Bifidobacterium adolescentis for promoting collagen synthesis in a host

Abstract

This invention discloses a metabiotic prepared from a strain of *Bifidobacterium adolescentis* that promotes host collagen synthesis, belonging to the fields of microbial technology and pharmaceutical technology. The metabiotic of *Bifidobacterium adolescentis* CCFM1386 provided by this invention has a good effect on promoting host collagen synthesis, improving skin moisture and elasticity, and simultaneously enhancing skin's antioxidant capacity and maintaining collagen homeostasis. Specifically, in vitro, bacterial lysate promotes the activity of LH1 enzyme in HSF cells; in vitro, it increases the content of COLⅠ and COLⅢ and the activity of LH1 enzyme in the self-fermentation supernatant; oral administration of inactivated bacterial cells increases collagen content; oral administration of the self-fermentation supernatant increases the activity of LH1, P4H, and LOX enzymes, promotes collagen synthesis, and increases collagen content. Therefore, this *Bifidobacterium adolescentis* has great application potential in the preparation of oral drugs that promote host collagen synthesis, as well as in the preparation of food, health products, or cosmetics.

Description

Technical Field

[0001] This invention relates to a metabiotic prepared from a strain of Bifidobacterium adolescentis that promotes collagen synthesis in the host, belonging to the fields of microbial technology and pharmaceutical technology. Background Technology

[0002] Collagen, the most abundant structural protein in the body, is widely distributed in various tissues such as skin, bone, cartilage, tendons, blood vessels, and internal organs. It plays a crucial role in maintaining tissue structural integrity, imparting mechanical properties to tissues, and regulating cellular physiological behavior. Maintaining the dynamic balance between collagen synthesis and degradation under various physiological and pathological conditions is essential for the normal functioning of tissues. Especially in areas such as skin and bone tissue repair, cartilage regeneration, and chronic wound healing, promoting the host's own collagen synthesis has become an important strategy for enhancing tissue regeneration capacity, repairing damage, and improving function.

[0003] Currently, several patented technologies have attempted to increase collagen content in host tissues through internal and external interventions, small molecule drugs, or plant extracts. For example, CN119700752A discloses the application of Bergapten or its isomers in the preparation of products that improve skin aging and / or promote skin repair. However, most existing studies remain at the level of total collagen detection. In particular, most studies have failed to systematically evaluate the expression and activity changes of key enzymes in collagen biosynthesis, such as prolyl hydroxylase (P4H), lysyl hydroxylase (LH1), and lysyl oxidase (LOX), which play a central role in the maturation, stabilization, and fiber formation of collagen molecules. The lack of detection of key regulatory nodes in the anabolic process makes it difficult to clarify the mechanism of action with existing technologies, limiting the scientific evaluation and optimization of intervention effects. At the same time, the identification and verification of regulatory targets (such as key molecules in upstream signaling pathways) are also relatively lacking, making strategies to promote collagen synthesis significantly insufficient in terms of mechanistic clarity and precise intervention. Summary of the Invention

[0004] To solve the above-mentioned technical problems, the present invention provides a Bifidobacterium adolescentis (Bifidobacterium adolescentis) Bifidobacterium adolescents is Application of CCFM1386 and its post-genes in the preparation of products that promote host collagen synthesis.

[0005] The first technical solution provided by this invention is a strain of Bifidobacterium adolescentis (Bifidobacterium adolescentis) Bifidobacterium adolescents is CCFM1386, the Bifidobacterium adolescentis CCFM1386, was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 10, 2025, with accession number: GDMCC No: 65746.

[0006] The Bifidobacterium adolescentis CCFM1386 strain was derived from samples from healthy individuals. Sequencing analysis of the strain and nucleic acid sequence alignment using NCBI confirmed the presence of Bifidobacterium adolescentis, hence the name Bifidobacterium adolescentis CCFM1386.

[0007] The colonies of Bifidobacterium adolescentis CCFM1386 on MRS solid medium were small, white, round, and with neat edges.

[0008] The second technical solution provided by the present invention is a microbial preparation containing the Bifidobacterium adolescentis CCFM1386 described in the first technical solution.

[0009] In some embodiments, the concentration of Bifidobacterium adolescentis CCFM1386 in the microbial preparation is not less than 1×10⁻⁶. 6 CFU / mL or ×10 6 CFU / g.

[0010] The third technical solution provided by the present invention is a metabiotic prepared using the Bifidobacterium adolescentis CCFM1386 described in the first technical solution or the microbial preparation described in the second technical solution.

[0011] In some embodiments, the metabiotic includes cell lysate, inactivated or dead cells, fermentation supernatant, or any of the above-mentioned powders prepared by drying.

[0012] In some embodiments, the inactivated or deadened cells are prepared by culturing the Bifidobacterium adolescentis CCFM1386 in a culture medium, collecting the bacterial cells in the cell culture medium, and obtaining inactivated bacterial cells after heat treatment.

[0013] In some embodiments, the heat treatment conditions are: 65-95°C for 10-30 min; preferably 65°C for 30 min.

[0014] In some embodiments, the preparation method of the bacterial lysate is as follows: after culturing the Bifidobacterium adolescentis CCFM1386 in a culture medium, the bacterial cells are collected, homogenized under high pressure, and the supernatant obtained by centrifugation is used to obtain the bacterial lysate.

[0015] In some embodiments, the fermentation supernatant is the supernatant obtained after centrifuging the Bifidobacterium adolescentis CCFM1386 in a culture medium.

[0016] The present invention also provides a fourth technical solution, which is a product containing the Bifidobacterium adolescentis CCFM1386 described in the first technical solution and / or the postbiotic described in the third technical solution.

[0017] In some embodiments, the product includes, but is not limited to, food, medicine, health products, or daily chemical products.

[0018] In some embodiments, the food product includes the above-described composition and conventional excipients.

[0019] In some embodiments, the conventional excipients include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

[0020] In some embodiments, the health product includes the above-described composition and conventional excipients.

[0021] In some embodiments, the conventional excipients include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

[0022] In some embodiments, the dosage form of the product includes at least one of creams, lotions, oils, liquids, gels, powders, and lyophilized products.

[0023] In some embodiments, the product is a probiotic powder.

[0024] In some embodiments, the bacterial powder is a solid powder of Bifidobacterium adolescentis CCFM1386 prepared by drying the liquid metabiotic.

[0025] In some embodiments, drying includes, but is not limited to, preparation by spray drying, vacuum freeze drying, fluidized bed drying, and vacuum drying.

[0026] The fifth technical solution provided by this invention is the application of Bifidobacterium adolescentis CCFM1386 described in the first technical solution, the microbial preparation described in the second technical solution, or the post-genetic agent described in the third technical solution in the preparation of products that promote collagen synthesis in skin fibroblasts.

[0027] In some embodiments, the product includes at least one of the following functions:

[0028] (1) Increase the content of collagen secreted by skin fibroblasts in vitro;

[0029] (2) Increase the activity of collagen synthase in skin fibroblasts in vitro.

[0030] The sixth technical solution provided by this invention is the application of Bifidobacterium adolescentis CCFM1386 described in the first technical solution, the microbial preparation described in the second technical solution, or the post-biotic described in the third technical solution in the preparation of anti-aging products.

[0031] In some implementations, the application includes at least one of the following functions:

[0032] (1) Increase the moisture and elasticity of the skin tissue in aging individuals;

[0033] (2) Increase the activity of antioxidant enzymes in the skin tissue of aging individuals;

[0034] (3) Reduce inflammation levels in aging individuals;

[0035] (4) Increase the collagen content in the skin tissue of aging individuals;

[0036] (5) Increase the activity of collagen synthase in the skin tissue of aging individuals.

[0037] In some embodiments, the symptoms associated with skin collagen loss include skin appearance, decreased skin collagen content, slowed collagen synthesis, and accelerated collagen degradation.

[0038] In some implementations, the aging includes skin aging.

[0039] In some embodiments, skin aging includes dry skin, decreased elasticity, sagging, wrinkle formation, oxidative damage, or collagen loss.

[0040] In some embodiments, the product contains at least 5 × 10⁻⁶ Bifidobacterium adolescentis CCFM1386. 7 CFU / mL.

[0041] In some embodiments, the dose of the postbiotic prepared from Bifidobacterium adolescentis CCFM1386 in the product is not less than 250 μg / kg.

[0042] In some embodiments, the product is a pharmaceutical or cosmetic product.

[0043] In some embodiments, the pharmaceutical product comprises the Bifidobacterium adolescentis CCFM1386, a drug carrier, and / or pharmaceutical excipients.

[0044] In some embodiments, the pharmaceutical excipient includes excipients and additives.

[0045] In some embodiments, the pharmaceutical excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, filter aids, and release inhibitors.

[0046] In some embodiments, the cosmetic contains Bifidobacterium adolescentis CCFM1386, matrix ingredients, and / or conventional excipients.

[0047] In some embodiments, the matrix raw materials include oily raw materials, waxy raw materials, synthetic oily raw materials, powdery raw materials, gelling raw materials, coagulants, and surfactants.

[0048] In some embodiments, the conventional excipients include one or more of the following: humectants, whitening agents, flavoring agents, adhesives, lubricants, preservatives, film-forming agents, antioxidants, emulsifiers, and cosmetic nutritional additives.

[0049] The seventh technical solution provided by the present invention is a method for promoting collagen synthesis in skin fibroblasts in vitro, wherein the method involves mixing and incubating the metagenerogen described in the third technical solution with skin fibroblasts.

[0050] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0051] The present invention is Bifidobacterium adolescentis ( Bifidobacterium adolescentis CCFM1386 and its prepared post-biotic oral formulations have the ability to promote host collagen synthesis, specifically manifested in:

[0052] (1) Increase the content of type I and type III collagen (COL I, III) secreted by human skin fibroblasts (HSF) in vitro;

[0053] (2) Enhance the activity of LH1 in human skin fibroblasts (HSF) in vitro;

[0054] (3) Improve skin moisture and elasticity in aging individuals;

[0055] (4) Increase the activity of CAT and SOD in the skin of aging individuals;

[0056] (5) Reduce the levels of CRP, IL-6 and TNF-α in the serum of aging individuals;

[0057] (6) Increase the levels of COLⅠ, Ⅲ, Ⅳ and VII in the skin of aging individuals;

[0058] (7) Enhance the activity of LH1, P4H and LOX in the skin of aging individuals;

[0059] Therefore, Bifidobacterium adolescentis ( Bifidobacterium adolescentis The post-genetic agents prepared by CCFM1386 have great application potential in products that promote host collagen synthesis.

[0060] Preservation of biological materials

[0061] Bifidobacterium adolescentis ( Bifidobacterium adolescentis CCFM1386, taxonomically named Bifidobacterium adolescentis It was deposited on January 10, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 65746, located at Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0062] Figure 1 Effects of post-biotics prepared for CCFM1386 on the levels of COLⅠ and COLⅢ secreted by HSF cells;

[0063] Figure 2 Effect of post-biotics prepared for CCFM1386 on LH1 enzyme activity in HSF cells;

[0064] Figure 3 Flowchart of mouse experiments;

[0065] Figure 4 Effects of post-biotics prepared for CCFM1386 on the content of COLⅠ, Ⅲ, Ⅳ and VII in mouse skin tissue;

[0066] Figure 5 Effects of post-biotics prepared for CCFM1386 on the activities of LH1, P4H and LOX enzymes in mouse skin tissue;

[0067] Figure 6 Effects of post-biotics prepared for CCFM1386 on CAT and SOD enzyme activities in mouse skin tissue;

[0068] Figure 7 Effects of post-biotics prepared for CCFM1386 on the levels of CRP, IL-6 and TNF-α in mouse serum;

[0069] Figure 8 Effects of post-biotics prepared for CCFM1386 on skin moisture and elasticity in mice;

[0070] Figure 9 MASSON staining for skin.

[0071] "**" indicates a statistically significant difference from the control group (P<0.01), and "***" indicates an extremely statistically significant difference from the control group (P<0.001).

[0072] "#" indicates a statistically significant difference from the model group (P<0.05), "##" indicates a statistically significant difference from the model group (P<0.01), "###" indicates an extremely statistically significant difference from the model group (P<0.001), and "####" indicates an extremely statistically significant difference from the model group (P<0.0001). Detailed Implementation

[0073] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0074] Raw materials used in the examples:

[0075] The human skin fibroblasts (HSF) involved in the following examples were purchased from the Kunming Cell Bank.

[0076] The BALB / c mice used in the following examples were purchased from Vital Rivers.

[0077] The Bifidobacterium adolescentis CCFM1386 involved in the following examples was a self-screened strain from the Food Biotechnology Center of Jiangnan University.

[0078] The ELISA kits used in the following examples were purchased from Nanjing Senbega Biotechnology Co., Ltd.

[0079] The D-galactose used in the following examples was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0080] The BCA protein concentration assay kits used in the following examples were purchased from Shanghai Beyotime Biotechnology Co., Ltd. The culture media used in the following examples are as follows:

[0081] Modified MRS liquid culture medium: yeast extract 5.0 g / L, peptone 10.0 g / L, glucose 20.0 g / L, anhydrous sodium acetate 2.0 g / L, diammonium citrate 2.0 g / L, dipotassium hydrogen phosphate 2.6 g / L, manganese sulfate monohydrate 0.25 g / L, magnesium sulfate heptahydrate 0.5 g / L, cysteine ​​1 g / L, and Tween-80 1 mL / L, pH 6.2–6.4.

[0082] Modified MRS solid culture medium: yeast extract 5.0 g / L, peptone 10.0 g / L, glucose 20.0 g / L, anhydrous sodium acetate 2.0 g / L, diammonium citrate 2.0 g / L, dipotassium hydrogen phosphate 2.6 g / L, manganese sulfate monohydrate 0.25 g / L, magnesium sulfate heptahydrate 0.5 g / L, Tween-80 1 mL / L, cysteine ​​1 g / L, and agar 20.0 g / L, pH 6.2–6.4.

[0083] Cell culture medium: 89% (v / v) DMEM medium + 10% (v / v) fetal bovine serum + 1% (v / v) 100× penicillin and streptomycin mixed solution (the mixed solution contains 10,000 U / mL penicillin and 10 mg / mL streptomycin).

[0084] Example 1: Cell resuscitation and culture

[0085] First, remove the frozen human skin fibroblast cell line (HSF), quickly thaw it in a 37°C water bath, then centrifuge at 1000 r / min for 3 min, discard the supernatant, add an appropriate volume of cell culture medium to resuspend the cells, place them in a culture dish, and incubate them in a 37°C incubator containing 5% CO2. When the cells regain their viability and grow for 1-2 days and reach 70%-80% confluence, passage the cells.

[0086] Example 2: Preparation of postbiotics from Bifidobacterium adolescentis CCFM1386

[0087] 1. Screening and identification of Bifidobacterium adolescentis CCFM1386

[0088] The strain samples were selected from healthy human samples. After pretreatment, they were stored in 20% glycerol at -80℃. After thawing, the samples were mixed and 0.5 mL was added to 4.5 mL of physiological saline. The samples were serially diluted with physiological saline and then plated onto MRS solid medium. The samples were anaerobically cultured at 37℃ for 48 h. Typical colonies of Bifidobacterium adolescentis were picked and streaked onto MRS solid medium for purification. Single colonies were transferred to MRS liquid medium for enrichment and stored in 30% glycerol to obtain the strain. The genome of the strain was extracted and amplified with 16S rDNA and sequenced (by Suzhou Genewiz Biotechnology Co., Ltd.). The results confirmed the strain to be Bifidobacterium adolescentis by NCBI sequence alignment. The strain was named Bifidobacterium adolescentis CCFM1386 and deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 10, 2025, with accession number GDMCC No: 65746.

[0089] 2. Preparation of postbiotics from Bifidobacterium adolescentis CCFM1386

[0090] (1) Bifidobacterium adolescentis CCFM1386 was streaked from the preservation tube and revived. It was cultured in a 37°C anaerobic workstation for 48 h using modified MRS solid medium to obtain single colonies. Single colonies were picked and inoculated into modified MRS liquid medium and cultured at 37°C for 12-18 h to obtain culture solution 1.

[0091] Culture medium 1 was inoculated into modified MRS liquid medium at an inoculation rate of 2% (v / v) and cultured anaerobically at 37°C for 12 h to obtain seed culture;

[0092] The seed culture was inoculated at 2% (v / v) into modified MRS liquid medium for expansion culture, and anaerobic culture was carried out at 37℃ for 18 h. The number of viable bacteria was recorded and bacterial culture a was obtained.

[0093] The bacterial culture a was centrifuged at 8000 r / min for 30 min, and the supernatant and bacterial sludge were collected. The supernatant was heat-treated (65 ℃, 30 min) and freeze-dried for later use to prepare the freeze-dried powder of Bifidobacterium adolescentis CCFM1386 fermentation supernatant (denoted as CCFM1386-S). The bacterial sludge was resuspended in 75% of the original bacterial culture volume of double-distilled water, and the resuspended liquid was heat-treated (65 ℃, 30 min), and then homogenized under high pressure (1000 MPa, 10P times) in a high-pressure homogenizer. After homogenization, the supernatant was collected by centrifugation at 8000 r / min for 30 min to obtain the bacterial cell lysate (denoted as CCFM1386-J).

[0094] The following postbiotics of Bifidobacterium adolescentis CCFM1386 were prepared by the above methods: bacterial lysate CCFM1386-J and fermentation supernatant CCFM1386-S.

[0095] Example 3: Effect of post-biotic prepared from Bifidobacterium adolescentis CCFM1386 on collagen content in HSF cells

[0096] (1) Take HSF cells in the logarithmic growth phase and use 1×10 5 Cells were seeded at a density of 100 cells / mL in 6-well plates and cultured overnight until cell attachment was achieved. The old culture medium was discarded, and the cells were washed three times with PBS. A control group and a metabiotic treatment group were set up.

[0097] Control group: Contains cell culture medium and HSF cells, but does not contain post-genetic agents;

[0098] The post-genetic treatment group contained cell culture medium and HSF cells, as well as post-genetic agents.

[0099] The metabiotic was resuspended in cell culture medium (the amount of resuspended metabiotic was the same as that fermented to a concentration of 5.0 × 10⁻⁶). 7The amount of metabiotic prepared from the bacterial culture at CFU / ml was equivalent, and 100 μL of metabiotic prepared from Bifidobacterium adolescentis CCFM1386 was added to each culture.

[0100] (2) The above well plates were incubated in an incubator at 37 °C for 24 h.

[0101] (3) After incubation, collect the cell culture supernatant and centrifuge at 1000 r / min for 5 min to collect the supernatant. Detect the COLⅠ and COLⅢ content using an ELISA kit.

[0102] (4) After washing the cells in the plate twice with PBS, add 1 ml of trypsin containing 0.25% EDTA and digest at 37°C for 30 s. Observe the cells under a microscope until most cells become rounded, then add 4 mL of DMEM complete culture medium to stop the digestion. Gently pipette to completely detach the cells from the culture dish, transfer the resulting cell suspension to a 15 mL centrifuge tube, centrifuge at 1000 r / min for 5 min, remove the supernatant, resuspend the cells with a mixture of protease inhibitors, and repeatedly freeze and thaw (rapid freezing at -80°C and slow thawing at 4°C) three times to obtain cell lysate samples.

[0103] (5) Use the BCA protein concentration assay kit to detect the total protein concentration of the sample in (4) and calibrate the above data.

[0104] The results are as follows Figure 1 As shown, compared with the control group, CCFM1386-S increased the content of COLⅠ secreted by HSF cells by approximately 20%, and CCFM1386-S significantly increased the content of COLⅢ secreted by HSF cells by approximately 18%. These results indicate that the post-biotic prepared by CCFM1386 can increase the content of collagen secreted by HSF cells and has the potential to regulate collagen synthesis and metabolism.

[0105] Example 4: Effect of post-biotic prepared from Bifidobacterium adolescentis CCFM1386 on collagen synthase activity in HSF cells

[0106] The preparation of cell lysate samples in the following examples is the same as in Example 3. The enzyme activity of lysine hydroxylase (LH1) in HSF cell lysates was detected by an ELISA kit.

[0107] LH1 plays a crucial role in collagen synthesis, being a key enzyme in the post-modification process of collagen. LH1 catalyzes the hydroxylation of lysine residues to generate hydroxylysine, a process essential for collagen stability and function. Hydroxylated lysine not only contributes to the cross-linking of collagen fibers, enhancing their mechanical strength, but also participates in the interaction of collagen with other extracellular matrix components, thus playing a vital role in maintaining tissue structure and function. The results of LH1 are as follows... Figure 2 As shown, compared with the control group (27.41 IU / g), the LH1 enzyme activity in HSF cells treated with CCFM1386-S increased to 34.96 IU / g, and the LH1 enzyme activity in HSF cells treated with CCFM1386-J increased to 41.72 IU / g. These results indicate that the post-genetic precursor prepared with CCFM1386 has the potential to promote collagen synthesis in the body.

[0108] Example 5: Effect of post-biotic prepared from Bifidobacterium adolescentis CCFM1386 on collagen content in the skin of aging mice

[0109] The preparation methods of the postgenes (CCFM1386-S and CCFM1386-J) of Bifidobacterium adolescentis CCFM1386 involved in the following examples are the same as in Example 2. The difference is that CCFM1386-J in this example specifically refers to the bacterial sludge obtained by centrifuging the bacterial solution a obtained in Example 2 at 8000 r / min for 30 min. The bacterial sludge is resuspended in double-distilled water at 75% of the original bacterial solution volume. The resuspended solution is heat-treated at 65 °C for 30 min without high-pressure homogenization.

[0110] (1) Twenty healthy male BALB / c mice aged 8 weeks were randomly divided into 4 cages, with 5 mice in each cage. The 4 cages were: 1 cage for the control group, 1 cage for the model group, 1 cage for the CCFM1386-S group, and 1 cage for the CCFM1386-J group.

[0111] Control group: Using physiological saline as a control;

[0112] Model group: using physiological saline as a control;

[0113] CCFM1386-S group: Bifidobacterium adolescentis CCFM1386 metabolite (fermentation supernatant) was used at a dose of 500 mg / kg mouse body weight;

[0114] CCFM1386-J group: Bifidobacterium adolescentis CCFM1386 postbiotic (inactivated bacterial cells) at a dose of 500 mg / kg mouse body weight;

[0115] In each of the above groups, inactivated bacterial cells or metabolites were inactivated using 1×10⁻⁶ cells / day. 9 Inactivated bacteria or metabolites are prepared from bacterial solutions containing an equal amount of live CFU.

[0116] The experiment lasted for 8 weeks: After one week of acclimatization, except for the control group, the other groups were subcutaneously injected with D-galactose (500 mg / kg) at a dose of 0.2 mL / mouse / day. Starting from the second week, each intervention group was administered postbiotic lyophilized powder (inactivated bacteria or fermentation supernatant) prepared from the corresponding strain, dissolved in physiological saline at the appropriate dose, and administered to the mice by gavage at a dose of 0.2 mL / mouse / day. The control group and the model group were administered an equal volume of physiological saline by gavage as controls until the end of the experiment. All groups had free access to water and food, and the experimental procedure was as follows: Figure 3 As shown.

[0117] After the experiment, mice were sacrificed and blood was collected from their eyeballs. After standing for 40 minutes, the blood was centrifuged at 3000 r / min for 20 minutes, and the blood supernatant was stored at -80℃. Skin tissue from the back was cut and homogenized with PBS at a weight-to-volume ratio of 1:9. The homogenate was centrifuged at 3000 r / min for 20 minutes, and the skin supernatant was collected and tested using an ELISA kit.

[0118] The levels of type I, III, IV, and VII collagen (COL I, III, IV, and VII) in mouse skin were detected using an ELISA kit. Figure 4 As shown.

[0119] Compared with the blank group (8.18 μg / mg), the content of COLⅠ in the skin of the model group decreased significantly to 3.44 μg / mg. The oral CCFM1386-S and CCFM1386-J groups significantly increased the content of COLⅠ compared with the model group, restoring the content of COLⅠ in the skin to 5.90 μg / mg and 5.23 μg / mg, respectively, which can upregulate the content of COLⅠ.

[0120] The COLⅢ content in the blank group was 1.94 μg / mg, which was 2.4 times that in the model group (0.81 μg / mg). Oral administration of CCFM1386-S significantly increased its content to 1.55 μg / mg compared with the model group, and CCFM1386-J also significantly increased its content to 1.41 μg / mg compared with the model group. Both can increase the COLⅢ content.

[0121] For COLⅣ, the content in the blank group was 15.29 μg / mg, and the content in the model group was 6.21 μg / mg. Compared with the model group, the oral CCFM1386-S group significantly upregulated the content of COLⅣ to 12.10 μg / mg, and the CCFM1386-J group also significantly upregulated the content of COLⅣ to 11.40 μg / mg compared with the model group.

[0122] For COLⅦ, the content in the blank group was 2.22 μg / mg, which was 2.6 times that of the model group (0.85 μg / mg). Only the oral CCFM1386-S group could significantly upregulate the content of COLⅦ compared with the model group, and the content in this group was restored to 1.98 μg / mg.

[0123] The results above indicate that the post-biotic (inactivated cells and supernatant) prepared from Bifidobacterium adolescentis CCFM1386 can significantly increase the collagen content in the skin of aging mice.

[0124] Example 6: Effects of Bifidobacterium adolescentis CCFM1386 and its prepared post-biotic on collagen synthase activity in aging mouse skin

[0125] The animal experimental design, gavage groups, and preparation of skin homogenate samples used in the ELISA kits in the following examples are the same as in Example 5.

[0126] LH1 catalyzes the hydroxylation of lysine residues, providing sites for glycosylation and influencing collagen fiber assembly and cross-linking; P4H catalyzes the hydroxylation of proline residues, promoting proper collagen folding and structural stability; LOX catalyzes the oxidative deamination of lysine and hydroxylysine residues, forming aldehyde derivatives, which then condense with adjacent lysine amino groups or hydroxyl groups to form covalent cross-links, thus stabilizing the fibrillary structure. Both are key catalytic enzymes in collagen synthesis. The enzyme activities of LH1, P4H, and LOX in mouse skin were detected using an ELISA kit, and the results are as follows: Figure 5 As shown.

[0127] Enzyme activities of LH1, P4H, and LOX were detected, showing a significant decrease in all three enzyme activities compared to the control group. After oral administration of CCFM1386-S, LH1 activity was 13.01 U / mg, significantly upregulated compared to the model group (8.58 U / mg); P4H activity was 30.45 U / mg, significantly upregulated compared to the model group (17.00 U / mg); and LOX activity increased to 13.43 U / mg, significantly higher than the model group's 8.48 U / mg.

[0128] The results above indicate that the post-biotic (fermentation supernatant) prepared from Bifidobacterium adolescentis CCFM1386 can increase the enzyme activity of LH1, P4H and LOX in the skin of aging mice and promote the synthesis of collagen.

[0129] Example 7: Effects of Bifidobacterium adolescentis CCFM1386 and its prepared postbiotic on the antioxidant capacity of skin in aging mice

[0130] The animal experimental design, gavage groups, and preparation of skin homogenate samples used in the ELISA kits in the following examples are the same as in Example 5.

[0131] The enzyme activities of superoxide dismutase (SOD) and catalase (CAT) in mouse skin were detected using an ELISA kit. Figure 6 As shown.

[0132] Regarding SOD enzyme activity, the SOD enzyme activity in the control group was 15.87 U / mg, while the SOD enzyme activity in the model group's skin significantly decreased to 7.35 U / mg. After oral administration of CCFM1386-S, the activity increased to 11.81 U / mg; after oral administration of CCFM1386-J, the activity increased to 12.31 U / mg. Compared with the model group, both significantly increased SOD enzyme activity.

[0133] Regarding CAT enzyme activity, compared with the control group (37.38 U / μg), the CAT enzyme activity in the skin of the model group was significantly decreased to 16.32 U / μg. Oral administration of CCFM1386-S and CCFM1386-J both upregulated the skin CAT enzyme activity to 23.62 U / μg and 25.10 U / μg, respectively, which were significantly different from those in the model group.

[0134] Oxidative stress has a significant impact on collagen synthesis and stability in skin tissue. Superoxide dismutase (SOD) can catalyze the synthesis and stability of superoxide anions (O2). - ) is converted into hydrogen peroxide (H2O2), and then CAT quickly decomposes H2O2 into water and oxygen, thereby effectively preventing the accumulation of ROS in cells, reducing the damage to collagen molecules caused by oxidative stress, and thus protecting the collagen structure.

[0135] In summary, the postbiotic (inactivated cells and supernatant) prepared from Bifidobacterium adolescentis CCFM1386 can increase the enzyme activity of CAT and SOD in the skin of aging mice, improve the skin's antioxidant capacity, and increase the content and stability of collagen in the host skin.

[0136] Example 8: Effects of Bifidobacterium adolescentis CCFM1386 and its prepared postbiotic on inflammation levels in aging mice

[0137] The animal experimental design, gavage groups, and serum sample preparation involved in the following examples are the same as in Example 5.

[0138] The levels of C-reactive protein (CRP) in mouse serum were detected using a biochemical analyzer, and the levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in mouse skin were detected using an ELISA kit. Figure 7 As shown.

[0139] As an acute-phase reactive protein, elevated CRP levels typically reflect an exacerbation of systemic inflammation. The inflammatory environment can accelerate collagen degradation and inhibit its synthesis. The CRP level in the control group was 4.58 mg / L, while the CRP level in the model group was 5.18 mg / L. Oral administration of CCFM1386-J significantly downregulated serum CRP levels to 4.98 mg / L.

[0140] IL-6 is an important pro-inflammatory cytokine that can promote the expression of matrix metalloproteinases (MMPs) by activating signaling pathways, thereby disrupting collagen fiber structure. In the IL-6 index, compared with the control group (10.54 μg / mg), the IL-6 content in the skin of the model group was significantly increased to 24.06 μg / mg. Oral administration of CCFM1386-J significantly downregulated the skin IL-6 content to 16.89 μg / mg.

[0141] Tumor necrosis factor-α (TNF-α), a potent pro-inflammatory cytokine, not only induces collagen breakdown but also inhibits fibroblast function and reduces collagen regeneration. In the TNF-α index, compared to the control group (54.59 ng / mg), the TNF-α content in the skin of the model group significantly increased to 112.29 μg / mg. Oral administration of CCFM1386-J significantly downregulated the skin TNF-α content to 82.95 μg / mg.

[0142] In summary, oral administration of CCFM1386-J can alleviate inflammation in aging mice to varying degrees and affect collagen synthesis and stability.

[0143] Example 9: Effects of Bifidobacterium adolescentis CCFM1386 and its prepared postbiotic on stratum corneum moisture content and skin elasticity in aging mice.

[0144] The animal experimental design and gavage groups involved in the following examples are the same as in Example 5. At the end of the experiment, the skin moisture content of the stratum corneum on the back of each mouse and the elasticity of the skin on the back of the mouse were measured using a skin moisture meter (equipped with a Corneometer CM825 stratum corneum moisture measurement probe) from CK GmbH, Germany. The results are as follows: Figure 8 As shown.

[0145] The moisture content of the stratum corneum is from Figure 8 It can be seen that, compared with the blank group of 78.83%, the water content of the model group was significantly reduced to 52.87%. The water content of the stratum corneum in the oral CCFM1386-S group (71.99%) was 19.12% higher than that in the model group, and the water content of the CCFM1386-J group (68.43%) was 15.56% higher than that in the model group.

[0146] Skin elasticity property R2 is composed of Figure 8 It can be seen that, compared with the blank group (80.80%), the skin elasticity of the model group was significantly reduced to 54.17%, the skin elasticity of the CCFM1386-S group (79.53%) was increased by 25.36% compared with the model group, and the skin elasticity of the CCFM1386-J group (75.20%) was increased by 21.03% compared with the model group.

[0147] Collagen is a major component that maintains skin structure and elasticity. Its loss leads to weakened skin support and decreased elasticity, resulting in signs of aging such as wrinkles and sagging. At the same time, a reduction in collagen also affects the skin's ability to retain moisture, causing the skin to become dry and rough, further accelerating skin aging.

[0148] In summary, the experimental results show that the postbiotic (inactivated cells and supernatant) prepared from Bifidobacterium adolescentis CCFM1386 can significantly increase the moisture content and elasticity of aging skin.

[0149] Example 10: MASSON staining analysis of skin tissue sections from aging mice by Bifidobacterium adolescentis CCFM1386 and its prepared metabiotic.

[0150] The animal experimental design and gavage groups involved in the following examples are the same as in Example 5. Mice were sacrificed at the end of the experiment, and skin from the shaved back area was taken for MASSON staining. The results are as follows: Figure 9 As shown.

[0151] The section results clearly show that the collagen fibers in the dermis of the control group mice are tightly and orderly arranged, staining a deep blue, and have a higher collagen content. The fiber bundles are intact and evenly distributed, with no obvious breakage or degeneration. Furthermore, the connection between the dermis and epidermis in the control group skin is tight, demonstrating good skin barrier function and elastic support. In contrast, the collagen fibers in the dermis of the model group mice are significantly lighter in staining, loosely and disordered, showing fiber breakage and discontinuity, and a significant reduction in collagen content. Compared with the model group, the post-regenerative effect of CCFM1386 significantly improved both the collagen content and structure in mouse skin.

[0152] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of Bifidobacterium adolescentis ( Bifidobacterium adolescentis CCFM1386, characterized in that, The Bifidobacterium adolescentis CCFM1386 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 10, 2025, with accession number GDMCC No: 65746.

2. A microbial preparation containing the Bifidobacterium adolescentis CCFM1386 as described in claim 1.

3. An epigenetic agent, characterized in that, The postbiotic prepared using Bifidobacterium adolescentis CCFM1386 as described in claim 1 or the microbial preparation as described in claim 2.

4. The epigenetic agent according to claim 3, characterized in that, The metabiotic includes bacterial lysate, inactivated or dead cells, fermentation supernatant, or any of the above-mentioned powders prepared by drying.

5. The epigenetic agent according to claim 4, characterized in that, The inactivated or deactivated cells are prepared as follows: after culturing the Bifidobacterium adolescentis CCFM1386 in a culture medium, the bacterial cells in the cell culture medium are collected and then heat-treated to obtain inactivated bacterial cells; The method for preparing the bacterial lysate is as follows: after culturing the Bifidobacterium adolescentis CCFM1386 in a culture medium, the bacterial cells are collected, homogenized under high pressure, centrifuged, and the supernatant is taken to obtain the bacterial lysate. The fermentation supernatant is the supernatant obtained after centrifuging the Bifidobacterium adolescentis CCFM1386 in a culture medium.

6. A medicine containing Bifidobacterium adolescentis CCFM1386 as described in claim 1 and / or the metabiotic as described in any one of claims 3 to 5.

7. The use of the Bifidobacterium adolescentis CCFM1386 according to claim 1, the microbial preparation according to claim 2, or the metabiotic according to any one of claims 3 to 5 in the preparation of a drug that promotes collagen synthesis in dermal fibroblasts, characterized in that, The drug has at least one of the following effects: (1) Increase the content of collagen secreted by skin fibroblasts in vitro; (2) Increase the activity of collagen synthase in skin fibroblasts in vitro.

8. The use of Bifidobacterium adolescentis CCFM1386 as described in claim 1, the microbial preparation as described in claim 2, or the metabiotic as described in any one of claims 3 to 5 in the preparation of anti-aging drugs.

9. The application according to claim 8, characterized in that, The application includes at least one of the following functions: (1) Increase the moisture and elasticity of the skin tissue in aging individuals; (2) Increase the activity of antioxidant enzymes in the skin tissue of aging individuals; (3) Reduce inflammation levels in aging individuals; (4) Increase the collagen content in the skin tissue of aging individuals; (5) Increase the activity of collagen synthase in the skin tissue of aging individuals.

10. A method for promoting collagen synthesis in skin fibroblasts in vitro, characterized in that, The method involves mixing and incubating the metagenerium described in any one of claims 3 to 5 with skin fibroblasts.

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