trypsin-EDTA and resuspend them in a single-cell suspension. Adjust the cell density to... Cells / mL, seeded in 96-well plates (100 μL per well, approximately...) Cells were pre-cultured in an incubator for 24 hours to allow them to adhere to the incubator.

[0260] 3. Grouping

[0261] Normal control group (BC): Cells were not exposed to UVB and no plum blossom bud extract was added.

[0262] Model control group (UVB): Cells were irradiated with UVB only, without the addition of plum blossom bud extract.

[0263] Sample treatment group (plum blossom bud extract): Cells were irradiated with UVB and then treated with the plum blossom bud extract obtained in Example 1 (120 μg / mL).

[0264] 4. UVB-induced photodamage

[0265] Before UVB irradiation, remove the culture medium from all wells and gently wash the cells twice with PBS to remove components such as serum that may interfere with the UVB effect. Add 100 μL of PBS to each well (to prevent drying), and then place the 96-well plate under a UVB light source. Calibrate the UVB dose using a UV radiometer at an irradiation intensity of 20 mJ / cm². Immediately after irradiation, remove the PBS and replace it with fresh complete culture medium (containing the appropriate extract or drug). Return the cells to the incubator and continue culturing for 24 hours to simulate the post-damage response.

[0266] 5. LDH release detection

[0267] After 24 hours of culture, collect 100 μL of supernatant from each well for LDH detection. Simultaneously, for the determination of total LDH (to calculate the release rate), separate cell lysis wells were prepared: 1% Triton X-100 was added to lyse cells, and after incubation for 30 minutes, the supernatant was collected. Following the LDH detection kit instructions, the supernatant was mixed with the LDH reaction mixture (containing sodium lactate, INT, coenzyme I, etc.) in a 96-well plate and incubated in the dark for 30 minutes. Stop solution was added, and the absorbance was measured at 490 nm using a microplate reader. The LDH release rate was calculated using the following formula:

[0268]

[0269] The results are as follows Figure 5 As shown in the results, 120 μg / mL of plum blossom buds can significantly inhibit the release of LDH, with an inhibition rate of 21.8%. This indicates that the extract can inhibit UVB-induced damage to HaCaT cells.

[0270] 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 the claims of this application.

Claims

1. A method for preparing a cluster extract, characterized by, Includes the following steps: S1. Mix dried plum blossom buds with water at a mass ratio of 1:30 to 1:10 to obtain the first mixture; S2. Culture Bacillus belye to obtain a seed culture of Bacillus belye; S3. Using the seed liquid as the fermentation liquid, add it to the first mixture at an inoculum amount of 3% to 8% of the volume of the first mixture for fermentation to obtain the second mixture; S4. Centrifuge and filter the second mixture, and dry the filtrate to obtain the plum blossom bud extract; Among them, the *Bacillus belyssus* strain is *Bacillus belyssus* BTN-BL13, classified and named as follows: Bacillus velezensis It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 3, 2025, with accession number GDMCC No: 67198; In step S3, the fermentation temperature is 25~40℃, the fermentation time is 16~36h, and the shaking speed is 100 rpm~220 rpm; In step S4, the second mixture is taken, centrifuged, and the supernatant is taken. The supernatant is then filtered through a ceramic membrane with a pore size of 0.1~10μm to obtain the filtrate.

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

3. The preparation method according to claim 1, characterized in that, Step S2 is as follows: The revived Bacillus berreatus BTN-BL13 strain is inoculated into LB medium and cultured at 25℃~40℃ for 18 h~48 h. After activating for 1~2 generations, the seed liquid is obtained.

4. The preparation method according to claim 3, characterized in that, The LB medium is either LB liquid medium or LB solid medium; The LB liquid culture medium includes tryptone, yeast extract, NaCl and ultrapure water, and the LB solid culture medium includes tryptone, yeast extract, NaCl, ultrapure water and agar powder.

5. A plum blossom bud extract, characterized in that, The plum blossom bud extract is prepared by the preparation method according to any one of claims 1 to 4.

6. The application of a plum blossom bud extract in skincare products, characterized in that, The plum blossom bud extract is prepared by the preparation method according to any one of claims 1 to 4.

7. A skincare product containing plum blossom bud extract, characterized in that, The plum blossom bud extract is prepared by the preparation method according to any one of claims 1 to 4.

8. The skincare product according to claim 7, characterized in that, The amount of the plum blossom bud extract added to the skin care product is 0.75-3% by weight.

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