Lactobacillus crispatus for relieving senescence by promoting collagen synthesis and metagen of lactobacillus crispatus

Abstract

The invention discloses lactobacillus crispatus for relieving senescence by promoting collagen synthesis and a metagen of the lactobacillus crispatus, and belongs to the technical field of microorganisms and medicines. The lactobacillus crispatus CCFM1462 metagen provided by the invention has a good skin anti-aging effect, the content of collagen is increased, meanwhile, the skin moisture can be increased, and the skin oxidation resistance can be improved. Specifically, the enzyme activity of MMP-3 and MMP-1 of HSF cells can be inhibited in vitro by thallus lysate, and the content of COL I and COL III and the enzyme activity of LH1 can be improved in vitro from fermentation of supernate; the enzyme activity of MMP-1 and MMP-3 can be inhibited by orally taking inactivated thalli; the self-fermented supernate is orally taken, so that the enzyme activity of LH1 and LOX can be improved, the enzyme activity of MMP-3 can be reduced, and the anabolism of collagen can be regulated and controlled.

Description

Technical Field

[0001] The invention relates to a Lactobacillus crispatus strain capable of alleviating aging by promoting collagen synthesis and a postbiotic thereof, and belongs to the technical field of microorganisms and medicine. Background Art

[0002] The skin is the largest organ in the human body. It not only serves as a barrier between the body and the external environment but also plays a vital role in maintaining appearance, protecting internal tissues, and regulating body temperature. With aging, the skin gradually shows signs of aging, primarily manifested by decreased skin elasticity, increased wrinkling, sagging, and dull skin tone. These phenomena are closely related to a decrease in collagen content in the skin. Collagen is the primary structural component of the dermis, accounting for approximately 70%-80% of the dermis' dry weight. It provides structural support and elasticity to the skin. However, with aging, the body's ability to synthesize collagen decreases. Furthermore, external factors such as ultraviolet light, oxidative stress, and environmental pollution accelerate collagen degradation, impairing the skin's structural integrity and accelerating the aging process.

[0003] To alleviate skin aging and increase collagen content, relevant technological inventions have emerged in recent years in the field of internal and external use products. For example, patent CN118120927A discloses an oral solution of small-molecule collagen peptides for skin aging, its preparation method, and its use. Using low-molecular-weight collagen peptides as the main ingredient, combined with vitamin C and other ingredients, oral supplementation of collagen raw materials promotes collagen synthesis in the body and combats skin aging. Regarding oral collagen supplementation, although some small-molecule peptides can be absorbed through the digestive tract and enter the blood circulation, the absorption process still faces issues such as degradation by digestive enzymes, inefficient gastrointestinal barrier transport, and rapid clearance from plasma, resulting in a low rate of effective delivery to target tissues. Even if successfully absorbed, they lack sufficient tissue targeting, making it difficult to accurately enrich in the skin's dermis. Furthermore, an individual's own collagen synthesis capacity is significantly affected by factors such as age, hormone levels, and nutritional status. Oral supplementation cannot directly guarantee a significant increase in the rate of collagen regeneration. Therefore, existing strategies still need further optimization and improvement in terms of effective delivery, ingredient stability, bioavailability, and targeted regulation. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a Lactobacillus crispatus CCFM1462 and its postbiotics for use in preparing products that increase collagen content and alleviate aging.

[0005] The first technical solution provided by the present invention is a strain of Lactobacillus crispatus CCFM1462, which has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on January 10, 2025, with the deposit number: GDMCCNo: 65751.

[0006] The Lactobacillus crispatus CCFM1462 was derived from reproductive tract 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 Lactobacillus crispatus, and was named Lactobacillus crispatus CCFM1462.

[0007] The colonies of the Lactobacillus crispatus CCFM1462 on the MRS solid culture medium are white flakes with a protruding center and irregular edges.

[0008] The second technical solution provided by the present invention is a microbial preparation containing the Lactobacillus crispatus CCFM1462 described in the first technical solution.

[0009] In certain embodiments, the concentration of Lactobacillus crispatus CCFM1462 in the microbial preparation is not less than 1×10 6 CFU / mL or ×10 6 CFU / g.

[0010] The third technical solution provided by the present invention is a postbiotic, which is prepared using the Lactobacillus crispatus CCFM1462 described in the first technical solution or the microbial preparation described in the second technical solution.

[0011] In certain embodiments, the postbiotics include bacterial lysate, inactivated or inactivated cells, fermentation supernatant, or a powder prepared by drying any of the above.

[0012] In certain embodiments, the inactivated or inactivated cells are prepared as follows: after culturing the Lactobacillus crispatus CCFM1462 in a culture medium, bacterial cells in the cell culture medium are collected, and the cells are heat-treated to obtain inactivated bacterial cells.

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

[0014] In certain embodiments, the bacterial lysate is prepared by culturing the Lactobacillus crispatus CCFM1462 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.

[0015] In certain embodiments, the fermentation supernatant is the supernatant obtained by centrifuging the Lactobacillus crispatus CCFM1462 after culturing the Lactobacillus crispatus CCFM1462 in a culture medium.

[0016] The present invention provides a fourth technical solution, which is a product containing the Lactobacillus crispatus CCFM1462 described in the first technical solution and / or the postbiotics described in the third technical solution.

[0017] In certain embodiments, the products include but are not limited to food, medicine, health care products or daily chemical products.

[0018] In certain embodiments, the food comprises the above composition and conventional auxiliary materials.

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

[0020] In certain embodiments, the health care product comprises the above composition and conventional excipients.

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

[0022] In certain embodiments, the dosage form of the product includes at least one of a cream, an emulsion, an oil, an aqueous solution, a gel, a powder, and a freeze-dried formulation.

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

[0024] In certain embodiments, the bacterial powder is a solid powder postbiotic prepared by drying the prepared liquid postbiotic of Lactobacillus crispatus CCFM1462.

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

[0026] The fifth technical solution provided by the present invention is the use of the postbiotics described in the third technical solution in the preparation of a product that promotes the synthesis of collagen in fibroblasts.

[0027] In certain embodiments, the product comprises at least one of the following effects:

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

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

[0030] (3) inhibiting collagen-degrading enzyme activity in skin fibroblasts in vitro.

[0031] The sixth aspect of the present application provides use of the Lactobacillus crispatus CCFM1462 of the first aspect, the microbial preparation of the second aspect, or the postbiotic of the third aspect in the preparation of a product for alleviating aging.

[0032] In some embodiments, the use comprises at least one of the following effects:

[0033] (1) increasing moisture and elasticity of skin tissue of an aging individual;

[0034] (2) increasing antioxidant enzyme activity of skin tissue of an aging individual;

[0035] (3) reducing inflammation level of an aging individual;

[0036] (4) increasing collagen content of skin tissue of an aging individual;

[0037] (5) increasing collagen-synthesizing enzyme activity of skin tissue of an aging individual;

[0038] (6) reducing collagen-degrading enzyme activity of skin tissue of an aging individual;

[0039] (7) increasing TGF-β content of skin tissue of an aging individual.

[0040] In some embodiments, the skin collagen loss-related symptoms include skin appearance, reduced collagen content, slowed collagen synthesis, and accelerated collagen degradation.

[0041] In some embodiments, the aging comprises skin aging.

[0042] In some embodiments, the skin aging comprises dry skin, reduced elasticity, sagging, wrinkle formation, oxidative damage, or collagen loss.

[0043] In some embodiments, the product contains the Lactobacillus crispatus CCFM1462 in an amount of not less than 5 x 10 7 CFU / mL.

[0044] In some embodiments, the product contains the postbiotic prepared from the Lactobacillus crispatus CCFM1462 in an amount of not less than 250 μg / kg.

[0045] In some embodiments, the product is a pharmaceutical product or a cosmetic product.

[0046] In some embodiments, the pharmaceutical product comprises the Lactobacillus crispatus CCFM1462, a pharmaceutical carrier, and / or a pharmaceutical excipient.

[0047] In certain embodiments, the pharmaceutical excipients include excipients and additives.

[0048] In certain embodiments, the pharmaceutical excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, and release retardants.

[0049] In certain embodiments, the cosmetic contains Lactobacillus crispatus CCFM1462, matrix raw materials and / or conventional excipients.

[0050] In certain embodiments, the matrix raw materials include oil raw materials, wax raw materials, synthetic oil raw materials, powdery raw materials, colloid raw materials, coagulants, and surfactants.

[0051] In certain embodiments, the conventional excipients include one or more of moisturizers, whitening agents, flavoring agents, adhesives, lubricants, preservatives, film agents, antioxidants, emulsifiers, and cosmetic nutritional additives.

[0052] The seventh technical solution provided by the present invention is a method for promoting the anabolism of collagen in skin fibroblasts in vitro, wherein the method comprises mixing the postbiotics described in the third technical solution with skin fibroblasts and incubating them.

[0053] Compared with the prior art, the present invention has the following beneficial effects:

[0054] The Lactobacillus crispatus CCFM1462 of the present invention and the postbiotics prepared therefrom have the ability to increase collagen content and alleviate aging, which is specifically reflected in:

[0055] (1) Increase the content of type I and type III collagen (COLⅠ, Ⅲ) secreted by human skin fibroblasts (HSF) in vitro;

[0056] (2) Increase LH1 enzyme activity in human skin fibroblasts (HSF) in vitro;

[0057] (3) Reduce the enzyme activities of MMP-1 and MMP-3 in human skin fibroblasts (HSF) in vitro

[0058] (4) Improve skin moisture and elasticity in aging individuals;

[0059] (5) Increase CAT enzyme activity in the skin of aging individuals;

[0060] (6) Reduce the level of CRP in the serum of aging individuals;

[0061] (7) Increase the levels of COL I, III, and IV in the skin of aging individuals;

[0062] (8) Increase the enzyme activities of LH1 and LOX in the skin of aging individuals;

[0063] (9) Reduce the enzyme activities of MMP-1 and MMP-3 in the skin of aging individuals;

[0064] (10) Increase the content of TGF-β in the skin of aging individuals.

[0065] Therefore, postbiotics prepared from Lactobacillus crispatus CCFM1462 have great application prospects in products that increase collagen content and alleviate aging.

[0066] Biomaterial Deposit

[0067] Lactobacillus crispatus CCFM1462, taxonomically named Lactobacillus crispatus, was deposited in Guangdong Provincial Microbiological Culture Collection on January 10, 2025, with the deposit number GDMCCNo: 65751, and the deposit address is Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 Effects of postbiotics prepared for CCFM1462 on the secretion of COLⅠ and COLⅢ by HSF cells;

[0069] Figure 2 Effects of postbiotics prepared for CCFM1462 on LH1 enzyme activity in HSF cells;

[0070] Figure 3 Effects of postbiotics prepared for CCFM1462 on MMP-1 and MMP-3 enzyme activities in HSF cells;

[0071] Figure 4 This is the flow chart of the mouse experiment;

[0072] Figure 5 Effects of postbiotics prepared for CCFM1462 on the levels of COLⅠ, Ⅲ, and Ⅳ in mouse skin tissue;

[0073] Figure 6 Effects of postbiotics prepared for CCFM1462 on LH1 and LOX enzyme activities in mouse skin tissue;

[0074] Figure 7 Effects of postbiotics prepared for CCFM1462 on MMP-1 and MMP-3 enzyme activities in mouse skin tissue;

[0075] Figure 8 Effects of postbiotics prepared for CCFM1462 on TGF-β content in mouse skin tissue;

[0076] Figure 9 Effects of postbiotics prepared for CCFM1462 on CAT enzyme activity in mouse skin tissue;

[0077] Figure 10 Effects of postbiotics prepared for CCFM1462 on CRP levels in mouse serum;

[0078] Figure 11 Effects of postbiotics prepared for CCFM1462 on skin hydration and elasticity in mice;

[0079] Figure 12 Masson-stained skin sections.

[0080] “#” indicates statistical difference compared with the control group (P<0.05), “##” indicates significant statistical difference compared with the control group (P<0.01), and “###” indicates extremely significant statistical difference compared with the control group (P<0.001).

[0081] “*” indicates statistical difference compared with the model group (P<0.05), “**” indicates significant statistical difference compared with the model group (P<0.01), “***” indicates extremely significant statistical difference compared with the model group (P<0.001); “****” indicates extremely significant statistical difference compared with the model group (P<0.0001). DETAILED DESCRIPTION

[0082] The following describes preferred embodiments of the present invention. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0083] The raw materials used in the embodiment are:

[0084] The present invention will be further described below with reference to specific embodiments.

[0085] The human skin fibroblasts (HSF) involved in the following examples were purchased from Kunming Cell Bank.

[0086] The BALB / c mice involved in the following examples were purchased from Weitonglihua Company.

[0087] The Lactobacillus crispatus CCFM1462 involved in the following examples is a strain screened by the Food Biotechnology Center of Jiangnan University.

[0088] The ELISA kits involved in the following examples were purchased from Nanjing Senbeijia Biotechnology Co., Ltd.

[0089] The D-galactose involved in the following examples was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0090] The BCA protein concentration determination kit involved in the following examples was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0091] The culture medium involved in the following examples is as follows:

[0092] Modified MRS liquid 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, and Tween-80 1 mL / L, pH 6.2-6.4.

[0093] 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, and agar 20.0 g / L, pH 6.2-6.4.

[0094] 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).

[0095] Example 1: Cell recovery and culture

[0096] 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.

[0097] Example 2: Preparation of postbiotics by Lactobacillus crispatus CCFM1462

[0098] 1. Screening and identification of Lactobacillus crispatus CCFM1462

[0099] The strain sample screening was from vaginal samples of 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 medium. It was cultured at 37°C for 48 hours, and the typical colonies of Lactobacillus crispatus were picked and streaked on MRS solid medium for purification. Single colonies were picked and transferred to MRS liquid 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 Lactobacillus crispatus by NCBI sequence alignment and named Lactobacillus crispatus CCFM1462. It was deposited in Guangdong Provincial Microbiological Culture Collection on January 10, 2025, with the deposit number GDMCC No: 65751.

[0100] 2. Preparation of postbiotics using Lactobacillus crispatus CCFM1462

[0101] (1) Lactobacillus crispatus CCFM1462 was streaked out of the preservation tube and cultured in a water-tight incubator at 37°C for 48 hours using a modified MRS solid medium to obtain a single colony; a single colony was selected and inoculated into a modified MRS liquid medium and cultured at 37°C for 12-18 hours to obtain culture fluid 1;

[0102] The culture solution 1 was inoculated into the modified MRS liquid medium at an inoculum volume of 2% (v / v), and cultured at 37°C for 12 h to obtain the seed solution;

[0103] The seed solution was inoculated into a modified MRS liquid medium at 2% (v / v) for expansion and cultured at 37° C. for 18 h. The number of viable bacteria was recorded to obtain bacterial solution a.

[0104] 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., 30 min), and freeze-dried for standby use to prepare a freeze-dried powder of the fermentation supernatant of Lactobacillus crispatus CCFM1462 (denoted as CCFM1462-S). The bacterial mud was resuspended in double-distilled water with 75% volume of the original bacterial solution, the resuspension was heat-treated (65° C., 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 CCFM1462-J).

[0105] The postbiotics of Lactobacillus crispatus CCFM1462 (bacterial lysate CCFM1462-J and fermentation supernatant CCFM1462-S) were prepared by the above means.

[0106] Example 3: Effect of postbiotics prepared by Lactobacillus crispatus CCFM1462 on collagen content in HSF cells

[0107] (1) HSF cells in the logarithmic growth phase were taken at 1×10 5 Cells were seeded with 100 μg / mL in 6-well plates and cultured overnight until they adhered. The old culture medium was discarded and the cells were rinsed three times with PBS. A blank group and a postbiotic-treated group were set up.

[0108] Blank group (control): contains cell culture medium and HSF cells, but no postbiotics;

[0109] Postbiotic treatment group: contains cell culture medium and HSF cells, as well as postbiotics.

[0110] Resuspend the postbiotics in cell culture medium (the amount of postbiotics after resuspension should be the same as that of fermentation to a concentration of 5.0×10 7 CFU / ml of bacterial solution), and 100 μL of postbiotics prepared from Lactobacillus crispatus CCFM1462 was added respectively.

[0111] (2) Incubate the above-mentioned well plates in an incubator at 37°C for 24 hours.

[0112] (3) After incubation, the cell culture supernatant was collected and centrifuged at 1000 rpm for 5 min. The COLⅠ and COLⅢ contents were detected using ELISA kits.

[0113] (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 seconds. Observe the cells under a microscope until most of the cells become round, then add 4 mL of DMEM complete medium to terminate 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 rpm for 5 minutes, remove the supernatant, resuspend the cells with a mixed protease inhibitor, and repeatedly freeze-thaw (-80°C quick freezing and 4°C slow thawing) three times to lyse the cells to obtain a cell lysate sample.

[0114] (5) Use the BCA protein concentration assay kit to detect the total protein concentration of the sample in (4) and calibrate the above data.

[0115] The results are as follows Figure 1As shown, compared with the control group, CCFM1462-S can increase the content of COL I secreted by HSF cells by about 29%, and CCFM1462-S can significantly increase the content of COL III secreted by HSF cells by about 21%. The results show that the postbiotic prepared by CCFM1462 can increase the content of collagen secreted by HSF cells, and has the potential to regulate collagen synthesis and metabolism.

[0116] Example 4: Effect of postbiotic prepared by Lactobacillus crispatus CCFM1462 on collagen synthesis enzyme activity of HSF cells

[0117] The cell lysate samples involved in the following examples are prepared in the same way as in Example 3. The enzyme activity of lysine hydroxylase (LH1) in the HSF cell lysate is detected by an ELISA kit.

[0118] LH1 plays an important role in collagen synthesis and is one of the key enzymes in the post-modification process of collagen. LH1 is responsible for catalyzing the hydroxylation of lysine residues to generate hydroxylysine, which is crucial for the stability and function of collagen. Hydroxylated lysine not only helps to cross-link collagen fibers, enhancing their mechanical strength, but also participates in the interaction of collagen with other extracellular matrix components, thereby playing an important role in maintaining tissue structure and function. The results of LH1 are as follows Figure 2 As shown, compared with the control group (81.48 IU / g), the enzyme activity of LH1 in HSF cells treated with CCFM1462-S can be increased to 102.96 IU / g. The results show that the postbiotic prepared by CCFM1462 has the potential to regulate collagen synthesis in the body.

[0119] Example 5: Effect of postbiotic prepared by Lactobacillus crispatus CCFM1462 on collagen degradation enzyme activity of HSF cells

[0120] The cell culture supernatant samples involved in the following examples are prepared in the same way as in Example 3. The enzyme activity of matrix metalloproteinases 1, 3 (MMP-1, 3) in the HSF cell culture supernatant is detected by an ELISA kit.

[0121] MMP-1 is a key enzyme for collagen degradation, and its substrates include type I and type III collagen, with cleavage sites usually located between Gly-Ile or Gly-Leu bonds, which can cut the intact collagen triple helix structure to form fragments of different lengths, which are more easily degraded by other MMPs. The results of MMP-1 enzyme activity are as follows Figure 3 As shown, compared with the control group, CCFM1462-J can reduce the enzyme activity of MMP-1 by about 27%.

[0122] 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 in the figure, compared with the control group, CCFM1462-J could significantly reduce the enzyme activity of MMP-3, down-regulating it by about 27%.

[0123] The results showed that postbiotics prepared by CCFM1462 have the potential to regulate collagen degradation in the body.

[0124] Example 6: Effect of postbiotics prepared from Lactobacillus crispatus CCFM1462 on collagen content in the skin of aging mice

[0125] The preparation method of the postbiotics (CCFM1462-S and CCFM1462-J) of Lactobacillus crispatus CCFM1462 involved in the following examples is the same as that in Example 2, except that CCFM1462-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 a bacterial mud, and the bacterial mud is resuspended with double distilled water containing 75% of the volume of the original bacterial liquid, and the resuspension is heat-treated at 65° C. for 30 min without high-pressure homogenization.

[0126] (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 CCFM1462-S group, and 1 cage for the CCFM1462-J group, respectively.

[0127] Blank group (control): physiological saline was used as a control;

[0128] Model group: normal saline was used as the control;

[0129] CCFM1462-S group: Lactobacillus crispatus CCFM1462 metabolite (fermentation supernatant) was used at a dose of 500 mg / kg mouse body weight;

[0130] CCFM1462-J group: Lactobacillus crispatus CCFM1462 postbiotics (inactivated bacteria) were used at a dose of 500 mg / kg mouse body weight;

[0131] 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.

[0132] 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.

[0133] 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.

[0134] The contents of type I, III, and IV collagen (COLⅠ, Ⅲ, and Ⅳ) in mouse skin were detected by ELISA kit. Figure 5 shown.

[0135] 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. Oral administration of CCFM1462-S and CCFM1462-J groups significantly increased the content of COLⅠ compared with the model group, restoring the content of COLⅠ in the skin to 5.21 μg / mg and 5.17 μg / mg, respectively. Both groups can increase the content of COLⅠ.

[0136] 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). Only oral administration of CCFM1462-J could significantly increase its content to 1.33μg / mg compared with the model group, and inactivated bacteria could increase the content of COLⅢ.

[0137] For COLⅣ, the content in the blank group was 15.29μg / mg, and the content in the model group was 6.21μg / mg. Only the oral CCFM1462-S group could significantly increase the content of COLⅣ compared with the model group, and this group restored the content to 11.98μg / mg.

[0138] The above results show that the postbiotics (inactivated bacteria and supernatant) prepared from Lactobacillus crispatus CCFM1462 can increase the collagen content in the skin of aging mice.

[0139] Example 7: Effects of Lactobacillus crispatus CCFM1462 and its postbiotics on collagen synthase activity in aging mouse skin

[0140] 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.

[0141] LH1 can catalyze the hydroxylation of lysine residues, thereby providing sites for glycosylation and affecting the assembly and cross-linking of collagen fibers; LOX can catalyze the oxidative deamination reaction of lysine and hydroxylysine residues to form aldehyde derivatives, which condense with adjacent lysine amino groups or hydroxyl groups of hydroxylysine to form covalent cross-links, thereby stabilizing the structure of the fibrils. Both are key catalytic enzymes in the process of collagen synthesis. The enzymatic activities of LH1 and lysyl oxidase (LOX) in mouse skin were detected by ELISA kit. The results are as follows: Figure 6 shown.

[0142] Testing of LH1 and LOX enzyme activities revealed that both LH1 and LOX activities were significantly decreased in the model group compared to the blank group. Oral administration of CCFM1462-J significantly increased LH1 activity to 14.81 U / mg, compared to 8.58 U / mg in the model group. Oral administration of CCFM1462-S significantly increased LOX activity to 13.14 U / mg, compared to 8.48 U / mg in the model group.

[0143] The above results show that the postbiotics (inactivated bacteria and supernatant) prepared from Lactobacillus crispatus CCFM1462 can increase the enzyme activities of LH1 and LOX in the skin of aging mice and promote the synthesis of collagen.

[0144] Example 8: Effects of Lactobacillus crispatus CCFM1462 and its prepared postbiotics on the activity of collagen degrading enzymes in the skin of aging mice

[0145] 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.

[0146] The enzyme activities of MMP-1 and MMP-3 in mouse skin were detected by ELISA kit. Figure 7 shown.

[0147] Oral administration of CCFM1462-J significantly downregulated MMP-1 activity compared to the model group (9.94 IU / mg in the model group and 7.98 IU / mg in the CCFM1462-J group, respectively), showing significant differences compared to the model group. Oral administration of CCFM1462-S, on the other hand, failed to downregulate MMP-1 activity, with an activity of 9.20 IU / mg.

[0148] 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 CCFM1462-J and CCFM1462-S significantly decreased MMP-3 activity in the skin to 3.52 IU / mg and 3.63 IU / mg, respectively.

[0149] The above results show that the postbiotics (inactivated bacteria and supernatant) prepared from Lactobacillus crispatus CCFM1462 can reduce the enzymatic activity of MMP-1 and MMP-3 in the skin of aging mice and inhibit the degradation of collagen, among which CCFM1462-J has a more significant effect.

[0150] Example 9: Effects of Lactobacillus crispatus CCFM1462 and its postbiotics on TGF-β content in the skin of aging mice

[0151] 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.

[0152] Oral administration of CCFM1462-S significantly increased TGF-β levels in mouse skin compared to the model group. The TGF-β levels in the model group were 22.45 ng / mg, while those in the CCFM1462-S group were 41.62 ng / mg, a significant difference from the model group. However, oral administration of CCFM1462-J, at 34.07 ng / mg, failed to significantly increase TGF-β levels in the skin of aging mice.

[0153] 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.

[0154] In summary, oral administration of CCFM1462-S can better increase the content of TGF-β, thereby regulating the content of collagen.

[0155] Example 10: Effects of Lactobacillus crispatus CCFM1462 and its prepared postbiotics on the antioxidant capacity of aging mouse skin

[0156] 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.

[0157] The activity of catalase (CAT) in mouse skin was detected by ELISA kit. Figure 9 shown.

[0158] Compared with the control group (37.38 U / μg), the CAT enzyme activity in the model group decreased significantly to 16.32 U / μg. Oral administration of CCFM1462-S and CCFM1462-J increased the CAT enzyme activity in the skin to 23.84 U / μg and 29.49 U / μg, respectively, which was significantly higher than that in the model group.

[0159] CAT enzyme activity plays a key role in skin health. It reduces oxidative stress by scavenging hydrogen peroxide, thereby protecting fibroblasts and promoting collagen synthesis. With aging, CAT enzyme activity decreases, leading to increased oxidative stress, inhibiting collagen synthesis, and accelerating collagen degradation, ultimately accelerating skin aging, manifested by decreased elasticity and wrinkle formation. Therefore, maintaining or increasing CAT enzyme activity can help reduce oxidative stress, protect collagen, and slow the aging process.

[0160] In summary, the postbiotics (inactivated bacteria and supernatant) prepared from Lactobacillus crispatus CCFM1462 can increase the CAT enzyme activity in the skin of aging mice, improve the antioxidant capacity of the skin, and delay skin aging.

[0161] Example 11: Effects of Lactobacillus crispatus CCFM1462 and its prepared postbiotics on CRP levels in serum of aging mice

[0162] The animal experimental design, gavage groups and serum sample preparation involved in the following examples are the same as those in Example 6.

[0163] The C-reactive protein (CRP) content in mouse serum was detected by biochemical analyzer. Figure 10 shown.

[0164] CRP is a sensitive and nonspecific indicator of inflammation. It is also an inflammation-related aging marker and is closely related to skin aging. The CRP content in the blank group was 4.58 mg / L, and the CRP content in the model group was 5.18 mg / L. Oral administration of CCFM1462-J significantly reduced the CRP content in serum (4.73 mg / L), while after oral administration of CCFM1462-S, the CRP content in serum was 5.00 mg / L, which was not significantly different from the model group.

[0165] In summary, oral administration of CCFM1462-J can alleviate the inflammation and aging conditions of aging mice to varying degrees, and has better anti-inflammatory and anti-aging effects than CCFM1462-S.

[0166] Example 12: Effects of Lactobacillus crispatus CCFM1462 and its 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 11 It 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 CCFM1462-S group (74.15%) increased by 21.28% compared with the model group. The water content in the CCFM1462-J group (69.46%) increased by 16.59% 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 CCFM1462-S group (68.27%) increased by 14.10% compared with the model group, and the skin elasticity of the CCFM1462-J group (78.47%) increased by 24.30% 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 Lactobacillus crispatus CCFM1462 can significantly increase the moisture content of aging skin, among which its inactivated bacteria (CCFM1462-J) can also increase the elasticity of the back skin of aging mice, thereby slowing down skin aging.

[0172] Example 13: MASSON staining analysis of skin tissue sections of aged mice using Lactobacillus crispatus CCFM1462 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 for MASSON staining. 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 CCFM1462 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. A strain of Lactobacillus crispatus CCFM1462, characterized in that: The Lactobacillus crispatus was deposited in the Guangdong Provincial Microbial Culture Collection Center on January 10, 2025, with the deposit number: GDMCC No: 65751.

2. A microbial preparation containing the Lactobacillus crispatus CCFM1462 according to claim 1.

3. A postbiotic, characterized in that: The postbiotics are prepared using the Lactobacillus crispatus CCFM1462 described in claim 1 or the microbial preparation described in claim 2.

4. The postbiotic according to claim 3, characterized in that The postbiotics include bacterial lysates, inactivated or inactivated cells, fermentation supernatants, or powders prepared by drying any of the above.

5. The postbiotic according to claim 4, characterized in that The inactivated or inactivated cells are prepared as follows: after culturing the Lactobacillus crispatus CCFM1462 in a culture medium, collecting bacterial cells in the cell culture solution, and subjecting the culture medium to heat treatment to obtain inactivated bacterial cells; The preparation method of the bacterial lysate comprises: culturing the Lactobacillus crispatus CCFM1462 in a culture medium, collecting bacterial cells, homogenizing under high pressure, and obtaining the bacterial lysate from the supernatant of the centrifugation; The fermentation supernatant is the supernatant obtained by centrifuging the Lactobacillus crispatus CCFM1462 in a culture medium.

6. A product containing the Lactobacillus crispatus CCFM1462 of claim 1 and / or the postbiotic of any one of claims 3 to 5, characterized in that: The products include food, medicine, health products or daily chemical products.

7. Use of the postbiotic according to any one of claims 3 to 5 in the preparation of a product for promoting collagen synthesis in fibroblasts, characterized in that: The product includes at least one of the following effects: (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.

8. Use of the Lactobacillus crispatus CCFM1462 according to claim 1, the microbial preparation according to claim 2, or the postbiotic according to any one of claims 3 to 5 in the preparation of a product that alleviates aging.

9. The use according to claim 8, 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.

10. A method for promoting collagen anabolism in skin fibroblasts in vitro, characterized in that: The method comprises mixing the postbiotic according to any one of claims 3 to 5 with skin fibroblasts and incubating the mixture.

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