Application of lactobacillus helveticus CCFM1440 in regulating and controlling anabolism of host collagen to relieve senescence

Abstract

The invention discloses application of lactobacillus helveticus CCFM1440 in regulating and controlling anabolism of host collagen to relieve senescence, and belongs to the technical field of microorganisms and the technical field of medicines. The metagen of the lactobacillus helveticus CCFM1440 provided by the invention has a good skin anti-aging effect, and can improve the moisture and elasticity of the skin, improve the antioxidant capacity of the skin, reduce the inflammation level of the organism and relieve aging. Specifically, the enzyme activity of LH1 and LOX of HSF cells and the content of COLIII are improved in vitro and in vitro through thallus lysate; the inactivated thalli are orally taken, so that the enzyme activity of LH1, P4H and LOX is improved, the enzyme activity of MMP-1 and MMP-3 is inhibited, and the collagen content is increased; the self-fermented supernate is orally taken, so that the enzyme activity of MMP-3 can be inhibited, the content of IL-6 and CRP can be reduced, and the skin moisture can be improved.

Description

Technical Field

[0001] The present invention relates to application of Lactobacillus helveticus CCFM1440 in regulating host collagen synthesis metabolism and alleviating aging, and belongs to the fields of microbial technology and medical technology. Background Art

[0002] As the largest organ in the human body, the skin undertakes multiple critical functions, including barrier protection, maintaining appearance, maintaining internal stability, and regulating body temperature. With aging and the long-term effects of adverse external factors, skin inevitably undergoes signs of aging, manifested by decreased elasticity, deeper wrinkles, sagging, and a dull, dull complexion. These changes are closely related to a decrease in collagen content in the skin. Collagen is the primary supporting protein in the dermis, accounting for approximately 70%-80% of the dermis' dry weight. It plays a vital role in maintaining the skin's structural stability and imparting its elasticity and toughness. With aging, the rate of new collagen synthesis gradually decreases. At the same time, external stimuli such as ultraviolet radiation, oxidative stress, free radical damage, and environmental pollution accelerate collagen degradation, leading to a loosening of the dermal structure, a decline in barrier function, and a significant acceleration of the skin aging process. Therefore, maintaining a stable collagen content and slowing its degradation rate have become important strategies for delaying skin aging and improving skin quality.

[0003] In order to alleviate skin aging and increase collagen content, relevant technical inventions have been made in the field of internal and external products in recent years. For example, patent CN111557882A discloses a composition and its application in the preparation of anti-aging skin care products. The composition includes sodium hyaluronate, vitamin C derivatives, peptide components and plant extracts, etc., which stimulate collagen synthesis by external application, improve skin elasticity, and slow down signs of aging. Although the above-mentioned technology has shown certain potential in promoting collagen content and alleviating skin aging, there are still many shortcomings that need to be improved. First, the macromolecular components (such as hyaluronic acid and certain peptides) in the external composition have poor permeability and are difficult to penetrate the skin surface to reach the dermis, which limits its effect of stimulating collagen synthesis. Secondly, the stability of plant extracts and vitamin components is poor, and they are easily affected by environmental factors such as light, temperature and oxidation and degrade and lose their effectiveness. In addition, there are differences in the reactions of different individuals to these components, and some people may experience irritation or allergic reactions due to sensitive skin. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a Lactobacillus helveticus CCFM1440 and its postbiotics for use in preparing a product that regulates host collagen anabolism and alleviates aging.

[0005] The first technical solution provided by the present invention is a strain of Lactobacillus helveticus CCFM1440, which has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 12, 2024, with the deposit number: GDMCCNo: 65124.

[0006] The Lactobacillus helveticus CCFM1440 is derived from fermented dairy products. 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 helveticus, and was named Lactobacillus helveticus CCFM1440.

[0007] The colonies of the Lactobacillus helveticus CCFM1440 on the MRS solid culture medium are smooth, white, and have small round protrusions.

[0008] The second technical solution provided by the present invention is a microbial preparation containing Lactobacillus helveticus CCFM1440 described in the first technical solution.

[0009] In certain embodiments, the concentration of Lactobacillus helveticus CCFM1440 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 prepared using the Lactobacillus helveticus CCFM1440 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 helveticus CCFM1440 in a culture medium, bacterial cells in the cell culture medium are collected, and the inactivated bacterial cells are obtained after heat treatment.

[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 helveticus CCFM1440 in a culture medium, 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 culturing the Lactobacillus helveticus CCFM1440 in a culture medium for a period of time and then centrifuging the culture medium.

[0016] The fourth technical solution provided by the present invention is a product, which contains the Lactobacillus helveticus CCFM1440 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 Lactobacillus helveticus CCFM1440 liquid postbiotic.

[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 Lactobacillus helveticus CCFM1440 described in the first technical solution, the microbial preparation described in the second technical solution, or the postbiotic described in the third technical solution in the preparation of products that alleviate aging.

[0027] In certain embodiments, the application comprises at least one of the following actions:

[0028] (1) Increase the moisture and elasticity of skin tissue in aging individuals;

[0029] (2) Increase the antioxidant enzyme activity in the skin tissue of aging individuals;

[0030] (3) reduce inflammation levels in aging individuals;

[0031] (4) Increase collagen content in skin tissue of aging individuals;

[0032] (5) Increase the activity of collagen synthase in the skin tissue of aging individuals;

[0033] (6) Reduce the activity of collagen-degrading enzymes in the skin tissue of aging individuals;

[0034] (7) Increase the TGF-β content in the skin tissue of aging individuals.

[0035] In certain embodiments, the symptoms associated with skin collagen loss include skin appearance, decreased skin collagen content, slowed collagen synthesis, and accelerated collagen degradation.

[0036] In certain embodiments, the aging comprises skin aging.

[0037] In certain embodiments, the skin aging includes skin dryness, decreased elasticity, sagging, wrinkling, oxidative damage, or collagen loss.

[0038] In certain embodiments, the content of Lactobacillus helveticus CCFM1440 in the product is not less than 5×10 7 CFU / mL.

[0039] In certain embodiments, the dosage of the postbiotics prepared by Lactobacillus helveticus CCFM1440 in the product is not less than 250 μg / kg.

[0040] In certain embodiments, the product is a pharmaceutical or cosmetic product.

[0041] In certain embodiments, the drug comprises the Lactobacillus helveticus CCFM1440, a drug carrier and / or a pharmaceutical excipient.

[0042] In certain embodiments, the pharmaceutical excipients include excipients and additives.

[0043] 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.

[0044] In certain embodiments, the cosmetic contains Lactobacillus helveticus CCFM1440, matrix raw materials and / or conventional excipients.

[0045] 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.

[0046] 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.

[0047] The sixth technical solution provided by the present invention is a method for regulating 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.

[0048] The seventh 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 for enhancing collagen synthesis in fibroblasts.

[0049] In certain embodiments, the application includes at least one of the following effects:

[0050] (1) Increase the content of type III collagen (COLⅢ) secreted by skin fibroblasts;

[0051] (2) Increase the enzyme activity of collagen synthase LH1 and LOX in skin fibroblasts.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] The Lactobacillus helveticus CCFM1440 of the present invention and the postbiotics prepared therefrom have the ability to regulate host collagen synthesis and metabolism and alleviate aging, which is specifically reflected in:

[0054] (1) Increase the content of type III collagen (COLⅢ) secreted by human skin fibroblasts (HSF) in vitro and increase the enzymatic activities of collagen synthases LH1 and LOX;

[0055] (2) Improve skin moisture and elasticity in aging individuals;

[0056] (3) Improve the enzyme activities of SOD, GSH-PX, and CAT in the skin of aging individuals;

[0057] (4) reduce the levels of CRP, IL-6, and TNF-α in the serum of aging individuals;

[0058] (5) Increase the levels of COLⅠ, III, IV, and VII in the skin of aging individuals;

[0059] (6) Increase the enzyme activities of LH1, P4H and LOX in the skin of aging individuals;

[0060] (7) Reduce the enzyme activities of MMP-1 and MMP-3 in the skin of aging individuals;

[0061] (8) Increase the content of TGF-β in the skin of aging individuals.

[0062] Therefore, postbiotics prepared from Lactobacillus helveticus CCFM1440 have great application prospects in products that regulate host collagen synthesis metabolism and alleviate aging.

[0063] Biomaterial Deposit

[0064] Lactobacillus helveticus CCFM1440, taxonomically named Lactobacillus helveticus, was deposited in the Guangdong Provincial Microbiological Culture Collection on September 12, 2024, with the deposit number GDMCC No: 65124, and the deposit address is Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 Effects of postbiotics prepared for CCFM1440 on the COLⅢ content, LH1 and LOX enzyme activities secreted by HSF cells;

[0066] Figure 2 This is the flow chart of the mouse experiment;

[0067] Figure 3 Effects of postbiotics prepared for CCFM1440 on the levels of COLⅠ, Ⅲ, Ⅳ and Ⅶ in mouse skin tissue;

[0068] Figure 4 Effects of postbiotics prepared for CCFM1440 on LH1, P4H, and LOX enzyme activities in mouse skin tissue;

[0069] Figure 5 Effects of postbiotics prepared for CCFM1440 on MMP-1 and MMP-3 enzyme activities in mouse skin tissue;

[0070] Figure 6 Effects of postbiotics prepared for CCFM1440 on TGF-β content in mouse skin tissue;

[0071] Figure 7 Effects of postbiotics prepared for CCFM1440 on SOD, GSH-PX, and CAT enzyme activities in mouse skin tissue;

[0072] Figure 8Effects of postbiotics prepared for CCFM1440 on the levels of CRP, IL-6, and TNF-α in mouse serum.

[0073] Figure 9 Effects of postbiotics prepared for CCFM1440 on skin hydration and elasticity in mice;

[0074] Figure 10 Masson-stained skin sections.

[0075] “*” indicates statistical difference compared with the control group (P<0.05).

[0076] “#” 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

[0077] 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.

[0078] The raw materials used in the embodiment are:

[0079] The human skin fibroblasts (HSF) involved in the following examples were purchased from Kunming Cell Bank.

[0080] The BALB / c mice involved in the following examples were purchased from Weitonglihua Company.

[0081] The Lactobacillus helveticus CCFM1440 involved in the following examples is a strain screened by the Food Biotechnology Center of Jiangnan University.

[0082] The ELISA kits involved in the following examples were purchased from Nanjing Senbeijia Biotechnology Co., Ltd.

[0083] The D-galactose involved in the following examples was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0084] The BCA protein concentration determination kit involved in the following examples was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0085] The culture medium involved in the following examples is as follows:

[0086] 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.

[0087] 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.

[0088] 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).

[0089] Example 1: Cell recovery and culture

[0090] 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, and place them in a culture dish. Place them in a 37℃ incubator containing 5% CO2 for culture. When the cells recover their vitality and grow for 1-2 days to reach 70%-80% fusion, cell passage is performed.

[0091] Example 2: Preparation of postbiotics by Lactobacillus helveticus CCFM1440

[0092] 1. Screening and identification of Lactobacillus helveticus CCFM1440

[0093] The strain sample screening was derived from fermented dairy products. After pretreatment, 0.5 mL of the sample was added to 4.5 mL of normal saline, and gradient dilution was performed with normal saline. The appropriate gradient dilution solution was selected and spread on MRS solid medium and cultured at 37 ° C for 48 h. The typical colony of Lactobacillus helveticus was picked and streaked on MRS solid medium for purification. A single colony was 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 helveticus by NCBI sequence alignment and named Lactobacillus helveticus CCFM1440. It was deposited in Guangdong Provincial Microbiological Culture Collection on September 12, 2024, with the deposit number GDMCC No: 65124.

[0094] 2. Preparation of postbiotics with Lactobacillus helveticus CCFM1440

[0095] (1) Lactobacillus helveticus CCFM1440 was streaked out of the preservation tube and cultured in a water-tight incubator at 37°C for 48 h 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 h to obtain culture fluid 1;

[0096] 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;

[0097] 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.

[0098] 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 helveticus CCFM1440 (denoted as CCFM1440-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 CCFM1440-J).

[0099] The postbiotics of Lactobacillus helveticus CCFM1440 (bacterial lysate CCFM1440-J and fermentation supernatant CCFM1440-S) were prepared by the above-mentioned means.

[0100] Example 3: Effect of postbiotics prepared by Lactobacillus helveticus CCFM1440 on collagen content in HSF cells

[0101] (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.

[0102] Blank group (control): contains cell culture medium and HSF cells, but no postbiotics;

[0103] Postbiotic treatment group: contains cell culture medium and HSF cells, as well as postbiotics.

[0104] 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 helveticus CCFM1440 were added respectively.

[0105] (2) Incubate the above-mentioned well plates in an incubator at 37°C for 24 hours.

[0106] (3) After the incubation, the cell culture supernatant was collected and centrifuged at 1000 rpm for 5 min. The COLⅢ content and LOX enzyme activity were detected using an ELISA kit.

[0107] (4) After washing the cells 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 cells become rounded, then add 4 ml of complete DMEM medium to terminate the digestion. Gently pipette the cells to completely detach them from the culture dish. Transfer the resulting cell suspension to a 15 ml centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Remove the supernatant and resuspend the cells with a mixed protease inhibitor. Repeatedly freeze and thaw (quickly freeze at -80°C and slowly thaw at 4°C) three times to lyse the cells to obtain a cell lysate sample. LH1 enzyme activity was detected using an ELISA kit.

[0108] (5) Use the BCA protein concentration assay kit to detect the total protein concentration of the sample in (4) and calibrate the above data.

[0109] The results of COLⅢ are as follows Figure 1 As shown, compared with the control group, CCFM1440-J can increase the content of COLⅢ secreted by HSF cells by about 16%, and can significantly increase the collagen content.

[0110] LH1 can hydroxylate lysine residues in collagen, enhancing the stability and cross-linking ability of collagen. Figure 1As shown, compared with the control group (27.41 IU / g), the enzyme activity of LH1 in HSF cells treated with CCFM1440-S can be increased to 35.66 IU / g, and the enzyme activity of LH1 in HSF cells treated with CCFM1440-J can be increased to 37.94 IU / g. Both can significantly increase the enzyme activity of LH1.

[0111] LOX can catalyze the oxidation of lysine residues during the maturation of collagen to form a cross-linked structure, thereby enhancing the strength and toughness of collagen fibers. Figure 1 As shown, compared with the control group (0.52 IU / mg), the LOX enzymatic activity in HSF cells treated with CCFM1440-J can be increased to 0.59 IU / mg, which can significantly increase the LOX enzymatic activity.

[0112] In summary, the postbiotics prepared by CCFM1440 can increase the content of collagen secreted by HSF cells and promote the synthesis of collagen, and have the potential to regulate collagen anabolism.

[0113] Example 4: Effect of postbiotics prepared from Lactobacillus helveticus CCFM1440 on collagen content in the skin of aging mice

[0114] The preparation method of the postbiotics (CCFM1440-S and CCFM1440-J) of Lactobacillus helveticus CCFM1440 involved in the following examples is the same as in Example 2, except that CCFM1440-J in this embodiment specifically refers to the bacterial solution 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 solution, and the resuspension is heat-treated at 65° C. for 30 min without high-pressure homogenization.

[0115] (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 CCFM1440-S group, and 1 cage for the CCFM1440-J group.

[0116] Blank group (control): physiological saline was used as a control;

[0117] Model group: normal saline was used as the control;

[0118] CCFM1440-S group: Lactobacillus helveticus CCFM1440 metabolite (fermentation supernatant) was used at a dose of 500 mg / kg mouse body weight;

[0119] CCFM1440-J group: Lactobacillus helveticus CCFM1440 postbiotics (inactivated bacteria) were used at a dose of 500 mg / kg mouse body weight;

[0120] 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.

[0121] 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 2 shown.

[0122] 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.

[0123] The contents of type Ⅰ, Ⅲ, Ⅳ, and Ⅶ collagen (COL Ⅰ, Ⅲ, Ⅳ, and Ⅶ) in mouse skin were detected by ELISA kit. Figure 3 shown.

[0124] 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. Only oral administration of CCFM1440-J could significantly increase its content to 6.06 μg / mg compared with the model group. Inactivated bacteria could increase the content of COLⅠ.

[0125] 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). The oral CCFM1440-J group significantly increased the content of COLⅢ compared with the model group, restoring the content of COLⅢ in the skin to 1.46μg / mg. Inactivated bacteria can increase the content of COLⅢ.

[0126] For COLⅣ, the content in the blank group was 15.29μg / mg, and the content in the model group was 6.21μg / mg. The oral CCFM1440-J group significantly increased the COLⅣ content compared with the model group, and the COLⅣ content in the skin was restored to 13.76μg / mg. Inactivated bacteria can increase the content of COLⅣ.

[0127] For COLⅦ, the content in the blank group was 2.22 μg / mg, which was 2.6 times that of the model group (0.85 μg / mg). The oral CCFM1440-J group could significantly increase the content of COLⅦ compared with the model group, and the content in this group was restored to 1.74 μg / mg.

[0128] The above results show that the postbiotics (inactivated bacteria and fermentation supernatant) prepared from Lactobacillus helveticus CCFM1440 can increase the collagen content in the skin of aging mice.

[0129] Example 5: Effects of Lactobacillus helveticus CCFM1440 and its prepared postbiotics on collagen synthase activity in aging mouse skin

[0130] 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 4.

[0131] 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 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, P4H and LOX in mouse skin were detected by ELISA kits, and the results are as follows. Figure 4 shown.

[0132] By testing the enzyme activities of LH1, P4H, and LOX, it was found that the enzyme activities of LH1, P4H, and LOX in the model group were significantly decreased compared with the blank group. After oral administration of CCFM1440-J, the enzyme activity of LH1 was 14.53 U / mg, which was significantly increased compared with the model group (8.58 U / mg); the enzyme activity of P4H was 26.51 U / mg, which was significantly increased compared with the model group (17.00 U / mg); and the enzyme activity of LOX increased to 17.16 U / mg, which was significantly increased compared with the enzyme activity of 8.48 U / mg in the model group.

[0133] The above results show that the postbiotics (inactivated bacteria and fermentation supernatant) prepared by Lactobacillus helveticus CCFM1440 can significantly increase the enzyme activities of LH1, P4H and LOX in the skin of aging mice and promote the synthesis of collagen.

[0134] Example 6: Effects of Lactobacillus helveticus CCFM1440 and its prepared postbiotics on the activity of collagen degrading enzymes in the skin of aging mice

[0135] 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 4.

[0136] The enzyme activities of MMP-1 and MMP-3 in mouse skin were detected by ELISA kit. Figure 5 shown.

[0137] MMP-1 is a key enzyme in collagen degradation. Its substrates include collagen types I and III. The cleavage site is usually located between Gly-Ile or Gly-Leu bonds. It can cut the intact collagen triple helix structure into fragments of varying lengths, making it more susceptible to degradation by other MMPs. Oral administration of CCFM1440-J significantly downregulated the enzymatic activity of MMP-1 compared to the model group. The MMP-1 enzymatic activity in the model group was 9.94 IU / mg, while that in the CCFM1440-J group was 7.99 IU / mg, which was significantly different from the model group. However, oral administration of CCFM1440-S had an enzymatic activity of 9.14 IU / mg, which failed to downregulate the enzymatic activity of MMP-1.

[0138] MMP-3 can convert inactive collagenase precursors (such as pro-MMP-1) into active forms through proteolytic cleavage, enhancing collagenase activity and degrading fiber fragments and proteins in the cell matrix, disrupting the stability of the collagen network. In terms of MMP-3, compared with the blank group (3.80 IU / mg), the MMP-3 enzyme activity in the model group skin increased significantly to 4.91 IU / mg. Oral administration of CCFM1440-J and CCFM1440-S can significantly downregulate the enzyme activity of MMP-3 in the skin to 4.01 IU / mg and 3.88 IU / mg, respectively. Both can reduce the enzyme activity of MMP-3.

[0139] The above results show that the postbiotics (inactivated bacteria and supernatant) prepared from Lactobacillus helveticus CCFM1440 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 CCFM1440-J has a more significant effect.

[0140] Example 7: Effects of Lactobacillus helveticus CCFM1440 and its prepared postbiotics on TGF-β content in aging mouse skin

[0141] The animal experiment design, gavage groups and skin homogenate sample preparation used in the following examples are the same as those in Example 4. The content of transforming growth factor-β (TGF-β) in mouse skin was detected by ELISA kit. Figure 6 shown.

[0142] Oral administration of CCFM1440-J 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 CCFM1440-J group were 37.16 ng / mg, a significant difference from the model group. However, oral administration of CCFM1440-S, at 27.79 ng / mg, failed to significantly increase TGF-β levels in the skin of aging mice.

[0143] 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.

[0144] In summary, oral administration of CCFM1440-S can better increase the content of TGF-β, thereby regulating the content of collagen.

[0145] Example 8: Effects of Lactobacillus helveticus CCFM1440 and its prepared postbiotics on the antioxidant capacity of aging mouse skin

[0146] 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 4.

[0147] The enzyme activities of SOD, GSH-PX and CAT in mouse skin were detected by ELISA kit. Figure 7 shown.

[0148] In aging mouse models, the activity of antioxidant enzymes such as SOD, GSH-PX, and CAT generally decreases, leading to a weakening of the body's antioxidant capacity and excessive accumulation of ROS. ROS can not only cause aging of the body, but also directly damage the molecular structure of collagen through oxidative reactions, thereby reducing the stability and function of collagen.

[0149] For SOD enzyme activity, such as Figure 7 As shown in the data, the SOD enzyme activity of the control group was 15.87 U / mg, and the SOD enzyme activity of the model group skin decreased significantly to 7.35 U / mg. After oral administration of CCFM1440-J, it could be increased to 13.89 U / mg, which can significantly improve the SOD enzyme activity compared with the model group.

[0150] For the enzyme activity of GSH-PX, Figure 7As shown in the results, compared with the control group (25.77 U / mg), the GSH-PX enzyme activity in the skin of the model group decreased significantly to 12.71 U / mg. After oral administration of CCFM1440-J, the GSH-PX enzyme activity was increased to 21.14 U / mg, which was significantly higher than that of the model group.

[0151] For CAT enzyme activity Figure 7 As shown in the results, 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 CCFM1440-J increased the CAT enzyme activity in the skin to 25.66 U / μg, which was significantly higher than that in the model group.

[0152] In summary, the postbiotics (inactivated bacteria) prepared from Lactobacillus helveticus CCFM1440 can increase the enzyme activities of SOD, GSH-PX, and CAT in the skin of aging mice, improve the skin's antioxidant capacity, maintain collagen stability, and delay skin aging.

[0153] Example 9: Effects of Lactobacillus helveticus CCFM1440 and its prepared postbiotics on inflammation levels in aging mice

[0154] The animal experimental design, gavage groups and serum sample preparation involved in the following examples are the same as those in Example 4.

[0155] The levels of C-reactive protein (CRP) in mouse serum were detected by biochemical analyzer, and the levels of interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) in mouse skin were detected by ELISA kit. Figure 8 shown.

[0156] CRP is a sensitive and nonspecific indicator of inflammation and an inflammation-related marker of aging, closely related to skin aging. The CRP level in the blank group was 4.58 mg / L, while the CRP level in the model group was 5.18 mg / L. Oral administration of CCFM1440-S and CCFM1440-J significantly reduced serum CRP levels to 4.90 mg / L and 5.08 mg / L, respectively.

[0157] Under chronic inflammatory conditions, proinflammatory cytokines IL-6 and TNF-α activate the NF-κB signaling pathway, inhibiting the synthesis of COL I and COL III in fibroblasts while also upregulating the expression of MMP-1 and MMP-3, accelerating collagen degradation. Regarding IL-6, the skin IL-6 content in the model group significantly increased to 24.06 μg / mg compared to the blank group (10.54 μg / mg). Oral administration of CCFM1440-S and CCFM1440-J significantly downregulated the skin IL-6 content to 21.13 μg / mg and 17.73 μg / mg, respectively. Regarding TNF-α, the skin TNF-α content in the model group significantly increased to 112.29 μg / mg compared to the blank group (54.59 ng / mg). Oral administration of CCFM1440J significantly downregulated the skin TNF-α content to 88.80 μg / mg.

[0158] In summary, oral administration of postbiotics (inactivated bacteria and supernatant) prepared from Lactobacillus helveticus CCFM1440 can alleviate the inflammation and aging of aging mice to varying degrees, among which CCFM1440-J has better anti-inflammatory and anti-aging effects.

[0159] Example 10: Effects of Lactobacillus helveticus CCFM1440 and its prepared postbiotics on the water content of the stratum corneum and skin elasticity of aged mice

[0160] The animal experiment design and gavage groups involved in the following examples are the same as those in Example 4. 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 9 shown.

[0161] The moisture content of the stratum corneum is determined by Figure 9 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 of the stratum corneum in the oral CCFM1440-J group (77.54%) increased by 24.67% compared with the model group. The water content in the CCFM1440-J group (75.63%) increased by 22.76% compared with the model group. Both can significantly improve skin moisture.

[0162] Skin elasticity R2 is determined by Figure 9 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 CCFM1440-J group (73.33%) increased by 19.16% compared with the model group, which was significant, while there was no significant difference between the skin elasticity of the CCFM1440-S group (57.40%) and the model group.

[0163] 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.

[0164] From the above experimental results, it can be seen that the postbiotics (inactivated bacteria and supernatant) prepared from Lactobacillus helveticus CCFM1440 can significantly increase the moisture content of aging skin, among which its inactivated bacteria (CCFM1440-J) can also increase the elasticity of the back skin of aging mice, thereby slowing down skin aging.

[0165] Example 11: MASSON staining analysis of skin tissue sections of aged mice using Lactobacillus helveticus CCFM1440 and its prepared postbiotics

[0166] The animal experiment design and gavage groups involved in the following examples are the same as those in Example 4. 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 10 shown.

[0167] 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 CCFM1440 significantly improved the collagen content and structure of mouse skin, among which the improvement effect of the inactivated bacteria was more significant.

[0168] 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 helveticus CCFM1440, characterized in that: The Lactobacillus helveticus was deposited in the Guangdong Provincial Microbial Culture Collection Center on September 12, 2024, with the deposit number: GDMCC No: 65124.

2. A microbial preparation containing the Lactobacillus helveticus CCFM1440 according to claim 1.

3. A postbiotic prepared using the Lactobacillus helveticus CCFM1440 of claim 1 or the microbial preparation of 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 helveticus CCFM1440 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 helveticus CCFM1440 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 helveticus CCFM1440 in a culture medium.

6. A product, characterized in that The product contains the Lactobacillus helveticus CCFM1440 according to claim 1 and / or the postbiotic according to any one of claims 3 to 5.

7. Use of the Lactobacillus helveticus CCFM1440 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.

8. The use according to claim 7, 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.

9. A method for regulating collagen synthesis and metabolism in skin fibroblasts in vitro, comprising mixing the postbiotic according to any one of claims 3 to 5 with skin fibroblasts and incubating the mixture.

10. Use of the postbiotic according to any one of claims 3 to 5 in preparing a product that enhances collagen synthesis in fibroblasts, characterized in that: The application includes at least one of the following functions: (1) Increase the content of type III collagen secreted by skin fibroblasts; (2) Increase the enzyme activity of collagen synthase LH1 and LOX in skin fibroblasts.

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