Weissella tamariscina strain, its fermentation product filtrate and its biological products

Abstract

This invention discloses *Wesleyanus esculenta* strain, its fermentation product filtrate, and its biological products, belonging to the field of cosmetic technology. This strain can be used in cosmetics, exhibiting excellent HAS inhibition ability and significant DPPH and ABTS free radical scavenging capabilities, achieving good antioxidant effects. Secondly, at various concentrations, its fermentation product filtrate can significantly downregulate the proportion of SA-β-Gal positive cells and significantly inhibit the gene expression of various matrix metalloproteinases, demonstrating a certain effect in reducing cellular aging markers. Furthermore, its fermentation product filtrate can inhibit tyrosinase activity in vitro and significantly inhibit melanin production in a zebrafish model, showing potential as a tyrosinase inhibitor or melanin production inhibitor. Its fermentation product filtrate can also significantly inhibit the secretion of the cellular inflammatory factor NO, exhibiting good soothing effects and a certain ability to regulate the skin microecology.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology, and specifically relates to a strain of *Wesleyanus edodes*, its fermentation product filtrate, and its biological products. Background Technology

[0002] Lactic acid bacteria are a common type of beneficial bacteria for humans. Numerous in vivo and in vitro studies have demonstrated that lactic acid bacteria often possess strong antioxidant capabilities. However, *Westernella esculenta* is a relatively new member of the lactic acid bacteria family, and has been studied less extensively. Although *Westernella esculenta* is a relatively new genus of lactic acid bacteria, it has demonstrated unique advantages in many aspects since its species status was established, particularly in the food industry.

[0003] (1) Articles “Screening, Identification and Characteristic Analysis of Antioxidant Active Weissella in Sichuan Traditional Pickled Vegetables”, “Metabolic profiles of Weissella The study "Spp. postbiotics with anti-microbial and antioxidant effects" indicates that *Westernella esculenta* is a beneficial lactic acid bacterium that combines antioxidant activity with good fermentation performance.

[0004] (2) Patent CN105255787B provides a strain of Weissella tamariscina XHR1 and its application, specifically a kimchi containing Weissella tamariscina XHR1.

[0005] (3) Patent CN109402010B provides a method for rapidly fermenting stinky mandarin fish using a mixed inoculation of Lactococcus lactis M10 and Weissella esculenta M3, and discloses a strain of Lactococcus lactis ( Lactococcus lactis M10 and a strain of Weissella esculenta ( Weissella cibaria M3, and used the Lactococcus lactis M10 and Weissella esculenta M3 to inoculate and rapidly ferment stinky mandarin fish.

[0006] (4) Patent CN113930367B discloses a strain of lactic acid bacteria with cholesterol-lowering properties and its application. The strain of Weissella esculenta provided has a high cholesterol-lowering ability and a strong antioxidant capacity, and also has good resistance to adverse gastrointestinal environments.

[0007] Currently, there are relatively few publicly available beneficial strains of Weissella tamariscina, and the efficacy of these strains is relatively limited, with most applications in the field of fermented foods. Summary of the Invention

[0008] The purpose of this invention is to provide a strain of *Wesleyanus edodes*, its fermentation product filtrate, and its biological products to solve the problems mentioned in the background art.

[0009] Specifically, this invention provides a strain of *Westernella edodes* derived from the leaves of *Prickly pear* fruit from Haba Snow Mountain in Yunnan Province, and is classified as... Weissella cibaria The strain of *Westernella taurida* BTN-HB-F2 was deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 65161, deposit date of September 20, 2024, and address of Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0010] The *Westernella esculenta* strain BTN-HB-F2 has a lactobacillus morphology, and its 16S rDNA sequence is shown in SEQ ID NO. 1 below:

[0011]

[0012] Specifically, the present invention also provides a fermentation product filtrate of a strain of *Westernella taurida*, wherein the fermentation product filtrate is a fermentation product filtrate of *Westernella taurida* strain BTN-HB-F2.

[0013] Specifically, the present invention also provides a biological product comprising a fermentation product filtrate of Weissella taurida strain BTN-HB-F2.

[0014] More specifically, the biological products are mainly used in the cosmetics field. It should be noted that cosmetics include makeup, skin care products, and other chemical industrial products or fine chemical products that are applied to any part of the human body surface, such as skin, hair, nails, lips, and teeth, by means of smearing, spraying, or other similar methods, for the purpose of cleaning, maintaining, beautifying, modifying and changing appearance or maintaining a good condition.

[0015] Furthermore, the biological product has at least one of the following effects: anti-skin oxidation, reduction of cell aging markers, inhibition of tyrosinase activity or melanin production, skin microecological regulation, and skin soothing.

[0016] Furthermore, the bioproduct contains fermentation product filtrate of *Westernella taurida* strain BTN-HB-F2, and the amount of fermentation product filtrate added to the bioproduct can be 0.09% to 3% (v / v).

[0017] Specifically, the present invention also provides a method for preparing a fermentation product filtrate of a strain of *Westernella esculenta*, comprising:

[0018] S1. Inoculate the BTN-HB-F2 strain of Weissella tauridii into the culture medium to activate the strain and obtain the fermentation seed liquid.

[0019] S2. Inoculate the fermentation seed liquid into the culture medium for fermentation culture to obtain the fermentation broth;

[0020] S3. Take the fermentation broth, centrifuge to collect the supernatant, and filter to obtain the fermentation product filtrate of Weissella esculenta strain BTN-HB-F2.

[0021] Further, in steps S1 and S2, the culture medium is MRS liquid culture medium, including bacterial peptone, yeast extract, beef extract, triammonium citrate, sodium acetate, glucose, magnesium sulfate heptahydrate, dipotassium hydrogen phosphate, manganese sulfate monohydrate, Tween and water, with a pH value of 6.2-6.4.

[0022] Further, in step S2, the fermentation seed liquid is inoculated into MRS liquid medium at an inoculation rate of 1-5% (v / v) and fermented at 25-37°C and 0-200 rpm for 24-48 h.

[0023] Further, in step S2, the fermentation seed liquid is inoculated into MRS liquid medium at an inoculation rate of 3% (v / v).

[0024] Further, in step S3, the fermentation broth is taken, centrifuged at 5000-8000 rpm for 15-30 min, the supernatant is taken, and the fermentation product filtrate is obtained after filtration through a 0.22 μm sterile filter membrane.

[0025] The present invention has the following beneficial effects:

[0026] This invention isolates and purifies a strain of *Westernella esculenta* BTN-HB-F2 from the leaves of *Cypripedium sarmentosum* in Haba Snow Mountain, Yunnan Province. This strain can be used in the cosmetics field and has at least one of the following effects: anti-skin oxidation, reduction of cell aging markers, inhibition of tyrosinase activity or melanin production, regulation of skin microecology, and skin soothing.

[0027] 1. It has the effects of reducing skin cell aging markers, inhibiting tyrosinase activity or melanin production, and regulating the skin microecology. It can be used as an active ingredient in skin care products and has practical application value.

[0028] 2. The fermentation product filtrate of the BTN-HB-F2 strain of Weissella esculenta provided by the present invention has excellent HAS inhibition ability, and at the same time has significant DPPH and ABTS free radical scavenging ability, thus achieving good antioxidant effects.

[0029] 3. The fermentation product filtrate of the BTN-HB-F2 strain of Weissella esculenta provided by the present invention can significantly downregulate the proportion of SA-β-Gal positive cells at various concentrations, significantly inhibit the overexpression of genes of various matrix metalloproteinases, and has the effect of reducing cell senescence markers.

[0030] 4. The fermentation product filtrate of the BTN-HB-F2 strain of Weissella esculenta provided by the present invention can significantly inhibit the production of melanin in zebrafish, showing a good melanin inhibition effect and having certain application prospects in skin whitening.

[0031] 5. The fermentation product filtrate of the BTN-HB-F2 strain of *Westernella tamariscina* provided by this invention can effectively inhibit the growth of *Staphylococcus aureus* and *Propionibacterium acnes*, and has a good skin microecological regulation ability.

[0032] 6. The fermentation product filtrate of the BTN-HB-F2 strain of *Westernella esculenta* provided by this invention can significantly inhibit the secretion of the inflammatory factor NO in cells and has a good soothing effect.

[0033] 7. The fermentation product filtrate of the BTN-HB-F2 strain of Weissella esculenta provided by this invention contains abundant natural active ingredients and no chemical reagents are added, ensuring safety and efficacy. Moreover, the strain culture and filtrate preparation methods are simple, with high batch stability, and have great potential for large-scale industrial production. Attached Figure Description

[0034] Figure 1 The results of relative cell viability testing in Example 5 of this invention;

[0035] Figure 2 The results of gene expression detection of multiple matrix metalloproteinases in Experiment Example 6 of this invention;

[0036] Figure 3 The results of tyrosinase inhibition rate detection in Experiment Example 7 of this invention;

[0037] Figure 4 The results of skin microecological regulation detection in Experiment Example 8 of this invention;

[0038] Figure 5 This is a diagram showing the results of the D-Gal-induced anti-aging SA-β-Gal staining experiment in HFF cells in Experiment Example 9 of this invention. (The diagram shows the results of the experiment.) Figure 5 In the figure, 'a' represents microscopic observations of HFF cells from different experimental groups after D-Gal induction. Figure 5 b in the figure represents the detection results of the proportion of SA-β-Gal positive cells in different experimental groups;

[0039] Figure 6 This is a graph showing the results of evaluating the melanin production inhibition efficacy of zebrafish in Experiment 10 of this invention. Figure 6 In the figure, 'a' represents the zebrafish observed under a stereomicroscope in each experimental group. Figure 6 In the figure, b represents the relative melanin content of zebrafish in each experimental group.

[0040] Figure 7 This is the result of the detection of the relative content of inflammatory factor NO in Experiment Example 11 of the present invention. Detailed Implementation

[0041] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] Unless otherwise defined or stated, all technical and scientific terms used herein have the same meaning as are familiar to a user skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention.

[0043] Unless otherwise expressly specified and limited, the term "or" as used in this invention includes the relationship of "and". "And" is equivalent to the Boolean logic operator "AND", and "or" is equivalent to the Boolean logic operator "OR", with "AND" being a subset of "OR".

[0044] In this invention, the terms "mainly composed of" and "composed of" are included in the terms "containing", "comprising" or "including".

[0045] <Culture Media Selection>

[0046] MRS liquid culture medium: 10.0 g bacterial peptone, 5.0 g yeast extract, 10.0 g beef extract, 2.0 g triammonium citrate, 5.0 g sodium acetate, 20.0 g glucose, 0.5 g magnesium sulfate heptahydrate, 2.0 g dipotassium hydrogen phosphate, 0.2 g manganese sulfate monohydrate, 1.0 mL Tween-80 and 1 L ultrapure water, with a pH of 6.2-6.4;

[0047] MRS solid medium: Add 2% agar powder to MRS liquid medium.

[0048] The culture medium used in the experiment was sterilized at 121°C for 15 min before use.

[0049] <Example 1> Isolation, purification and identification of bacterial strains

[0050] (1) Collect leaves of *Cypripedium sarmentosum* from Haba Snow Mountain in Yunnan, place them in sterile bags, and bring them back to the laboratory for strain isolation;

[0051] (2) Rinse the collected leaves of the prickly pear fruit with running water, soak the leaves in sterile water, shake continuously at 180 rpm for 1.5 h, and plate the suspension obtained from each plant part onto MRS solid medium.

[0052] (3) Incubate at 37°C for 24 h, pick out single colonies with different colony morphological characteristics from MRS solid medium, and streak them on the surface of MRS solid medium for purification.

[0053] (4) Observe the single colonies on different plates after purification under a microscope, select different strains with lactobacillus morphology for identification, screen out Weissella taurida BTN-HB-F2 strain, and preserve the selected strain.

[0054] <Example 2> Preparation of fermentation product filtrate of *Westernella esculenta* strain BTN-HB-F2

[0055] (1) The preserved Weissella esculenta BTN-HB-F2 strain was inoculated onto MRS agar plates and cultured at 37°C for 24 h to complete the strain recovery;

[0056] (2) Select a single colony from the revived strain and inoculate it into MRS liquid medium for activation. Incubate at 37°C for 24 h to obtain fermentation seed liquid;

[0057] (3) The fermentation seed liquid was inoculated into MRS liquid medium at an inoculation rate of 1% (v / v) for large-scale fermentation. The fermentation temperature was 25℃ and the fermentation time was 24 h. At the same time, the shaking speed was set to 0 rpm to obtain the fermentation broth.

[0058] (4) After centrifuging the fermentation broth at 5500 rpm for 15 min, the supernatant was obtained; after filtering the supernatant through a 0.22 μm sterile filter membrane, the fermentation product filtrate of BTN-HB-F2 of Weissella esculenta was obtained.

[0059] <Example 3> Preparation of fermentation product filtrate of *Westernella esculenta* strain BTN-HB-F2

[0060] In this embodiment, fermentation step (3) differs from that in Example 2. Specifically, the fermentation seed liquid is inoculated into MRS liquid culture medium at 2% (v / v) for large-scale fermentation. The fermentation temperature is 30°C, the fermentation time is 48 h, and the shaking speed is set to 200 rpm. The remaining conditions are the same as in Example 2.

[0061] <Example 4> Preparation of fermentation product filtrate of *Westernella esculenta* strain BTN-HB-F2

[0062] In this embodiment, fermentation step (3) differs from that in Example 2. Specifically, the fermentation seed liquid is inoculated into MRS liquid culture medium at 3% (v / v) for large-scale fermentation. The fermentation temperature is 37°C, the fermentation time is 48 h, and the shaking speed is set to 200 rpm. The remaining conditions are the same as in Example 2.

[0063] <Comparative Example 1> Preparation of fermentation product filtrate of lactic acid bacteria strain CICC6055

[0064] In this embodiment, the lactic acid bacteria strain CICC6055 was used instead of the *Westernella esculenta* BTN-HB-F2 strain in Example 4. The lactic acid bacteria strain CICC6055 was purchased commercially. All other conditions were the same as in Example 4.

[0065] <Comparative Example 2> Preparation of fermentation product filtrate of *Westernella esculenta* strain D19

[0066] In this embodiment, the *Westernophora sinensis* strain D19 was used instead of the *Westernophora sinensis* strain BTN-HB-F2 in Example 4; the other conditions were the same as in Example 4.

[0067] <Comparative Example 3> Preparation of fermentation product filtrate of Bacillus strain D12

[0068] In this embodiment, Bacillus strain D12 was used instead of the Weissella esculenta strain BTN-HB-F2 in Example 4; the other conditions were the same as in Example 4.

[0069] <Comparative Example 4> Preparation of fermentation product filtrate of Bacillus strain D17

[0070] In this embodiment, Bacillus strain D17 was used instead of Weissella esculenta strain BTN-HB-F2 in Example 4; the other conditions were the same as in Example 4.

[0071] <Experimental Example 1> Determination of bacterial biomass

[0072] 200 μL of the fermentation product filtrate from Examples 2-4 was placed in a 96-well plate, and the absorbance was measured at a wavelength of 600 nm. The results are shown in Table 1.

[0073] <Experimental Example 2> DPPH Free Radical Scavenging Ability Test

[0074] Referring to the "Cosmetic - Experimental Method for Free Radical (DPPH) Scavenging (T / SHRH006-2018)", the DPPH free radical scavenging ability of the fermentation product filtrates prepared in Examples 2-4 and Comparative Examples 1-4 was tested respectively.

[0075] (1) Preparation of the drug: Prepare a 0.1 mg / mL DPPH solution with anhydrous ethanol and store it in the dark.

[0076] (2) Experimental procedure: Set up a sample group, a sample blank control group, a control group, and a control blank control group, with 3 replicates in each group; In the sample group, take 150 μL of the fermentation product filtrate of Examples 2-4 and Comparative Examples 1-4 into a 96-well plate as the test sample, and then add the test sample and DPPH solution in an equal volume of 1:1, shake well, react at room temperature in the dark for 30 min, and measure the absorbance at a wavelength of 517 nm; In the sample blank control group, replace the DPPH solution with an equal volume of anhydrous ethanol, and other conditions are the same as the sample group; In the control group, replace the fermentation product filtrate with an equal volume of ultrapure water, and other conditions are the same as the sample group; In the control blank control group, replace the fermentation product filtrate with an equal volume of ultrapure water, replace the DPPH solution with anhydrous ethanol, and other conditions are the same as the sample group.

[0077] (3) Calculation formula: DPPH free radical scavenging rate = [1 - (Asample - A sampleblank ) / (A control -A controlblank )] x 100%.

[0078] In the formula: A sample Indicates the absorbance of the sample group; A sampleblank A represents the absorbance of the sample blank control group; control Indicates the absorbance of the control group; A controlblank This indicates the absorbance of the control group (blank).

[0079] The results of the analysis of the fermentation product filtrates of Examples 2-4 and Comparative Examples 1-4 are shown in Table 1.

[0080] <Experimental Example 3> ABTS Free Radical Scavenging Ability Test

[0081] (1) Preparation of drugs: Accurately weigh 28.4 mg of ABTS and dissolve it in 10 mL of ultrapure water. Weigh 6.7 mg of potassium persulfate and dissolve it in 10 mL of ultrapure water. Mix the ABTS solution and potassium persulfate solution at a volume ratio of 1:1. After standing overnight at room temperature in the dark, the ABTS stock solution is obtained. Dilute the ABTS stock solution so that its absorbance at a wavelength of 734 nm is 0.7±0.1 to obtain the ABTS working solution. The ABTS working solution should be prepared fresh for use.

[0082] (2) Experimental procedure: Set up a sample group and a control group. In the sample group, the fermentation product filtrate of Examples 2-4 and Comparative Examples 1-4 was used as the test sample. 180 μL of ABTS working solution and 20 μL of the test sample were added to a 96-well plate. After reacting for 180 min, the absorbance of the reaction system at a wavelength of 734 nm was measured. In the control group, ultrapure water was used instead of fermentation product filtrate, and other conditions were the same as those in the sample group.

[0083] (3) Calculation formula: ABTS free radical scavenging rate = [1 - (A sample / A control ) ] x 100%.

[0084] In the formula: A sample Indicates the absorbance of the sample group; A control This indicates the absorbance of the control group.

[0085] The results of the analysis of the fermentation product filtrates of Examples 2-4 and Comparative Examples 1-4 are shown in Table 1.

[0086] <Experimental Example 4> Hyaluronidase (HAS) Inhibition Capacity Test

[0087] (1) Preparation of reagents: Dissolve and prepare 0.25 mg / mL hyaluronidase solution and 1 mg / mL sodium hyaluronate solution in 0.1 mM acetate buffer; prepare 12.5 mM calcium chloride solution and 0.4 M sodium hydroxide solution in ultrapure water; take 6.1 g potassium borate, add water to 50 g, prepare 4 M potassium borate solution, and let stand overnight before use; take 0.8 g p-dimethylaminobenzaldehyde, add 20 mL acetic acid, and then add 5 mL concentrated hydrochloric acid (store away from light) as color reagent.

[0088] (2) Set up groups:

[0089] Sample group (Group A): The samples to be tested are the fermentation product filtrates prepared in Examples 2-4 and Comparative Examples 1-4;

[0090] The blank sample group (Group B) consists of the fermentation product filtrate prepared in Examples 2-4 and Comparative Examples 1-4.

[0091] The control group (Group C) and the blank control group (Group D) both used ultrapure water as the sample solvent.

[0092] (3) Experimental steps:

[0093] According to the grouping requirements, add 0.5 mL of the corresponding test sample to groups A, B, C and D respectively; then add 0.5 mL of hyaluronidase solution to groups A and C, and add 0.5 mL of acetate buffer to groups B and D, and incubate at 37℃ for 20 min.

[0094] Each group was added with 100 μL of calcium chloride and incubated at 37°C for 20 min.

[0095] Group A and Group C were each added with 0.5 mL of sodium hyaluronate, while Group B and Group D were each added with 0.5 mL of acetate buffer. The mixtures were incubated at 37°C for 40 min.

[0096] Each group was incubated with 100 μL sodium hydroxide and 100 μL potassium borate, respectively, in a water bath at 85°C for 5 min, followed by an ice bath for 2 min, and then placed at room temperature for 5 min.

[0097] After the reaction solution is mixed evenly, 100 μL of each group of reaction solution is added to a 96-well plate, and 100 μL of colorimetric solution is added to each well.

[0098] Place the 96-well plate quickly into the microplate reader and shake at 37°C for 1 minute; measure the absorbance at 585 nm wavelength after 10 minutes.

[0099] (4) The formula for calculating the hyaluronidase inhibition rate is: HAS inhibition rate = [(CD)-(AB)] / (CD) ×100%.

[0100] In the formula: A, B, C, and D represent the absorbance of groups A, B, C, and D measured at a wavelength of 585 nm, respectively.

[0101] The HAS inhibition rate test results of the fermentation product filtrates of Examples 2-4 and Comparative Examples 1-4 are shown in Table 1.

[0102] <Experimental Example 5> Cell Proliferation Efficacy Test

[0103] (1) Selection of cells and culture medium: Human foreskin fibroblasts (HFF cells) were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences; the culture medium was DMEM complete medium (containing 10% FBS fetal bovine serum).

[0104] (2) Experimental steps: Selected HFF cells with good morphology and in the logarithmic growth phase were seeded into 96-well plates and incubated in an incubator for 24 h; a sample group, a control group, and a zeroing group were set up. In the sample group, different concentrations of the fermentation product filtrate of Example 4 were taken as test samples and added to the HFF cell well plates. The test samples were diluted with DMEM complete medium, including six concentrations of 0.09%, 0.1875%, 0.38%, 0.75%, 1.5%, and 3%, with three replicates for each concentration gradient. They were incubated in an incubator for 24 h. After removing the supernatant of DMEM complete medium, MTT test solution was added to each well and incubated in an incubator for another 2 h. The absorbance at 570 nm wavelength was measured using a microplate reader. In the control group, the test samples in the sample group were replaced with complete medium, and the other conditions were the same as those in the sample group. In the zeroing group, no cells were seeded, only complete medium was added, and the other conditions were the same as those in the sample group. Finally, the relative cell viability was calculated.

[0105] (3) The formula for calculating relative cell viability is: Relative cell viability = (A sample - A blank ) / (A control -A blank ) × 100%.

[0106] In the formula: A sample Indicates the absorbance of the sample group; A control : Control group absorbance; A blank : Absorbance of the zeroing group.

[0107] The results of the relative cell viability test for each group are as follows: Figure 1 As shown, *** indicates a significant difference between the sample group and the control group at p<0.001.

[0108] <Experimental Example 6> Inhibition rate test of matrix metalloproteinases (MMP1, MMP3 and MMP9)

[0109] (1) Selection of cells and culture medium: Human foreskin fibroblasts (HFF cells) were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences; the culture medium was DMEM complete medium (containing 10% FBS fetal bovine serum).

[0110] (2) Experimental procedure: Select HFF cells in the logarithmic growth phase with good morphology and seed them in 6-well plates at 2.5 × 10⁻⁶ m². 5 Cells / wells were incubated in an incubator for 24 h. Sample, control, and blank groups were set up, with 3 replicate wells in each group. In the sample group, DMEM complete medium containing 3% fermentation product filtrate of Example 4 was added. After treating the cells for 4 h, 400 μM H2O2 solution was added for induction treatment for 2 h. DMEM complete medium containing 3% fermentation product filtrate of Example 4 was added again, and the cells were incubated in a 37 ℃, 5% CO2 incubator for another 24 h. No fermentation product filtrate was added to the control group, and the other conditions were the same as the sample group. No fermentation product filtrate was added to the blank group, and H2O2 solution was not used for induction. The other conditions were the same as the sample group.

[0111] (3) Gene expression level test: RNA was extracted from cells in the sample group, control group, and blank group, and the RNA content was measured. Qualified RNA was reverse transcribed into cDNA, and gene expression level was measured by real-time PCR. The calculation method adopted was 2... -ΔΔCt The internal reference gene is β-actin.

[0112] (4) Data processing: Experimental data were processed using GraphPad and statistically analyzed using One-way ANDNA (and nonparametric) Turkey: Compare all pairs of cells. Each experiment was repeated at least three times. Data are expressed as Mean ± SEM. The mRNA levels of MMP1, MMP3, and MMP9 in HFF cells increased under H2O2 induction, but after intervention with the fermentation product filtrate obtained in Example 4, the expression levels of the above matrix metalloproteinases decreased, and senescence was alleviated. The experimental results are as follows: Figure 2 As shown, ### indicates a significant difference between the control group and the blank at p<0.001, and *** indicates a significant difference between the sample group and the control group at p<0.001.

[0113] <Experimental Example 7> Tyrosinase Inhibition Test

[0114] (1) Preparation of drugs: Add 45.5 mL of 0.1 M citric acid monohydrate to 154.5 mL of 0.2 M Na2HPO4·12H2O to prepare a pH 6.8 disodium hydrogen phosphate-citric acid buffer solution, and use the buffer solution to prepare a 100 u / mL tyrosinase solution and a 1 mg / mL levodopa solution.

[0115] (2) Set up groups:

[0116] Sample group (Group A): The samples to be tested are the fermentation product filtrates prepared in Examples 2-4 and Comparative Examples 1-3;

[0117] The blank sample group (Group B) consisted of the fermentation product filtrates prepared in Examples 2-4 and Comparative Examples 1-3, and kojic acid at concentrations of 0.008 mg / mL, 0.04 mg / mL, 0.2 mg / mL, and 1 mg / mL.

[0118] The control group (Group C) used disodium hydrogen phosphate-citric acid buffer as the test sample.

[0119] The control group (Group D) and the test sample were disodium hydrogen phosphate-citric acid buffer.

[0120] (3) Experimental steps:

[0121] Add 1 mL of the sample to be tested to groups A, B, C, and D respectively; then add 0.5 mL of tyrosinase solution to groups A and C, and 0.5 mL of disodium hydrogen phosphate-citrate buffer to groups B and D, and incubate in a water bath at 37°C for 10 min.

[0122] Add 2 mL of levodopa solution to each group and react for 5 min;

[0123] Pipette 200 μL of each reaction solution into a 96-well plate and quickly measure the absorbance at 475 nm using an ELISA reader.

[0124] (4) Formula for calculating tyrosinase inhibition rate: Tyrosinase inhibition rate (%) = [(CD)-(AB)] / (CD) ×100%.

[0125] In the formula: A, B, C, and D represent the absorbance of groups A, B, C, and D measured at a wavelength of 475 nm, respectively.

[0126] The results of the tyrosinase inhibition rate test of the fermentation product filtrates of Examples 2-4 and Comparative Examples 1-4 are shown in Table 1. No tyrosinase inhibition ability is indicated by "ns".

[0127] In addition, kojic acid at concentrations of 0.008 mg / mL, 0.04 mg / mL, 0.2 mg / mL, and 1 mg / mL were used as test samples, and the tests were performed according to the above steps. The test results were compared with those in Example 4. Figure 3 As shown.

[0128] Table 1

[0129]

[0130] <Experimental Example 8> Skin Microecological Regulation Capacity Test

[0131] The effects of the samples on the inhibitory ability of Staphylococcus aureus and Propionibacterium acnes (ATCC 6919 and ATCC 11827) on the skin were tested respectively.

[0132] (1) Solution preparation:

[0133] Staphylococcus aureus and Propionibacterium acnes (ATCC 6919 and ATCC 11827) were cultured on BHI solid medium. After culturing at 37°C for 24 h, the bacterial cells were resuspended in BHI liquid medium and the turbidity was adjusted to 0.5 to obtain the test solution for the model strains.

[0134] (2) Set up groups:

[0135] Sample group (Group A): The sample to be tested was the fermentation product filtrate prepared in Example 4. 1 mL of the model strain test solution and 1 mL of the sample to be tested were added to a 96-well plate, respectively. After incubation at 37℃ for 24 h, 200 μL of the mixture was taken into a 96-well plate and the absorbance at 600 nm was measured.

[0136] The blank sample group (Group B) was the fermentation product filtrate prepared in Example 4. 1 mL of the model strain test solution and 1 mL of the test sample were added to a 96-well plate, and 200 μL was immediately taken into the 96-well plate to measure the absorbance at 600 nm.

[0137] In the control group (Group C), the test sample was sterile PBS solution. 1 mL of model strain test solution and 1 mL of sterile PBS were added to a 96-well plate, respectively. After incubation at 37℃ for 24 h, 200 μL of the mixture was taken into a 96-well plate and the absorbance at 600 nm was measured.

[0138] In the control group (Group D), the test sample was a sterile PBS solution. 1 mL of the model strain test solution and 1 mL of sterile PBS were added to a 96-well plate, and 200 μL of the mixture was immediately added to the 96-well plate to measure the absorbance at a wavelength of 600 nm.

[0139] (3) The formula for calculating the microbial inhibition rate is: Microbial inhibition rate (%) = [(CD)-(AB)] / (CD) ×100%.

[0140] In the formula: A, B, C, and D are the absorbance of groups A, B, C, and D measured at a wavelength of 600 nm, respectively.

[0141] In the ATCC 6919 test method for Propionibacterium acnes, the culture time before measuring A1 and B1 is 3 days, and the other conditions are the same as those for the Staphylococcus aureus test method; in the ATCC 11827 test method for Propionibacterium acnes, the culture time before measuring A1 and B1 is 7 days, and the other conditions are the same as those for the Staphylococcus aureus test method.

[0142] The microbial inhibition rate test results of the fermentation product filtrate prepared in Example 4 against Staphylococcus aureus and Propionibacterium acnes (ATCC 6919 and ATCC 11827) are as follows: Figure 4 As shown.

[0143] <Experimental Example 9> SA-β-Gal Cell Senescence Detection

[0144] (1) Cell seeding: Selected HFF cells with good morphology and in the logarithmic growth phase were seeded in 24-well plates at a density of 4 × 10⁶ cells / well. 4 Cells / well were incubated in an incubator for 24 h.

[0145] (2) Experimental grouping and administration: A blank group, an induction control group and a sample group were set up. The test samples of the sample group were complete culture medium containing 1% and 3% of the fermentation product filtrate in Example 4. Each group was set up with 3 replicate wells. D-galactose (D-Gal) at a concentration of 20 mg / ml was added to the sample group and senescence induction treatment was carried out in an incubator at 37 ℃ and 5 % CO2 for 48 h. The induction control group used complete culture medium instead of fermentation product filtrate, and the other conditions were the same as the sample group. The blank group did not undergo senescence induction treatment, and the other conditions were the same as the induction control group.

[0146] (3) SA-β-Gal staining: Cell senescence was detected according to the instructions of the cell senescence β-galactosidase staining kit; the staining results were observed and photographed under a regular optical microscope, and the images were analyzed and the number of positive cells and the total number of cells were counted using ImageJ software. The proportion of positive cells to the total number of cells was calculated using the formula: proportion of positive cells = number of positive cells / total number of cells × 100%.

[0147] (4) Data processing: Experimental data were processed using GraphPad and analyzed using a two-tailed Student's-st-test. Each group had at least three replicates. All data were expressed as mean ± SEM. ### represents a significant difference between the induction control group and the blank group at p<0.001. *** and ** represent significant differences between the sample group and the control group at p<0.001 and p<0.01, respectively.

[0148] In this experimental case, the results of the D-Gal-induced anti-aging SA-β-Gal staining experiment in HFF cells are as follows: Figure 5 As shown in the figure. The microscopic observation results of each group of HFF cells are as follows. Figure 5 As shown in a, the results of the SA-β-Gal positive cell proportion detection in each group are as follows: Figure 5 As shown in b, the proportion of SA-β-Gal positive cells in the fermentation product filtrate of Example 4 with 1% and 3% concentrations decreased significantly, indicating that the fermentation product filtrate has significant anti-aging effects.

[0149] <Experiment 10> Evaluation of melanin production inhibition efficacy in zebrafish

[0150] Zebrafish selection: Select wild-type zebrafish of the AB lineage.

[0151] Embryo Collection: Zebrafish were kept according to "The Zebrafish Book", maintaining a water temperature of approximately 28.5 ℃, with 14 hours of light and 10 hours of darkness per day, and fed twice a day, morning and evening. The night before collecting the embryos, one female and two male fish were placed in the spawning tank and separated by a partition. The embryos were collected the following morning after spawning. The embryos were placed in egg water and cultured in a 28.5 ℃ light incubator.

[0152] (3) Set up groups:

[0153] (a) Blank group: The sample to be tested is standard dilution water;

[0154] (b) Positive control group: The test sample was 0.2 mM propylthiouracil (PTU);

[0155] (c) Sample group: The sample to be tested is the fermentation product filtrate prepared in Example 4 with a concentration of 0.05%.

[0156] (4) Test sample treatment: According to the experimental requirements, a sufficient number of 6 hpf zebrafish embryos with consistent development were pre-selected. The zebrafish embryos were randomly assigned to six-well plates, 15 per well. The standard dilution water in the six-well plate was removed without damaging the embryos. Then, 3 ml of the corresponding test sample was quickly added. After thorough mixing, the culture plate was covered and wrapped with aluminum foil. The plate was incubated in a biochemical incubator at 28°C in the dark until the endpoint (51 hpf).

[0157] (5) Observation and photography: After incubation, zebrafish were randomly selected from each group of zebrafish with normal phenotype and behavior, fixed with 3% methylcellulose, observed and photographed under a stereomicroscope. All zebrafish photography results were completed under the same instrument and environmental conditions, and the zebrafish body positions were kept consistent.

[0158] (6) Detection results: The results of the zebrafish melanin production inhibition efficacy evaluation in this experimental example are as follows: Figure 6 As shown in the figure. The results of zebrafish observation under a stereomicroscope in the blank group, positive control group, and sample group are as follows. Figure 6 As shown in a; the relative melanin content of zebrafish in the blank group, positive control group, and sample group is as follows. Figure 6 As shown in b, ** indicates a significant difference between the sample group and the blank control group at p < 0.01; *** indicates a significant difference between the positive control group and the blank control group at p < 0.001.

[0159] <Experimental Example 11> Evaluation of soothing effects using RAW264.7 cells

[0160] (1) Cells and culture conditions: Mouse macrophages RAW264.7 were selected and DMEM medium containing 10% FBS fetal bovine serum was used as the cell culture medium.

[0161] (2) ELISA method for detecting the inhibitory effect of test substances on TNF-α and IL-6:

[0162] RAW264.7 cells in logarithmic growth phase and with good morphology were seeded into 24-well plates and incubated for 24 h. A blank control group, an LPS-induced stimulation group, a positive control group, and a sample group were set up. The sample group was supplemented with 1% (v / v) of the fermentation product filtrate prepared in Example 4. The positive control group was supplemented with dexamethasone (dex). The blank control group and the LPS-induced stimulation group were both supplemented with DMEM medium. All cell groups were incubated at 37°C in a 5% CO2 incubator for 2 h. Then, except for the blank control group, LPS was added to each well, and the cells were incubated for 24 h. Cell supernatants were collected from each group, and the NO content in the collected cell supernatants was detected according to the instructions of the nitric oxide assay kit.

[0163] (3) Data Processing: Experimental data were analyzed using GraphPad Prism software. One-way ANOVA was used to compare the LPS-induced stimulation group with each other group, with at least three replicates for each group. Data are expressed as mean ± SEM. # indicates the ratio of the blank control group to the LPS-induced stimulation group; * indicates the ratio of other groups to the LPS-induced stimulation group; * or # indicates p < 0.05; ** or ## indicates p < 0.01; *** or ### indicates p < 0.001; ns indicates no statistical significance. The experimental results are as follows: Figure 7 As shown.

[0164] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A strain of *Westernella esculenta*, characterized in that, The strain of *Westernophora esculenta* is *Westernophora esculenta* (…). Weissella cibaria The BTN-HB-F2 strain was deposited at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC No: 65161, and the deposit date was September 20, 2024.

2. A fermentation product filtrate of a strain of *Westernella esculenta*, characterized in that, The fermentation product filtrate is the fermentation product filtrate of the *Westernella esculenta* strain BTN-HB-F2 as described in claim 1; the fermentation product filtrate is prepared in the following manner: S1. The *Westernella tauridica* strain BTN-HB-F2 as described in claim 1 is inoculated into a culture medium to activate the strain and obtain a fermentation seed liquid. S2. Inoculate the fermentation seed liquid into the culture medium for fermentation culture to obtain fermentation broth; S3. Take the fermentation broth, centrifuge to collect the supernatant, filter to obtain the fermentation product filtrate of Weissella esculenta strain BTN-HB-F2. In steps S1 and S2, the culture medium is MRS liquid culture medium, which includes bacterial peptone, yeast extract, beef extract, triammonium citrate, sodium acetate, glucose, magnesium sulfate heptahydrate, dipotassium hydrogen phosphate, manganese sulfate monohydrate, Tween and water, with a pH of 6.2-6.

4. In step S2, the fermentation seed liquid is inoculated into MRS liquid culture medium at an inoculation rate of 1-5%, and fermented at 25-37℃ and 0-200rpm for 24-48 h.

3. A biological product, characterized in that, Fermentation product filtrate containing the *Westernella esculenta* strain as described in claim 2.

4. A biological product according to claim 3, characterized in that, It has at least one of the following effects: anti-skin oxidation, anti-skin aging, skin whitening, skin microecological regulation, and skin soothing.

5. The application of the biological product according to claim 3 in the field of cosmetics.

6. A method for preparing the fermentation product filtrate of a strain of *Westernella esculenta*, characterized in that, Includes the following steps: S1. The *Westernella tauridica* strain BTN-HB-F2 as described in claim 1 is inoculated into a culture medium to activate the strain and obtain a fermentation seed liquid. S2. Inoculate the fermentation seed liquid into the culture medium for fermentation culture to obtain fermentation broth; S3. Take the fermentation broth, centrifuge to collect the supernatant, filter and obtain the fermentation product filtrate of Weissella taurida BTN-HB-F2 strain. In steps S1 and S2, the culture medium is MRS liquid culture medium, which includes bacterial peptone, yeast extract, beef extract, triammonium citrate, sodium acetate, glucose, magnesium sulfate heptahydrate, dipotassium hydrogen phosphate, manganese sulfate monohydrate, Tween and water, with a pH of 6.2-6.

4. In step S2, the fermentation seed liquid is inoculated into MRS liquid culture medium at an inoculation rate of 1-5%, and fermented at 25-37℃ and 0-200rpm for 24-48 h.

7. A method for preparing the fermentation product filtrate of a strain of *Westernella esculenta* according to claim 6, characterized in that, In step S2, the fermentation seed liquid is inoculated into MRS liquid medium at an inoculation rate of 3% and cultured at 37°C and 200 rpm for 48 h.

8. A method for preparing the fermentation product filtrate of a strain of *Westernella esculenta* according to claim 6, characterized in that, In step S3, the fermentation broth is taken and centrifuged at 5000-8000 rpm for 15-30 min. The supernatant is then filtered through a 0.22 μm sterile filter membrane to obtain the fermentation product filtrate.

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