Application of bifidobacterium adolescentis CCFM1302 in promoting synthesis of host collagen

Abstract

The invention discloses application of bifidobacterium adolescentis CCFM1302 in promoting synthesis of host collagen, and belongs to the technical field of microorganisms and medicines. The metagen of the bifidobacterium adolescentis CCFM1302 disclosed by the invention has a good effect of regulating and controlling synthesis and degradation of collagen, the moisture and elasticity of the skin are improved, the antioxidant capacity of the skin is improved, the inflammation level of an organism is reduced, and the stable state of collagen is maintained. The method specifically comprises the following steps: in vitro promoting the enzyme activity of HSF cell LH1 by a thallus lysate, inhibiting the enzyme activity of MMP-3, increasing the content of COL I and COL III and the enzyme activity of LH1, P4H and LOX from the outside of a fermented supernatant, and inhibiting the enzyme activity of MMP-3; the inactivated thalli are orally taken to increase the collagen content and the enzyme activity of LH1 and inhibit the enzyme activity of MMP-1 and MMP-3; the fermentation supernatant is orally taken to inhibit enzyme activity of MMP-3 and MMP-1 and degradation of collagen, and the content of TGF-beta is increased.

Description

TECHNICAL FIELD

[0001] The application relates to the application of Bifidobacterium adolescentis CCFM1302 in promoting collagen synthesis of a host, and belongs to the technical fields of microorganisms and medicine. BACKGROUND

[0002] Collagen is the most crucial structural protein in the connective tissue of the body, accounting for a major proportion of the total amount of extracellular matrix proteins, and widely exists in the skin, skeleton, cartilage, tendon and blood vessel wall, and plays an important role in supporting, connecting and protecting tissues and organs. Collagen not only endows tissues with necessary mechanical strength and elasticity, but also participates in the maintenance of tissue homeostasis and regeneration repair by regulating cell adhesion, migration, proliferation and differentiation. Under normal physiological conditions, the synthesis and decomposition of collagen maintain a dynamic balance, but under the conditions of injury, aging and diseases, the balance is easily broken, leading to damage of tissue structure and function. Therefore, for the problems of skin aging, osteoporosis, cartilage degeneration and difficult-to-heal wounds, activating the collagen neogenesis mechanism of the host and restoring the tissue microenvironment have become an important research direction for promoting regenerative medicine and tissue repair.

[0003] At present, a number of patent technologies attempt to improve the collagen content in the host tissue by means of internal and external intervention, application of small molecule drugs or plant extracts. For example, CN119258200A discloses an anti-aging composition for increasing dermal collagen and a preparation method thereof, and the composition comprises Melissa officinalis extract, Pistacia chinensis flower extract, mulberry extract, collagen tripeptide and Sargassum fusiforme polysaccharide. Although the composition provided in the patent CN119258200A aims to promote the increase of dermal collagen content through the synergistic effect of multiple natural extracts and collagen tripeptide in theory, there are still some deficiencies. First, the plant extract components in the composition are complex, the specific active ingredients and their action mechanisms are not clear, and the plant-derived components have the problems of large batch difference and insufficient activity stability, which are easily affected by extraction process, storage conditions and environmental factors, leading to fluctuations in the effect of the final product. Especially, the natural extracts contain a variety of small molecule impurities and potential allergens, which are easy to cause skin or systemic allergic reactions, affecting the universality and safety of the user population. Secondly, although the small molecular weight of collagen tripeptide is helpful for absorption, the oral route still faces common problems such as digestion and decomposition, plasma clearance and insufficient skin tissue targeting, and the actual efficiency of promoting skin collagen neogenesis is limited. In addition, the active ingredients in the composition are easy to decompose under light, temperature change and oxidation conditions, leading to a decrease in activity and a shortening of the product shelf life, and the storage conditions are relatively high. SUMMARY

[0004] In response to the above-mentioned prior art, the present invention provides a use of Bifidobacterium adolescentis CCFM1302 and its postbiotics in the preparation of products for regulating collagen synthesis.

[0005] The present invention provides a strain of Bifidobacterium adolescentis CCFM1302, which is deposited in Guangdong Provincial Microbiological Culture Collection Center with the collection number: GDMCC No: 63176.

[0006] The Bifidobacterium adolescentis CCFM1302 was derived from samples of healthy people. The strain was sequenced and analyzed, and the obtained sequence was compared with the nucleic acid sequence in NCBI. The result showed that it was Bifidobacterium adolescentis, and was named Bifidobacterium adolescentis CCFM1302.

[0007] The colonies of Bifidobacterium adolescentis CCFM1302 on the MRS solid culture medium are white, round, raised, relatively small, and have neat edges.

[0008] The first technical solution provided by the present invention is the use of Bifidobacterium adolescentis CCFM1302, a microbial preparation containing the Bifidobacterium adolescentis CCFM1302, or a postbiotic prepared from the Bifidobacterium adolescentis CCFM1302 in the preparation of anti-aging products. The preservation number of the Bifidobacterium adolescentis CCFM1302 is GDMCC No: 63176.

[0009] The present invention also provides a postbiotic prepared by applying the Bifidobacterium adolescentis CCFM1302.

[0010] In one embodiment, the postbiotics include bacterial lysate, killed or inactivated cells, fermentation supernatant, or any of the above powders prepared by drying.

[0011] In one embodiment, the inactivated or inactivated cells are prepared as follows: the Bifidobacterium adolescentis CCFM1302 is cultured in a culture medium for a period of time, bacterial cells in the cell culture medium are collected, and inactivated bacterial cells are obtained after heat treatment.

[0012] In one embodiment, the heat treatment conditions are: 65° C., 30 min.

[0013] In one embodiment, the bacterial lysate is prepared by culturing the Bifidobacterium adolescentis CCFM1302 in a culture medium for a period of time, collecting bacterial cells, homogenizing under high pressure, and obtaining the bacterial lysate from the supernatant of the centrifugation.

[0014] In one embodiment, the fermentation supernatant is the supernatant obtained by culturing the Bifidobacterium adolescentis CCFM1302 in a culture medium for a period of time and then centrifuging the culture medium.

[0015] In one embodiment, the product includes but is not limited to food, medicine, health care product or daily chemical product.

[0016] In one embodiment, the food comprises the above composition and conventional auxiliary materials.

[0017] In one embodiment, the conventional excipients include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional enhancers.

[0018] In one embodiment, the health care product comprises the above composition and conventional excipients.

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

[0020] In one embodiment, the dosage form of the product includes at least one of creams, lotions, oils, aqueous solutions, gels, powders, and freeze-dried solutions.

[0021] In one embodiment, the product is a probiotic powder.

[0022] In one embodiment, the bacterial powder is a solid powder postbiotic prepared by drying the prepared liquid postbiotic of Bifidobacterium adolescentis CCFM1302.

[0023] In one embodiment, drying includes but is not limited to spray drying, vacuum freeze drying, fluidized bed drying, and vacuum drying.

[0024] In one embodiment, the application includes at least one of the following functions:

[0025] (1) Increase the moisture and elasticity of skin tissue in aging individuals;

[0026] (2) Increase the antioxidant enzyme activity in the skin tissue of aging individuals;

[0027] (3) reduce inflammation levels in aging individuals;

[0028] (4) Increase collagen content in skin tissue of aging individuals;

[0029] (5) Increase the activity of collagen synthase in the skin tissue of aging individuals;

[0030] (6) Reduce the activity of collagen-degrading enzymes in the skin tissue of aging individuals;

[0031] (7) increasing the TGF-β content of the skin tissue of the aging individual.

[0032] In an embodiment, the skin collagen loss-related symptom comprises skin appearance, decreased skin collagen content, slowed collagen synthesis, and accelerated collagen degradation.

[0033] In an embodiment, the aging comprises skin aging.

[0034] In an embodiment, the skin aging comprises dry skin, decreased elasticity, laxity, wrinkle formation, oxidative damage, or collagen loss.

[0035] In an embodiment, the product contains Bifidobacterium adolescentis CCFM1302 in an amount of not less than 5 x 10 7 CFU / mL.

[0036] In an embodiment, the product contains Bifidobacterium adolescentis CCFM1302 prepared postbiotic in an amount of not less than 250 μg / kg.

[0037] In an embodiment, the product is a pharmaceutical product or a cosmetic product.

[0038] In an embodiment, the pharmaceutical product further comprises a pharmaceutical carrier and / or a pharmaceutical excipient.

[0039] In an embodiment, the pharmaceutical excipient comprises an excipient and an additional agent.

[0040] In an embodiment, the pharmaceutical excipient comprises a solvent, a propellant, a solubilizer, a co-solvent, an emulsifier, a coloring agent, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending agent, a coating material, a fragrance, an anti-adhesion agent, an integrating agent, a penetration enhancer, a pH regulator, a buffer, a plasticizer, a surfactant, a foaming agent, an antifoaming agent, a thickening agent, a complexing agent, a humectant, an absorbent, a diluent, a flocculating agent and a deflocculating agent, a filter aid, and a release retardant.

[0041] In an embodiment, the cosmetic product further comprises a base material and / or a conventional excipient,

[0042] In an embodiment, the base material comprises an oil-based material, a wax-based material, a synthetic oil-based material, a powder-based material, a gum-based material, a coagulant, and a surfactant.

[0043] In an embodiment, the conventional excipient comprises one or more of a humectant, a whitening agent, a flavoring agent, a binder, a lubricant, a preservative, a film-forming agent, an antioxidant, an emulsifier, and a cosmetic nutrient additive.

[0044] The second technical solution provided by the present invention is the use of postbiotics prepared by Bifidobacterium adolescentis CCFM1302 in the preparation of products that promote collagen synthesis in skin fibroblasts. The preservation number of Bifidobacterium adolescentis CCFM1302 is: GDMCCNo: 63176.

[0045] In one embodiment, the application includes at least one of the following functions:

[0046] (1) Increase the collagen content secreted by skin fibroblasts in vitro;

[0047] (2) Increase the activity of collagen synthase in skin fibroblasts in vitro;

[0048] (3) Inhibit the activity of collagen degrading enzymes in skin fibroblasts in vitro.

[0049] The third technical solution provided by the present invention is a method for promoting collagen synthesis of skin fibroblasts in vitro, wherein the method comprises mixing postbiotics prepared by Bifidobacterium adolescentis CCFM1302 with skin fibroblasts, wherein the preservation number of Bifidobacterium adolescentis CCFM1302 is GDMCC No: 63176.

[0050] Beneficial effects

[0051] The Bifidobacterium adolescentis CCFM1302 of the present invention and the postbiotics prepared therefrom have the ability to regulate collagen synthesis and degradation, which is specifically reflected in:

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

[0053] (2) Increase the enzymatic activities of LH1, P4H, and LOX in human skin fibroblasts (HSF) in vitro;

[0054] (3) Reduce the enzyme activity of MMP-3 in human skin fibroblasts (HSF) in vitro

[0055] (4) Improve skin moisture and elasticity in aging individuals;

[0056] (5) Improve the enzyme activities of SOD and GSH-PX in the skin of aging individuals;

[0057] (6) Reduce the levels of CRP and IL-6 in the serum of aging individuals;

[0058] (7) Increase the levels of COL I, III, and IV in the skin of aging individuals;

[0059] (8) Increase LH1 enzyme activity in the skin of aging individuals;

[0060] (9) Reduce the enzyme activities of MMP-1 and MMP-3 in the skin of aging individuals;

[0061] (10) Increase the content of TGF-β in the skin of aging individuals.

[0062] Therefore, postbiotics prepared from Bifidobacterium adolescentis CCFM1302 have great application prospects in products that regulate collagen synthesis and degradation.

[0063] Biomaterial Deposit

[0064] Bifidobacterium adolescentis CCFM1302, taxonomically named Bifidobacterium adolescentis, was deposited in the Guangdong Provincial Microbial Culture Collection on February 16, 2023, with the collection number GDMCC No: 63176. The collection address is Building 59, No. 100 Xianlie Middle Road, Guangzhou City, and has been disclosed in patent CN116426419A. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 Effects of postbiotics prepared for CCFM1302 on the secretion of COLⅠ and COLⅢ by HSF cells;

[0066] Figure 2 Effects of postbiotics prepared for CCFM1302 on LH1, P4H, and LOX enzyme activities in HSF cells;

[0067] Figure 3 Effects of postbiotics prepared for CCFM1302 on MMP-3 enzyme activity in HSF cells;

[0068] Figure 4 This is the flow chart of the mouse experiment;

[0069] Figure 5 Effects of postbiotics prepared for CCFM1302 on the levels of COLⅠ, Ⅲ, and Ⅳ in mouse skin tissue;

[0070] Figure 6 Effects of postbiotics prepared for CCFM1302 on LH1 enzyme activity in mouse skin tissue;

[0071] Figure 7 Effects of postbiotics prepared for CCFM1302 on MMP-1 and MMP-3 enzyme activities in mouse skin tissue;

[0072] Figure 8 Effect of the postbiotic prepared for CCFM1302 on the content of TGF-β in the skin tissue of mice;

[0073] Figure 9 Effect of the postbiotic prepared for CCFM1302 on the activities of SOD and GSH-PX enzymes in the skin tissue of mice;

[0074] Figure 10 Effect of the postbiotic prepared for CCFM1302 on the content of CRP and IL-6 in the serum of mice;

[0075] Figure 11 Effect of the postbiotic prepared for CCFM1302 on the moisture and elasticity of the skin of mice;

[0076] Figure 12 MASSON staining section of the skin.

[0077] “*” indicates a statistical difference (P < 0.05) from the control group, “**” indicates a significant statistical difference (P < 0.01) from the control group, “***” indicates a very significant statistical difference (P < 0.001) from the control group, and “****” indicates a very significant statistical difference (P < 0.0001) from the control group.

[0078] “#” indicates a statistical difference (P < 0.05) from the model group, “##” indicates a significant statistical difference (P < 0.01) from the model group, “###” indicates a very significant statistical difference (P < 0.001) from the model group, and “####” indicates a very significant statistical difference (P < 0.0001) from the model group. DETAILED DESCRIPTION

[0079] The preferred embodiments of the present application are described below, and it should be understood that the embodiments are for better explaining the present application and are not used to limit the present application.

[0080] Raw materials used in the embodiments:

[0081] The human skin fibroblasts (HSF) involved in the following embodiments are purchased from the Kunming Cell Bank.

[0082] The BALB / c mice involved in the following embodiments are purchased from Vivotecnia.

[0083] The Bifidobacterium adolescentis CCFM1302 involved in the following embodiments is from the Food Biotechnology Center of Jiangnan University.

[0084] The ELISA kits involved in the following examples were purchased from Nanjing Senbeijia Biotechnology Co., Ltd.

[0085] The D-galactose involved in the following examples was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0086] The BCA protein concentration determination kit involved in the following examples was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0087] The culture medium involved in the following examples is as follows:

[0088] Modified MRS liquid 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 hydrogen 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.

[0089] Modified MRS solid medium: yeast powder 5.0 g / L, peptone 10.0 g / L, glucose 20.0 g / L, anhydrous sodium acetate 2.0 g / L, diammonium hydrogen 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.

[0090] Cell culture medium: 89% (v / v) DMEM medium + 10% (v / v) fetal bovine serum + 1% (v / v) 100× mixed solution of penicillin and streptomycin (the penicillin content in the mixed solution is 10000 U / mL, and the streptomycin concentration is 10 mg / mL).

[0091] Example 1 Cell recovery and culture

[0092] First, take out the frozen human skin fibroblast cell line (HSF), quickly thaw it in a 37℃ water bath, then centrifuge it at 1000r / min for 3min, discard the supernatant, add an appropriate volume of cell culture medium to resuspend the cells, place them in a culture dish, and culture them in a 37℃ incubator containing 5% CO2. When the cells recover their vitality and grow for 1-2 days and reach 70%-80% confluence, cell passage is performed.

[0093] Example 2: Preparation of postbiotics using Bifidobacterium adolescentis CCFM1302

[0094] 1. Screening and identification of Bifidobacterium adolescentis CCFM1302

[0095] The strain sample screening was derived from samples from healthy people. After pretreatment, they were stored in a -80°C refrigerator in 20% glycerol. After thawing, they were mixed and 0.5 mL of sample was added to 4.5 mL of normal saline. The sample was gradiently diluted with normal saline. The appropriate gradient dilution was selected and spread on MRS solid culture medium. It was anaerobically cultured at 37°C for 48 hours, and typical colonies of Bifidobacterium adolescentis were picked and streaked on MRS solid culture medium for purification. Single colonies were picked and transferred to MRS liquid culture medium for enrichment and preserved in 30% glycerol to obtain the strain. The strain genome was extracted for 16S rDNA amplification and sequencing (performed by Suzhou Jinweizhi Biotechnology Co., Ltd.). The results were identified as Bifidobacterium adolescentis by NCBI sequence alignment and named Bifidobacterium adolescentis CCFM1302. It was deposited in Guangdong Provincial Microbiological Culture Collection on February 16, 2023, with a deposit number of GDMCC No: 63176, and has been disclosed in patent CN116426419A.

[0096] 2. Preparation of postbiotics with Bifidobacterium adolescentis CCFM1302

[0097] (1) Resuscitating Bifidobacterium adolescentis CCFM1302 from a sterile tube by streaking, culturing in a modified MRS solid medium at 37°C in an anaerobic workstation for 48 h to obtain a single colony; picking a single colony and inoculating it into a modified MRS liquid medium, culturing at 37°C for 12-18 h to obtain culture medium 1;

[0098] The culture solution 1 was inoculated into the modified MRS liquid medium at an inoculum volume of 2% (v / v), and cultured anaerobically at 37°C for 12 h to obtain the seed solution;

[0099] The seed solution was inoculated into a modified MRS liquid medium at 2% (v / v) for expansion, and anaerobically cultured at 37°C for 18 hours. The number of viable bacteria was recorded to obtain bacterial solution a.

[0100] The bacterial solution a was centrifuged at 8000 r / min for 30 min, the supernatant and bacterial mud were collected, the supernatant was heat-treated (65°C for 30 min), and freeze-dried for later use to prepare a freeze-dried powder of the fermentation supernatant of Bifidobacterium adolescentis CCFM1302 (denoted as CCFM1302-S). The bacterial mud was resuspended in double-distilled water with 75% of the volume of the original bacterial solution, the resuspension was heat-treated (65°C for 30 min), and then homogenized in a high-pressure homogenizer (1000 MPa, 10 times). After homogenization, the supernatant was centrifuged at 8000 r / min for 30 min to obtain a bacterial lysate (denoted as CCFM1302-J).

[0101] The postbiotics of Bifidobacterium adolescentis CCFM1302 (bacterial lysate CCFM1302-J and fermentation supernatant CCFM1302-S) were prepared by the above-mentioned means.

[0102] Example 3: Effect of the postbiotic prepared by Bifidobacterium adolescentis CCFM1302 on the collagen content of HSF cells

[0103] (1) The HSF cells in logarithmic growth phase were inoculated on 6-well plates at 1 x 10 5 cells / mL, and the cells were cultured overnight to adhere. The old culture medium was discarded, and the cells were washed with PBS for 3 times. Blank and postbiotic treatment groups were set up;

[0104] Blank group (control): containing cell culture medium and HSF cells, but not containing postbiotic;

[0105] Postbiotic treatment group: containing cell culture medium and HSF cells, and containing postbiotic.

[0106] The postbiotic was resuspended in cell culture medium (the amount of resuspended postbiotic was equivalent to the amount of postbiotic prepared from bacteria with a concentration of 5.0 x 10 7 CFU / ml), and 100 μL of postbiotic prepared by Bifidobacterium adolescentis CCFM1302 was added.

[0107] (2) The above-mentioned hole plates were incubated at 37°C for 24 h.

[0108] (3) After incubation, the cell culture supernatant was collected and centrifuged at 1000 r / min for 5 min to obtain the supernatant. The COL I and COL III contents were detected by ELISA kit.

[0109] (4) The cells in the plate were washed with PBS for 2 times, 1 ml of trypsin containing 0.25% EDTA was added, and the cells were digested at 37°C for 30 s. The cells were observed under a microscope until most of the cells were rounded, and 4 mL of DMEM complete medium was added to terminate the digestion. The cells were completely detached from the culture dish by gentle blowing, and the formed cell suspension was transferred to a 15 mL centrifuge tube and centrifuged at 1000 r / min for 5 min. The supernatant was removed, the cells were resuspended with protease cocktail inhibitor, and the cells were repeatedly frozen and thawed (-80°C freezing and 4°C thawing) for three times to obtain cell lysate samples.

[0110] (5) The total protein concentration of the samples in (4) was detected by BCA protein concentration determination kit, and the above data was calibrated.

[0111] The results are shown in Table 1. Figure 1As shown in the results, compared with the control group, CCFM1302-S increased the content of COLⅠ secreted by HSF cells by approximately 43%, and CCFM1302-S significantly increased the content of COLⅢ secreted by HSF cells by approximately 27%. These results indicate that postbiotics prepared with CCFM1302 can increase the content of collagen secreted by HSF cells and have the potential to regulate collagen synthesis metabolism.

[0112] Example 4: Effect of postbiotics prepared from Bifidobacterium adolescentis CCFM1302 on collagen synthase activity in HSF cells

[0113] The preparation of cell lysate samples involved in the following examples is the same as that in Example 3.

[0114] LH1 can catalyze the hydroxylation of lysine residues, thereby providing sites for glycosylation and affecting the assembly and cross-linking of collagen fibers; P4H can catalyze the hydroxylation of proline residues, promoting the correct folding and structural stability of collagen; LOX can catalyze the oxidative deamination reaction of lysine and hydroxylysine residues to form aldehyde derivatives, and condense with the adjacent lysine amino group or the hydroxyl group of hydroxylysine to form covalent cross-links to stabilize the structure of the fibrils. Both are key catalytic enzymes in the process of collagen synthesis. The enzymatic activities of LH1 and P4H in HSF cell lysates and the enzymatic activity of LOX in cell culture supernatant were detected by ELISA kits. The results are as follows: Figure 2 shown.

[0115] The results of LH1 are as follows Figure 2 As shown, compared with the control group (27.41 IU / g), the enzyme activity of LH1 in HSF cells treated with CCFM1302-S can be increased to 58.02 IU / g, and the enzyme activity of LH1 in HSF cells treated with CCFM1302-J can be increased to 45.55 IU / g.

[0116] P4H enzyme activity results Figure 2 As shown, compared with the control group (185.72 IU / g), the enzyme activity of P4H in HSF cells after CCFM1302-S treatment can be increased to 208.09 IU / g, which can significantly increase the enzyme activity of P4H in HSF cells. The enzyme activity of P4H after CCFM1302-J treatment is 172.23 IU / g, which has no improvement effect compared with the control group.

[0117] LOX enzyme activity results Figure 2 As shown, compared with the control group (0.52 IU / mg), the LOX enzymatic activity in HSF cells treated with CCFM1302-S can be increased to 0.61 IU / mg, which can significantly increase the LOX enzymatic activity.

[0118] Taken together, the above results indicate that the postbiotics prepared by CCFM1464 can increase the enzymatic activity of collagen synthase in HSF cells and have the potential to enhance collagen synthesis in the body.

[0119] Example 5: Effect of postbiotics prepared from Bifidobacterium adolescentis CCFM1302 on the activity of collagen degrading enzymes in HSF cells

[0120] The preparation of the cell culture supernatant samples involved in the following examples is the same as that in Example 3. The enzyme activity of matrix metalloproteinase 3 (MMP-3) in the HSF cell culture supernatant was detected by ELISA kit.

[0121] MMP-3 can convert inactive collagenase precursors (such as pro-MMP-1) into active forms through proteolytic cleavage, enhance the activity of collagenase, and degrade fiber fragments and proteins in the cell matrix, destroying the stability of the collagen network. Figure 3 As shown, compared with the control group (1.69 U / mg), CCFM1302-S could significantly reduce the enzyme activity of MMP-3 to 1.47 U / mg, and CCFM1302-J could significantly reduce the enzyme activity of MMP-3 to 1.26 U / mg.

[0122] The results showed that postbiotics prepared by CCFM1462 have the potential to regulate collagen degradation in the body.

[0123] Example 6: Effect of postbiotics prepared from Bifidobacterium adolescentis CCFM1302 on collagen content in the skin of aging mice

[0124] The preparation method of the postbiotics (CCFM1302-S and CCFM1302-J) of Bifidobacterium adolescentis CCFM1302 involved in the following examples is the same as that in Example 2, except that CCFM1302-J in this embodiment specifically refers to the bacterial liquid a obtained in Example 2, which is centrifuged at 8000 r / min for 30 min to obtain the bacterial sludge, and the bacterial sludge is resuspended with double distilled water with 75% volume of the original bacterial liquid, and the resuspension is heat-treated at 65°C for 30 min without high-pressure homogenization.

[0125] (1) Twenty 8-week-old healthy male BALB / c mice were randomly divided into 4 cages with 5 mice in each cage. The 4 cages were: 1 cage for the blank group (control), 1 cage for the model group (codel), 1 cage for the CCFM1302-S group, and 1 cage for the CCFM1302-J group.

[0126] Blank group (control): physiological saline was used as a control;

[0127] Model group: normal saline was used as the control;

[0128] CCFM1302-S group: using Bifidobacterium adolescentis CCFM1302 metabolite (fermentation supernatant) at a dose of 500 mg / kg mouse body weight;

[0129] CCFM1302-J group: Bifidobacterium adolescentis CCFM1302 postbiotics (inactivated bacteria) were used at a dose of 500 mg / kg mouse body weight;

[0130] Among them, the inactivated bacteria or metabolites in the above groups were 1×10 9 Inactivated bacteria or metabolites prepared from a bacterial solution with an amount of CFU of viable bacteria.

[0131] The experiment lasted for 8 weeks: After the mice adapted for one week, all groups except the blank group 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 used the corresponding strain-prepared postbiotic freeze-dried powder (inactivated bacteria or fermentation supernatant) dissolved in normal saline at a corresponding dose, and the mice were gavaged at a dose of 0.2 mL / mouse / day. The blank group and the model group were gavaged with the same amount of normal saline as a control until the end of the experiment. All groups had free access to water and food. The experimental process is as follows: Figure 4 shown.

[0132] After the experiment, the mice were killed and blood was collected from the 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°C. The back skin tissue was cut and ground into a homogenate at a weight-to-volume ratio of 1:9 with PBS. The homogenate was centrifuged at 3000 r / min for 20 minutes, and the skin supernatant was collected for detection using an ELISA kit.

[0133] The contents of type Ⅰ, Ⅲ, Ⅳ and Ⅶ collagen (COL Ⅰ, Ⅲ, Ⅳ, Ⅶ) in mouse skin were detected by ELISA kit. Figure 5 shown.

[0134] Compared with the blank group (8.18 μg / mg), the COLⅠ content in the skin of the model group decreased significantly to 3.44 μg / mg. The oral CCFM1302-J group significantly increased the COLⅠ content compared with the model group, restoring the COLⅠ content in the skin to 5.27 μg / mg.

[0135] The content of COLⅢ in the blank group was 1.94μg / mg, which was 2.4 times that of COLⅢ in the model group (0.81μg / mg). Oral administration of CCFM1302-J could significantly increase its content to 1.39μg / mg compared with the model group.

[0136] For COLⅣ, the content in the blank group was 15.29μg / mg, and the content in the model group was 6.21μg / mg. Oral administration of CCFM1302-S group can significantly increase the content of COLⅣ to 12.18μg / mg compared with the model group, and CCFM1302-J group can also significantly increase the content of COLⅣ to 10.42μg / mg compared with the model group.

[0137] The above results show that the postbiotics (inactivated bacteria and supernatant) prepared from Bifidobacterium adolescentis CCFM1302 can significantly increase the collagen content in the skin of aging mice.

[0138] Example 7: Effects of Bifidobacterium adolescentis CCFM1302 and its prepared postbiotics on collagen synthase activity in aging mouse skin

[0139] The animal experiment design, gavage groups, and preparation of skin homogenate samples used in the ELISA kits involved in the following examples are the same as those in Example 6.

[0140] The enzyme activity of LH1 in mouse skin was detected by ELISA kit. Figure 6 shown.

[0141] Compared with the blank group (19.64 U / mg), the LH1 enzyme activity in the model group decreased significantly to 8.58 U / mg. After oral administration of CCFM1302-S, the LH1 enzyme activity was 13.46 U / mg, which was significantly increased compared with the model group.

[0142] The above results show that the postbiotics (fermentation supernatant) prepared by Bifidobacterium adolescentis CCFM1302 can increase the enzyme activity of LH1 in the skin of aging mice and promote the synthesis of collagen.

[0143] Example 8: Effects of Bifidobacterium adolescentis CCFM1302 and its prepared postbiotics on the activity of collagen degrading enzymes in the skin of aging mice

[0144] The animal experiment design, gavage groups, and preparation of skin homogenate samples used in the ELISA kits involved in the following examples are the same as those in Example 6.

[0145] The enzyme activities of MMP-1 and MMP-3 in mouse skin were detected by ELISA kit. Figure 7 shown.

[0146] MMP-1 is a key enzyme in collagen degradation. Its substrates include collagen types I and III. Its cleavage site is typically located between Gly-Ile or Gly-Leu bonds, allowing it to cleave intact collagen triple helices into fragments of varying lengths, making them more susceptible to degradation by other MMPs. Oral administration of CCFM1302-S and CCFM1302-J significantly downregulated MMP-1 activity compared to the model group. The model group had an MMP-1 activity of 9.94 IU / mg, while the CCFM1302-S and CCFM1302-J groups had MMP-1 activities of 8.45 and 9.07 IU / mg, respectively, significantly different from the model group.

[0147] In terms of MMP-3 activity, compared to the blank control group (3.80 IU / mg), the MMP-3 activity in the model group increased significantly to 4.91 IU / mg. Oral administration of CCFM1440-J and CCFM1440-S significantly decreased MMP-3 activity in the skin to 3.54 IU / mg and 3.75 IU / mg, respectively.

[0148] The above results show that the postbiotics (inactivated bacteria and supernatant) prepared from Bifidobacterium adolescentis CCFM1302 can reduce the enzymatic activities of MMP-1 and MMP-3 in the skin of aging mice and inhibit the degradation process of collagen.

[0149] Example 9: Effects of Bifidobacterium adolescentis CCFM1302 and its prepared postbiotics on TGF-β content in aging mouse skin

[0150] The animal experiment design, gavage groups and skin homogenate sample preparation used in the following examples are the same as those in Example 6. The content of transforming growth factor-β (TGF-β) in mouse skin was detected by ELISA kit. Figure 8 shown.

[0151] Oral administration of CCFM1302-S can significantly increase the content of TGF-β in mouse skin compared with the model group. The TGF-β content in the model group was 22.45 ng / mg, while the TGF-β content in the CCFM1302-S group was 36.22 ng / mg, which was significantly different from the model group.

[0152] TGF-β is the cytokine most closely associated with collagen metabolism, promoting collagen synthesis by promoting fibroblast proliferation and migration. The TGF-β / Smads pathway is a classic pathway for TGF-β signaling and a key hub in regulating collagen synthesis. Activating this pathway not only directly regulates collagen gene transcription but also enhances collagen accumulation and stability in the extracellular matrix by modulating the gene expression of MMPs.

[0153] In summary, the postbiotics prepared by oral administration of Bifidobacterium adolescentis CCFM1302 can increase the content of TGF-β, thereby regulating the content of collagen.

[0154] Example 10: Effects of Bifidobacterium adolescentis CCFM1302 and its prepared postbiotics on the antioxidant capacity of aging mouse skin

[0155] The animal experiment design, gavage groups, and preparation of skin homogenate samples used in the ELISA kits involved in the following examples are the same as those in Example 6.

[0156] The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) in mouse skin were detected by ELISA kits. Figure 9 shown.

[0157] 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 skin dropped significantly to 7.35 U / mg. After oral administration of CCFM1302-J, it increased to 12.66 U / mg, which can significantly improve SOD enzyme activity.

[0158] Regarding GSH-PX enzyme activity, compared with the control group (25.77 U / μg), the GSH-PX enzyme activity in the model group skin decreased significantly to 12.71 U / μg. Oral administration of CCFM1302-S increased the GSH-PX enzyme activity in the skin to 18.75 U / μg, which was significantly higher than that in the model group.

[0159] Oxidative stress has an important impact on collagen synthesis and stability in skin tissue. SOD can catalyze the oxidation of superoxide anions (O2 - ) is converted into hydrogen peroxide (H2O2). GSH-PX can also effectively remove H2O2 and other reactive oxygen species in the body, thereby effectively preventing the accumulation of reactive oxygen species in cells, reducing the damage to collagen molecules caused by oxidative stress, and thus protecting the collagen structure.

[0160] In summary, the postbiotics (inactivated bacteria and supernatant) prepared from Bifidobacterium adolescentis CCFM1302 can increase the enzyme activities of GSH-PX and SOD in the skin of aging mice, improve the antioxidant capacity of the skin, and increase the content and stability of collagen in the host skin.

[0161] Example 11: Effects of Bifidobacterium adolescentis CCFM1302 and postbiotics prepared therefrom on inflammation levels in aging mice The animal experimental design, gavage groups, and serum sample preparation involved in the following examples are the same as those in Example 6.

[0162] The C-reactive protein (CRP) content in mouse serum was detected by biochemical analyzer, and the interleukin-6 (IL-6) content in mouse skin was detected by ELISA kit. Figure 10 shown.

[0163] Elevated levels of CRP, an acute phase protein, typically reflect worsening systemic inflammation. The inflammatory environment can accelerate collagen degradation and inhibit collagen synthesis. The CRP level in the blank group was 4.58 mg / L, while that in the model group was 5.18 mg / L. Oral administration of CCFM1302-J significantly reduced serum CRP levels to 5.03 mg / L.

[0164] IL-6 is an important pro-inflammatory cytokine that can promote the expression of matrix metalloproteinases (MMPs) by activating signaling pathways, thereby destroying collagen fiber structure. Compared with the blank group (10.54 μg / mg), the IL-6 content in the skin of the model group increased significantly to 24.06 μg / mg. Oral administration of CCFM1302-S significantly reduced the IL-6 content in the skin to 19.44 μg / mg.

[0165] In summary, the postbiotics (inactivated bacteria and supernatant) prepared from Bifidobacterium adolescentis CCFM1302 can alleviate the inflammation of aging mice to varying degrees and affect the anabolism and stability of collagen.

[0166] Example 12: Effects of Bifidobacterium adolescentis CCFM1302 and its prepared postbiotics on the water content of the stratum corneum and skin elasticity of aged mice

[0167] The animal experiment design and gavage groups involved in the following examples are the same as those in Example 6. At the end of the experiment, the skin moisture tester (with stratum corneum moisture measurement probe Corneometer CM825) of German CK Company was used to detect the stratum corneum moisture content and the elasticity of the mouse back skin. The results are as follows: Figure 11 shown.

[0168] The moisture content of the stratum corneum is determined by Figure 11It can be seen that compared with 78.83% in the blank group, the water content in the model group was significantly reduced to 52.87%. The water content in the stratum corneum of the oral CCFM1302-S group (69.25%) increased by 16.38% compared with the model group. The water content in the CCFM1302-J group (74.41%) increased by 21.54% compared with the model group.

[0169] Skin elasticity R2 is determined by Figure 11 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 CCFM1302-S group (69.53%) increased by 15.36% compared with the model group, and the skin elasticity of the CCFM1302-J group (70.50%) increased by 16.33% compared with the model group.

[0170] Collagen is the main component that maintains the skin's structure and elasticity. Its loss can weaken the skin's support and reduce its elasticity, leading to signs of aging such as wrinkles and sagging. At the same time, a decrease in collagen can also affect the skin's ability to retain water, causing it to lose moisture, become dry and rough, and further exacerbate skin aging.

[0171] From the above experimental results, it can be seen that the postbiotics (inactivated bacteria and supernatant) prepared from Bifidobacterium adolescentis CCFM1302 can significantly increase the moisture content and elasticity of aging skin.

[0172] Example 13: MASSON staining analysis of skin tissue sections of aged mice using Bifidobacterium adolescentis CCFM1302 and postbiotics prepared therefrom

[0173] The animal experiment design and gavage groups involved in the following examples are the same as those in Example 6. At the end of the experiment, the mice were killed, and the skin of the shaved back was removed and sent to Wuhan Saiweier Biotechnology Co., Ltd. for MASSON staining and scanning using a slice scanner. The results are as follows: Figure 12 shown.

[0174] It can be clearly seen from the slice results that the collagen fibers in the dermis of the control group mice are tightly and orderly arranged, stained darker blue, and have a higher collagen content. The fiber bundle structure is complete and evenly distributed, with no obvious breakage or degeneration. In addition, the dermis and epidermis of the skin in the control group are tightly connected, showing good skin barrier function and elastic support. The collagen fibers in the dermis of the model group mice were stained significantly lighter, arranged loosely and disorderly, with fiber breakage and faults, and the collagen content was significantly reduced. Compared with the model group, the postbiotics of CCFM1302 significantly improved the collagen content and structure of mouse skin.

[0175] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Use of Bifidobacterium adolescentis CCFM1302, a microbial preparation containing the Bifidobacterium adolescentis CCFM1302, or a postbiotic prepared from the Bifidobacterium adolescentis CCFM1302 in the preparation of anti-aging drugs, characterized in that: The preservation number of the Bifidobacterium adolescentis CCFM1302 is: GDMCC No: 63176.

2. The use according to claim 1, characterized in that The postbiotics include bacterial lysates, inactivated or inactivated cells, fermentation supernatants, or powders prepared by drying any of the above.

3. The use according to claim 2, characterized in that The inactivated or inactivated cells are prepared by the following method: culturing the Bifidobacterium adolescentis CCFM1302 in a culture medium for a period of time, collecting the bacterial cells in the cell culture medium, and obtaining inactivated bacterial cells after heat treatment; the preparation method of the bacterial lysate is as follows: culturing the Bifidobacterium adolescentis CCFM1302 in a culture medium, collecting the bacterial cells, high-pressure homogenizing, and obtaining the bacterial lysate from the supernatant of the centrifugation.

4. The use according to claim 1, characterized in that The application includes at least one of the following functions: (1) Increase the moisture and elasticity of skin tissue in aging individuals; (2) Increase the antioxidant enzyme activity in the skin tissue of aging individuals; (3) reduce inflammation levels in aging individuals; (4) Increase collagen content in skin tissue of aging individuals; (5) Increase the activity of collagen synthase in the skin tissue of aging individuals; (6) Reduce the activity of collagen-degrading enzymes in the skin tissue of aging individuals; (7) Increase the TGF-β content in the skin tissue of aging individuals.

5. The use according to claim 1 or 4, characterized in that Symptoms associated with skin collagen loss include skin appearance, decreased skin collagen content, slower collagen synthesis, and faster collagen degradation.

6. The use according to claim 1 or 4, characterized in that The aging includes skin aging; optionally, the skin aging includes skin dryness, decreased elasticity, sagging, wrinkles, oxidative damage or collagen loss.

7. The use according to claim 4, characterized in that The content of Bifidobacterium adolescentis CCFM1302 in the drug is not less than 5×10 7 CFU / mL; In the medicine, the dosage of the postbiotics prepared by Bifidobacterium adolescentis CCFM1302 is not less than 250 μg / kg.

8. Application of postbiotics prepared from Bifidobacterium adolescentis CCFM1302 in the preparation of products that promote collagen synthesis in skin fibroblasts, characterized in that: The preservation number of the Bifidobacterium adolescentis CCFM1302 is: GDMCC No: 63176.

9. The use according to claim 8, characterized in that The application includes at least one of the following functions: (1) Increase the collagen content secreted by skin fibroblasts in vitro; (2) Increase the activity of collagen synthase in skin fibroblasts in vitro; (3) Inhibit the activity of collagen degrading enzymes in skin fibroblasts in vitro.

10. A method for promoting collagen synthesis in skin fibroblasts in vitro, characterized in that: The method comprises mixing the postbiotics prepared by Bifidobacterium adolescentis CCFM1302 with skin fibroblasts, wherein the preservation number of Bifidobacterium adolescentis CCFM1302 is GDMCC No: 63176.

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