Bifidobacterium adolescentis strain ms-ylohas18, bacterial agent thereof, and application of the same in preparing products for preventing or improving skin photoaging

Abstract

The present application relates to the technical field of microorganisms, and particularly relates to a bifidobacterium adolescentis MS-YLOHAS18, a bacterial agent thereof and application of the bacterial agent in preparation of a product for preventing or improving skin photoaging. The bifidobacterium adolescentis (Bifidobacterium adolescentis) MS-YLOHAS18 is preserved in the China General Microbiological Culture Collection Center, has a preservation number of CGMCC No.39539, a preservation date of June 30, 2026, and an address of the preservation organization of No.3, Beichen West Road, Chaoyang District, Beijing. Bifidobacterium adolescentis The present application provides a bacterial agent containing the bifidobacterium adolescentis MS-YLOHAS18. The present application also provides application of the bifidobacterium adolescentis MS-YLOHAS18 in preparation of a product for preventing or improving skin photoaging. The live bacterial preparation, the inactivated bacterial preparation and the lysate of the strain all have good anti-aging effects.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Bifidobacterium adolescentis MS-YLOHAS18 and its bacterial agent, and its application in the preparation of products for preventing or improving skin photoaging. Background Technology

[0002] Skin aging is a complex process resulting from the combined effects of genetic factors, metabolic changes, and the external environment. Among these, photoaging caused by long-term ultraviolet (UV) radiation is the main form of exogenous skin aging. UV radiation can induce the skin to produce excessive reactive oxygen species, reduce the activity of antioxidant enzymes such as superoxide dismutase, and cause damage to lipids, proteins, and nucleic acids. At the same time, it can promote the expression of matrix metalloproteinases and accelerate collagen degradation, ultimately manifesting as increased wrinkles, decreased elasticity, dullness, and weakened barrier function.

[0003] In recent years, probiotics and their derivatives have been increasingly used in skin care. Bifida ferment lysate has certain antioxidant, skin repair, and anti-aging effects, but its active ingredients are usually released only once, making it difficult to continuously produce metabolites. Existing technologies also disclose the applications of several strains of Bifidobacterium adolescentis and their fermentation filtrate, lysate, and extracts in antioxidation, promoting fibroblast proliferation, inhibiting matrix metalloproteinase expression, and improving skin condition.

[0004] However, the skincare efficacy of microorganisms exhibits significant strain specificity. Current technologies lack systematic comparisons of live, intact, inactivated, and lysate Bifidobacterium adolescentis under the same bacterial equivalents and experimental conditions, making it difficult to determine the differences in the effects of different bacterial morphologies on skin photoaging. Furthermore, current efficacy evaluations largely remain at the in vitro cellular level or rely on single-indicator detection, failing to systematically assess anti-aging effects from multiple dimensions such as macroscopic phenotype, oxidative stress, collagen metabolism, and cellular aging. They also lack follow-up on the persistence of efficacy after discontinuation of treatment, resulting in an incomplete scientific evaluation system for probiotic anti-aging skincare products. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a strain of Bifidobacterium adolescentis MS-YLOHAS18, its bacterial agent, and its application in the preparation of products for preventing or improving skin photoaging.

[0006] The technical solution of this invention is as follows: In a first aspect, the present invention provides a strain of Bifidobacterium adolescentis MS-YLOHAS18, wherein Bifidobacterium adolescentis ( Bifidobacterium adolescentis MS-YLOHAS18 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39539, deposited on June 30, 2026. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0007] Furthermore, the 16S rDNA sequence of the Bifidobacterium adolescentis MS-YLOHAS18 is shown in SEQ ID NO.3.

[0008] Secondly, the present invention provides a bacterial agent containing Bifidobacterium adolescentis MS-YLOHAS18.

[0009] Furthermore, the viable count in the bacterial agent is ≥1.0 × 10⁻⁶. 9 CFU / g.

[0010] Furthermore, the preparation method of the bacterial agent is as follows: Stored *Bifidobacterium adolescentis* MS-YLOHAS18 is activated on MRS solid medium. The activated bacteria are inoculated into MRS liquid medium at a mass percentage of 1%, and then cultured at 37°C for 24 hours to obtain a bacterial suspension. After centrifugation, the bacterial cells are collected, washed with sterile physiological saline, and resuspended in 15% reconstituted skim milk to obtain a suspension. The concentration of the suspension is adjusted to 1.0~2.0 × 10⁻⁶. 9 CFU / mL was used to obtain a bacterial suspension; the bacterial suspension was freeze-dried to obtain bacterial powder; the bacterial powder was mixed with maltodextrin to prepare a bacterial agent containing Bifidobacterium adolescentis MS-YLOHAS18.

[0011] Thirdly, the present invention provides a bacterial agent containing a metabiotic of Bifidobacterium adolescentis MS-YLOHAS18; the metabiotic includes inactivated Bifidobacterium adolescentis MS-YLOHAS18 or lysate of Bifidobacterium adolescentis MS-YLOHAS18.

[0012] Furthermore, the preparation method of the bacterial agent is as follows: A suspension of *Bifidobacterium adolescentis* MS-YLOHAS18 is inactivated by heating in a 100°C water bath for 30 minutes; the inactivated sample is spread onto MRS solid medium and anaerobically cultured at 37°C for 48 hours. After verifying the absence of viable bacterial growth, the inactivated bacteria are collected by centrifugation, washed with sterile physiological saline, and resuspended in 15% (w / w) reconstituted skim milk, resulting in a bacterial concentration of 1.0 × 10⁻⁶. 9 The bacterial agent containing Bifidobacterium adolescentis MS-YLOHAS18 postbiotic was prepared by lyophilizing and mixing it with maltodextrin at CFU / mL.

[0013] Furthermore, the preparation method of the bacterial agent is as follows: take the suspension of Bifidobacterium adolescentis MS-YLOHAS18 and inactivate it by heating in a water bath at 100℃ for 30 minutes; resuspend the inactivated bacterial body in a buffer solution, add lysozyme at a ratio of 0.1% by mass / volume, and enzymatically hydrolyze in a water bath at 37℃ for 4 hours; circulate and homogenize the enzymatic hydrolysate 3 times to fully break the bacterial cells; centrifuge the homogenate, collect the supernatant, and filter it through a 0.22μm microporous membrane; obtain Bifidobacterium adolescentis MS-YLOHAS18 lysate, and mix the obtained lysate with a cosmetic matrix at a volume ratio of 1:9 to obtain the lysate preparation.

[0014] Fourthly, the present invention provides the application of Bifidobacterium adolescentis MS-YLOHAS18 in the preparation of products for preventing or improving skin photoaging.

[0015] Furthermore, the prevention or improvement of skin photoaging includes at least one of the following: reducing skin wrinkles, improving skin elasticity, improving skin radiance, increasing skin antioxidant enzyme activity, reducing skin oxidative damage, increasing the content of type I and / or type III collagen in the skin, inhibiting matrix metalloproteinase expression, increasing TIMP-1 expression, and reducing skin cell aging markers.

[0016] Furthermore, the application is administered topically to the skin.

[0017] The beneficial effects of this invention are as follows: (1) The present invention provides a strain of Bifidobacterium adolescentis MS-YLOHAS18. Experiments have shown that the live bacterial preparation, inactivated bacterial preparation and lysate of this strain have good anti-aging effects.

[0018] (2) The fermentation process of the Bifidobacterium adolescentis MS-YLOHAS18 strain provided by this invention is stable (viable count ≥1×10⁻⁶). 9 The preparation method (CFU / g) is simple and controllable, providing a safe, efficient, and long-lasting new solution for the development of anti-aging skincare products.

[0019] (3) This invention systematically evaluated the effects of UVB-induced photoaging in a mouse model from multiple dimensions, including macroscopic phenotypes (wrinkle area, skin elasticity, skin tone brightness), oxidative stress (SOD, GSH-Px, CAT, MDA, ROS), collagen metabolism (type I / III collagen, MMP-1 / MMP-3, TIMP-1), and cellular senescence (SA-β-gal, p16, p53), covering the main pathways of skin aging. The experimental results showed that all three forms of Bifidobacterium adolescentis MS-YLOHAS18 preparations had anti-photoaging effects, with the efficacy ranking as live bacteria > inactivated bacteria > lysate. This conclusion points the way for the development of probiotic anti-aging skin care products.

[0020] (4) The comprehensive efficacy index of the live bacteria preparation of Bifidobacterium adolescentis MS-YLOHAS18 of the present invention still reached 98% after 28 days of drug withdrawal, which is better than the lysate group (75%) and the inactivated bacteria group (88%). This proves that the live bacteria preparation has metabolic activity and colonization ability, which can achieve the long-term anti-aging effect of applying it while it is applied and continuing after drug withdrawal. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a colony morphology diagram of Bifidobacterium adolescentis MS-YLOHAS18 in Example 1 of the present invention.

[0023] Figure 2 This is a microscopic image of the cell morphology of Bifidobacterium adolescentis MS-YLOHAS18 in Example 1 of the present invention. Detailed Implementation

[0024] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0025] The components and preparation methods of the various culture media used in the embodiments of this invention are as follows: (1) Composition and preparation method of MRS solid culture medium: 10g peptone, 5g beef powder, 5g sodium acetate trihydrate, 2g dipotassium hydrogen phosphate heptahydrate, 1mL Tween 80, 0.05g manganese sulfate tetrahydrate, 2g triammonium citrate, 20g glucose, 0.2g magnesium sulfate heptahydrate, 5g CaCO3, 15g agar, 1000mL distilled water; After mixing the above raw materials, adjust the pH to 6.8, heat and mix well, and sterilize at 121℃ and 0.1MPa for 20min; Pour the sterilized culture medium into petri dishes, let it cool and use.

[0026] (2) Composition and preparation method of MRS liquid culture medium: 10g peptone, 5g beef powder, 5g sodium acetate trihydrate, 2g dipotassium hydrogen phosphate heptahydrate, 1mL Tween 80, 0.05g manganese sulfate tetrahydrate, 2g triammonium citrate, 20g glucose, 0.2g magnesium sulfate heptahydrate, 1000mL distilled water; After mixing the above raw materials, adjust the pH to 6.8, heat and mix well, and sterilize at 121℃ and 0.1MPa for 20min.

[0027] The main raw materials used in the embodiments of this invention are as follows: Maltodextrin: Purchased from Baolingbao Biotechnology Co., Ltd.

[0028] Example 1 Screening, purification and identification of Bifidobacterium adolescentis MS-YLOHAS18 1. Sampling: In August 2022, samples were taken from the intestines of infants in Yangzhou City, Jiangsu Province. After collection, the samples were transported back to the laboratory via cold chain for sample preparation.

[0029] 2. Separation, primary screening, and purification: (1) Add the above 1g sample to an Erlenmeyer flask containing sterile physiological saline, stir and shake at 4℃ for 30min, and set aside; (2) Dilute the solution from step (1) to prepare samples with different concentration gradients, each being 10... -1 10 -2 10 -3 10 -4 10 -5 10 -6 and 10 -7 ,spare; (3) Cultivation: The solutions of different dilution concentrations in step (2) were spread on solid culture medium using a spreading stick and cultured at 37°C under anaerobic conditions for 48 hours. (4) Colony selection: Select colonies according to the following colony characteristics: Colony characteristics: 1~2mm in diameter, rounded colonies with neat edges, slightly white with a raised center, and a large calcium dissolution zone.

[0030] (5) Isolation and purification: Based on the appearance and morphology, 22 single colonies were selected from the colony characteristics in step (4), and inoculated onto MRS solid medium by streak method. They were cultured at 37°C under anaerobic conditions for 48 hours. The above operation was repeated 2-3 times. Single colonies were picked and stored in glycerol tubes at -70°C as candidate strains.

[0031] 4. Strain identification The strain MS-YLOHAS18 was sent for identification by Jinan Tianyi Biotechnology Co., Ltd. The primers used in the identification process are as follows: Primer sequences: 27F: 5-AGAGTTTGATCCTGGCTCAG-3 (SEQ ID NO. 1) 1492R: 5-CTACGGCTACCTTGTTACGA-3 (SEQ ID NO.2) Perform PCR amplification and sequencing.

[0032] BLAST comparison results showed that strain MS-YLOHAS18 was similar to Bifidobacterium adolescentis (… Bifidobacterium of a young man The homology between the strain and *Bifidobacterium adolescentis* reached over 99.9%, and the identification results determined that the strain was *Bifidobacterium adolescentis*. Bifidobacterium adolescentis ).

[0033] The 16S rDNA sequence of strain MS-YLOHAS18 is shown in SEQ ID NO.3. Its full-length 16S rDNA gene sequence is as follows: Bifidobacterium adolescentis ( Bifidobacterium adolescentis MS-YLOHAS18 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39539, deposited on June 30, 2026. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0034] Example 2 Preparation of Bifidobacterium adolescentis MS-YLOHAS18 bacterial agent (1) Preparation of Bifidobacterium adolescentis MS-YLOHAS18 live bacterial preparation Stored Bifidobacterium adolescentis MS-YLOHAS18 was activated on MRS solid medium. The activated bacteria were then inoculated into MRS liquid medium at a 1% (w / w) inoculum and cultured at 37°C for 24 h to obtain a bacterial suspension. After centrifugation, the bacterial cells were collected, washed with sterile physiological saline, and resuspended in 15% (w / w) reconstituted skim milk to obtain a suspension. The concentration of the suspension was adjusted to 1.0~2.0×10⁻⁶. 9 CFU / mL was used to obtain a bacterial suspension; the bacterial suspension was freeze-dried to obtain bacterial powder; the bacterial powder was mixed with maltodextrin to prepare Bifidobacterium adolescentis MS-YLOHAS18 live bacterial powder. The live bacterial powder contained ≥1.0 × 10⁻⁶ live bacteria. 9 CFU / g.

[0035] (2) Preparation of inactivated Bifidobacterium adolescentis MS-YLOHAS18 preparation A suspension of *Bifidobacterium adolescentis* MS-YLOHAS18 was inactivated by heating in a 100°C water bath for 30 minutes. The inactivated sample was then spread onto MRS solid medium and anaerobically cultured at 37°C for 48 hours. After confirming the absence of viable bacterial growth, the inactivated cells were collected by centrifugation, washed with sterile physiological saline, and resuspended in 15% (w / w) reconstituted skim milk, resulting in a bacterial concentration of 1.0 × 10⁻⁶ cells / ml. 9 The inactivated Bifidobacterium adolescentis MS-YLOHAS18 powder was prepared by lyophilizing and mixing it with maltodextrin at a concentration of CFU / mL.

[0036] (3) Preparation of Bifidobacterium adolescentis MS-YLOHAS18 lysate Bifidobacterium adolescentis MS-YLOHAS18 was activated on MRS solid medium, and single colonies were picked and inoculated into MRS liquid medium and cultured under anaerobic conditions at 37°C until OD500. 600The culture medium was increased to 1.5–2.0 to obtain the fermentation broth. The broth was centrifuged at 8000 rpm for 10–15 min, and the bacterial precipitate was collected. The precipitate was washed 2–3 times with sterile physiological saline and then inactivated by heating in a 100°C water bath for 30 min. The inactivated bacterial cells were then resuspended in buffer solution at a concentration of 1.0 × 10⁻⁶ cells / mL. 9 CFU / mL, lysozyme was added at a ratio of 0.1% (w / v), and the mixture was hydrolyzed in a water bath at 37℃ for 4 hours with shaking. The hydrolysate was homogenized three times at 800 bar using a high-pressure homogenizer to ensure complete cell disruption. The homogenate was centrifuged at 12000 r / min for 20-30 min, and the supernatant was collected and filtered through a 0.22 μm microporous membrane to obtain Bifidobacterium adolescentis MS-YLOHAS18 lysate. The obtained lysate was mixed with a cosmetic matrix at a ratio of 1:9 (v / v) to obtain the lysate preparation.

[0037] (4) The steps for detecting the quality of lysate are as follows: 1) Cell disruption rate: Direct microscopic counting was used. Samples of lysate before and after cell disruption treatment were taken, appropriately diluted with sterile physiological saline, and 10 μL of the diluted solution was added to a hemocytometer. The cells were then observed and counted under an optical microscope (400×). Each sample was counted three times, and the average value was taken. The cell disruption rate was calculated using the following formula: Cell disruption rate (%) = (N0 - N1) / N0 × 100%, where N0 is the number of intact cells before treatment, and N1 is the number of remaining intact cells after treatment.

[0038] 2) Residual number of intact bacteria: Verification was performed using a combination of microscopic counting and plate colony counting. After appropriate dilution of the lysate sample, 10 μL was added to a hemocytometer, and the number of intact bacteria was counted under an optical microscope (400×). Simultaneously, the diluted solution was spread onto MRS solid culture plates and anaerobically incubated at 37°C for 48 h before counting the colonies. Residual number of intact bacteria (%) = (Number of intact bacteria after treatment / Total number of bacteria before treatment) × 100%.

[0039] 3) Protein content: Determined using the BCA (diquinoline carboxylic acid) method. Prepare BCA working solution (volume ratio of reagent A: reagent B = 50:1). Add 20 μL of different concentrations of BSA standards (0, 0.125, 0.25, 0.5, 1.0, 2.0 mg / mL) and appropriately diluted lysate samples to 96-well plates. Add 200 μL of BCA working solution to each well, mix thoroughly, and incubate at 37℃ for 30 min. Measure the absorbance at 562 nm using a microplate reader. Calculate the protein concentration in the sample based on the standard curve.

[0040] 4) Total sugar content: The phenol-sulfuric acid method was used to determine the total sugar content using a kit from Thermo Fisher Scientific (catalog number: ER0292). 1.0 mL of different concentrations of glucose standard solution (0, 20, 40, 60, 80, 100 μg / mL) and appropriately diluted lysate samples were placed in test tubes. 1.0 mL of 5% phenol solution and 5.0 mL of concentrated sulfuric acid were added sequentially, and the mixture was thoroughly shaken and allowed to stand at room temperature for 30 min. The absorbance was measured at 490 nm using a UV-Vis spectrophotometer, and the total sugar content in the sample was calculated based on the glucose standard curve.

[0041] (5) Nucleic acid content: determined by ultraviolet spectrophotometry. Take an appropriate amount of lysate sample, dilute it appropriately with sterile deionized water, add 1.0 mL to a quartz cuvette, and measure the absorbance value at a wavelength of 260 nm using an ultraviolet-visible spectrophotometer (with sterile deionized water as a blank control).

[0042] Nucleic acid concentration is calculated using the following formula: Nucleic acid content (mg / mL) = A 260 × dilution factor × 0.05, where A 260 The absorbance value is at 260 nm, and 0.05 is the conversion factor (1 OD). 260 (equivalent to 50g / mL nucleic acid).

[0043] The detection results of the lysates in this embodiment are shown in Table 1 below.

[0044] Table 1 - Results of Lysate Quality Testing

[0045] Example 3 Macroscopic phenotypic evaluation experiment of skin photoaging 1. Experimental grouping and modeling SPF-grade HR-1 hairless female mice were selected. Forty mice were randomly divided into five groups of eight each: a blank control group, a model control group, a live bacteria intervention group, an inactivated bacteria intervention group, and a lysate intervention group. The grouping scheme is shown in Table 2. The administration regimen was as follows: 50 μL was applied once daily for 28 consecutive days. The blank control group and the model control group were treated with the same amount of matrix.

[0046] Table 2 - Experimental Groups

[0047] 2. Macroscopic phenotypic evaluation experiment of skin photoaging The effects of live bacteria, inactivated bacteria, and lysate preparations on improving the macroscopic phenotype of skin photoaging were evaluated by detecting macroscopic indicators such as skin wrinkle area, elasticity coefficient, and skin color brightness in mice.

[0048] The formula for calculating the improvement rate is as follows:

[0049] The changes in the area of ​​skin wrinkles in each group of mice are shown in Table 3 below.

[0050] Table 3 - Changes in skin wrinkle area in mice (mm) 2 , ±s, n=8)

[0051] As shown in Table 3, after 28 days of UVB irradiation, the wrinkle area in the model control group increased to 52.6 ± 5.8 mm. 2 The model was successfully established. Compared with the model group, the wrinkle area in the live bacteria intervention group, inactivated bacteria intervention group, and lysate intervention group was 22.8±3.5 mm. 2 33.2±4.1mm 2 and 38.4±4.6mm 2 The improvement rates were 56.7%, 36.9%, and 27.0%, respectively, and all three formulations effectively inhibited the formation of skin wrinkles caused by UVB. The efficacy ranking was: live bacteria intervention group > inactivated bacteria intervention group > lysate intervention group.

[0052] The changes in the skin elasticity coefficient of mice in each group are shown in Table 4 below.

[0053] Table 4 - Changes in the skin elasticity coefficient of mice (mN / mm²) ±s, n=8)

[0054] As shown in Table 4, after 28 days of continuous UVB irradiation, the elasticity coefficient of the model control group decreased to 1.05±0.08 mN / mm, indicating a significant loss of skin elasticity. Compared with the model group, the elasticity coefficients of the live bacteria, inactivated bacteria, and lysate intervention groups were 1.56±0.11 mN / mm, 1.38±0.10 mN / mm, and 1.28±0.09 mN / mm, respectively, with improvement rates of 48.6%, 31.4%, and 21.9%. All three preparations significantly improved the decrease in skin elasticity caused by UVB, with the live bacteria preparation showing the best recovery effect and continuous improvement from day 14 to day 28. The efficacy ranking was: live bacteria intervention group > inactivated bacteria intervention group > lysate intervention group.

[0055] Skin color brightness L in each group of mice The value changes are shown in Table 5 below.

[0056] Table 5 - Mouse skin color brightness (L) Value change

[0057] As shown in Table 5, after 28 days of UVB irradiation, the L value of the model control group decreased to 48.2±2.5, indicating significant skin darkening. Compared with the model group, the L values ​​of the live bacteria, inactivated bacteria, and lysate intervention groups were 58.5±1.9, 55.8±2.0, and 53.6±2.2, respectively, with improvement rates of 72.8%, 53.5%, and 38.2%. All three preparations effectively improved UVB-induced skin darkening, with the live bacteria preparation showing the best brightening effect, increasing L value from 14 to 28 days. The values ​​continued to rise. The efficacy ranking was: live bacteria intervention group > inactivated bacteria intervention group > lysate intervention group.

[0058] Example 4 Skin oxidative stress level detection experiment The total SOD activity assay kit (Shanghai Beyotime Biotechnology Co., Ltd., S0101S), glutathione peroxidase assay kit (Shanghai Beyotime Biotechnology Co., Ltd., S0056), catalase assay kit (Shanghai Beyotime Biotechnology Co., Ltd., S0051), lipid peroxidation (MDA) assay kit (Shanghai Beyotime Biotechnology Co., Ltd., S0131S), and reactive oxygen species assay kit (DCFH-DA) (Shanghai Beyotime Biotechnology Co., Ltd., S0033S) were used to detect SOD, GSH-Px, CAT (antioxidant indicators), MDA, and ROS (oxidative damage indicators) in mouse skin tissue homogenates in each group according to the kit instructions. The test data are shown in Table 6 below.

[0059] Table 6 - Indicators of oxidative stress in mouse skin ( ±s, n=8)

[0060] As shown in Table 6, the activities of SOD, GSH-Px, and CAT in the model control group were significantly lower than those in the blank group, while the levels of MDA and ROS were significantly higher, indicating that UVB irradiation successfully induced oxidative stress damage in the skin. Generally, higher SOD / GSH-Px / CAT ratios and lower MDA / ROS ratios are better. All three formulations have antioxidant effects, with live bacteria showing the best results. In the live bacteria group, all indicators recovered to near-normal levels (SOD recovered by 89.1%, and MDA decreased by 62.3%). The efficacy ranking was: live bacteria intervention group > inactivated bacteria intervention group > lysate intervention group.

[0061] Example 5 Quantitative Experiment on Skin Collagen Synthesis and Degradation Pathways The types I and III collagen contents in mouse skin tissue homogenates were detected using the Mouse Collagen Type I, Col I ELISA Kit (Wuhan Huamei Biotechnology Co., Ltd., CSB-E08083m) and the Mouse Collagen Type III, Col III ELISA Kit (Wuhan Huamei Biotechnology Co., Ltd., CSB-E07925m) according to the kit instructions. The test data are shown in Table 7 below.

[0062] Table 7 - Collagen content in mouse skin (μg / mg tissue protein) ±s, n=8)

[0063] As shown in Table 7, compared with the blank control group, the type I and type III collagen contents in the model control group decreased by 57.2% and 33.6%, respectively, and the type I / type III ratio decreased from 2.23 to 1.44, indicating that UVB irradiation significantly inhibited collagen synthesis. All intervention groups effectively increased collagen content, and all three preparations effectively promoted collagen synthesis, with live bacteria showing the best effect. The efficacy ranking was: live bacteria > inactivated bacteria > lysate.

[0064] The protein expression levels of matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-3 (MMP-3), and their natural inhibitor tissue inhibitor of metalloproteinases-1 (TIMP-1) in mouse skin tissue of each group were detected by Western blot. The test data are shown in Table 8 below.

[0065] Table 8 - Expression of collagen degradation-related proteins in mouse skin (relative gray values) ±s, n=8)

[0066] MMP-1 / MMP-3 is a collagen-degrading enzyme (lower is better), while TIMP-1 is its natural inhibitor (higher is better). Table 8 shows that all intervention groups effectively inhibited MMP-1 / MMP-3 expression and increased TIMP-1 levels. In the live bacteria intervention group, the TIMP-1 / (MMP-1+MMP-3) ratio recovered to 75.2% of the normal level. The efficacy ranking was: live bacteria > inactivated bacteria > lysates.

[0067] Example 6 Skin cell aging marker detection experiment The positive rate of senescent cells in the skin tissue of mice in each group was detected by aging-associated β-galactosidase (SA-β-gal) staining; the protein expression levels of aging-associated markers p16 and p53 in the skin tissue of mice in each group were detected by immunohistochemistry (IHC). The test data are shown in Table 9 below.

[0068] Table 9 - Indicators of aging in mouse skin cells ( ±s, n=8)

[0069] SA-β-gal, p16, and p53 are all aging markers (lower is better). Table 9 shows that the SA-β-gal positivity rate and p16 and p53 expression in the model control group were significantly higher than in the blank group, indicating that UVB irradiation successfully induced skin cell aging. All intervention groups effectively reduced the above indicators. Among them, the SA-β-gal positivity rate in the live bacteria group was only 23.7% of that in the model group, significantly better than the lysate group (74.0%) and the inactivated bacteria group (50.8%).

[0070] Example 7 Long-term anti-aging effect verification experiment after drug withdrawal To evaluate the persistence of anti-aging efficacy after drug withdrawal, the comprehensive anti-aging efficacy index of the skin of mice in each group was measured on days 7, 14, and 28 after drug withdrawal, and the efficacy retention rate was calculated. The comprehensive efficacy value of each group on day 28 (before drug withdrawal) was taken as 100% as the baseline, and the percentage of the efficacy value at each time point relative to the baseline value was the efficacy retention rate at that time point. The test data are shown in Table 10 below.

[0071] Table 10 - Retention rate of anti-aging efficacy after discontinuation of medication (%)

[0072] As shown in Table 10, with the efficacy value on day 28 of modeling (before drug withdrawal) as 100% baseline, the comprehensive efficacy index at each time point after drug withdrawal reflects the dynamic changes in efficacy after drug withdrawal. The comprehensive efficacy index of the live bacteria group reached 98.0% 28 days after drug withdrawal, significantly better than the lysate group (75.0%) and the inactivated bacteria group (88.0%), demonstrating that the live bacteria preparation still has a continuous regulatory effect after drug withdrawal, and its efficacy maintenance effect is the best.

[0073] In summary, based on the above animal experimental results, the following conclusions can be drawn: (1) Among all the test indicators, the anti-aging efficacy of live Bifidobacterium adolescentis MS-YLOHAS18 was significantly better than that of intact inactivated bacteria and fermentation product lysate. The efficacy of the three forms was ranked as follows: live bacteria > inactivated bacteria > lysate.

[0074] (2) The anti-aging advantages of live bacteria preparations may be related to the following characteristics: live bacteria have complete metabolic activity, can continuously produce beneficial metabolites and promote the skin's microecological balance; live bacteria can colonize the skin surface to form a protective barrier, with better stability and longer-lasting efficacy (the comprehensive efficacy index was 98.0% after 28 days of drug withdrawal, while that of lysates was only 75.0%); live bacteria and their metabolites can reach the deep epidermis to the superficial dermis and act on fibroblasts. The active ingredients in lysates are released only once and cannot be continuously replenished. After 28 days of drug withdrawal, the comprehensive efficacy index was only 75.0%, significantly lower than that of the live bacteria group (98.0%), indicating that the long-term effect of lysates is insufficient. The efficacy of inactivated bacteria is between that of live bacteria and lysates, indicating that although the bacteria retain their complete structure after inactivation, they lose their metabolic activity and are less effective than live bacteria.

[0075] Although the present invention has been described in detail with reference to the accompanying drawings and preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. A strain of Bifidobacterium adolescentis MS-YLOHAS18, characterized in that, The Bifidobacterium adolescentis ( Bifidobacterium adolescentis MS-YLOHAS18 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39539, deposited on June 30, 2026. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. The Bifidobacterium adolescentis MS-YLOHAS18 as described in claim 1, characterized in that, The 16S rDNA sequence of the Bifidobacterium adolescentis MS-YLOHAS18 is shown in SEQ ID NO.

3.

3. A microbial agent, characterized in that, The bacterial agent contains Bifidobacterium adolescentis MS-YLOHAS18 as described in claim 1.

4. The microbial agent as described in claim 3, characterized in that, The number of viable bacteria in the bacterial agent is ≥1.0×10 9 CFU / g.

5. The microbial agent as described in claim 3 or 4, characterized in that, The preparation method of the bacterial agent is as follows: Stored Bifidobacterium adolescentis MS-YLOHAS18 is activated on MRS solid medium. The activated bacteria are inoculated into MRS liquid medium at a mass percentage of 1%, and then cultured at 37℃ for 24 hours to obtain a bacterial suspension. After centrifugation, the bacterial cells are collected, washed with sterile physiological saline, and resuspended in 15% reconstituted skim milk to obtain a suspension. The concentration of the suspension is adjusted to 1.0 × 10⁻⁶. 9 ~2.0×10 9 CFU / mL was used to obtain a bacterial suspension; the bacterial suspension was freeze-dried to obtain bacterial powder; the bacterial powder was mixed with maltodextrin to prepare a bacterial agent containing Bifidobacterium adolescentis MS-YLOHAS18.

6. A microbial agent, characterized in that, The bacterial agent contains a metagene of Bifidobacterium adolescentis MS-YLOHAS18 as described in claim 1; the metagene includes inactivated Bifidobacterium adolescentis MS-YLOHAS18 or lysate of Bifidobacterium adolescentis MS-YLOHAS18.

7. The microbial agent as described in claim 6, characterized in that, The preparation method of the bacterial agent is as follows: A suspension of *Bifidobacterium adolescentis* MS-YLOHAS18 was inactivated by heating in a 100°C water bath for 30 minutes. The inactivated cells were collected by centrifugation, washed with sterile physiological saline, and resuspended in 15% (w / w) reconstituted skim milk, resulting in a cell concentration of 1.0 × 10⁻⁶. 9 The bacterial agent containing Bifidobacterium adolescentis MS-YLOHAS18 postbiotic was prepared by lyophilizing and mixing it with maltodextrin at CFU / mL.

8. The microbial agent as described in claim 6, characterized in that, The preparation method of the bacterial agent is as follows: Take the suspension of Bifidobacterium adolescentis MS-YLOHAS18 and inactivate it by heating in a water bath at 100℃ for 30 minutes; resuspend the inactivated bacteria in buffer solution, add lysozyme at a ratio of 0.1% by weight / volume, and enzymatically hydrolyze in a water bath at 37℃ for 4 hours with shaking; circulate and homogenize the enzymatic hydrolysate 3 times to fully break down the bacteria. Centrifuge the homogenized liquid, collect the supernatant, and filter it through a 0.22μm microporous membrane to obtain Bifidobacterium adolescentis MS-YLOHAS18 lysate. Mix the obtained lysate with the cosmetic matrix at a volume ratio of 1:9 to obtain the lysate preparation.

9. The use of Bifidobacterium adolescentis MS-YLOHAS18 as described in claim 1 in the preparation of products for preventing or improving skin photoaging.

10. The application as described in claim 9, characterized in that, The prevention or improvement of skin photoaging includes at least one of the following: reducing skin wrinkles, improving skin elasticity, improving skin radiance, increasing the activity of skin antioxidant enzymes, reducing skin oxidative damage, increasing the content of type I and / or type III collagen in the skin, inhibiting the expression of matrix metalloproteinases, and increasing the expression of TIMP-1.

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