Plant lactobacillus plantarum ccfm1354 targeting anti-glycan and anti-aging to improve skin health and postbiotic

Abstract

The application discloses a plant lactiplantibacillus plantarum CCFM1354 targeted to anti-sugar anti-aging and improving skin health and a postbiotic thereof, and belongs to the technical field of microorganisms and the technical field of medicines. The application screens a plant lactiplantibacillus plantarum CCFM1354, and provides application of the plant lactiplantibacillus plantarum CCFM1354 and the postbiotic prepared from the plant lactiplantibacillus plantarum CCFM1354 in anti-sugar anti-aging and improving skin health. The strain can be used for preparing external use or oral products targeted to anti-sugar anti-aging and improving skin health, and has great application prospect.

Description

Technical Field

[0001] This invention relates to a plant-derived Lactobacillus CCFM1354 that targets anti-glycation, anti-aging, and improves skin health, and its post-biotics, belonging to the fields of microbial technology and pharmaceutical technology. Background Technology

[0002] Glycosylation, also known as the Maillard reaction, occurs widely in the human body. It refers to the non-enzymatic condensation reaction between the carbonyl group on reducing sugars (such as glucose) and the free amino group on macromolecules such as proteins, lipids, or nucleic acids. This process irreversibly generates stable adducts, namely advanced glycation end products (AGEs). The reactive nature of AGEs readily triggers subsequent glycation damage in the body. On the one hand, AGEs capture and cross-link adjacent proteins, affecting their normal functional properties. On the other hand, they bind to AGE-specific receptors (RAGEs) on the cell surface, activating multiple signal transduction pathways and inducing further oxidative stress and inflammatory responses.

[0003] As the largest organ in the body, the skin suffers the most severe damage from glycation; and among the adverse effects of glycation on the skin, aging is the most obvious. AGEs (Advanced Glycation End Products) gradually accumulate in the skin with age, affecting long-lived skin proteins such as collagen and elastin, and interfering with post-translational modifications of extracellular matrix proteins. The link between glycation and skin health is very close; glycation damage results in skin that is more prone to increased dryness, decreased elasticity, wrinkles, and pigmentation. Therefore, anti-glycation efforts to alleviate skin aging and sub-health have attracted increasing attention.

[0004] Currently, representative anti-glycation ingredients mainly include carnosine, lipoic acid, niacinamide, and plant extracts rich in flavonoids and polyphenols. Their anti-glycation effects are primarily achieved by reducing the production of AGEs (Advanced Glycation End Products). However, their anti-glycation effects are relatively limited in addressing the ongoing glycation damage caused by AGEs in the body. CN116350531A discloses an anti-glycation application of chrysanthemum green extract D, verifying that this extract has anti-glycation properties. CN116570544A discloses an anti-glycation emulsion and its preparation method, which incorporates plant extracts such as raspberry, turmeric, and grape seed to achieve an anti-glycation effect in the skincare emulsion. CN116459172A provides an anti-glycation application of vitexin. While these patents cover the anti-glycation effects of different plant extracts, they primarily focus on reducing AGE content, with insufficient utilization of anti-glycation methods for other processes of sugar damage. Therefore, the following anti-glycation pathways of probiotics and their post-biotics are proposed: probiotics and their post-biotics inhibit the formation of AGEs, thus hindering glycation reactions; probiotics and their post-biotics inhibit the binding of AGE-RAGE and reduce the cross-linking of AGEs with proteins, thus preventing direct biochemical reactions caused by AGEs; probiotics and their post-biotics inhibit the activation of subsequent glycation signaling pathways and reduce downstream continuous cascade reactions in the body. Summary of the Invention

[0005] This invention aims to provide a plant lactobacillus ( Lactiplantibacillus plantarum (and its preparation of post-glycation derivatives that can target anti-glycation, anti-aging, and improve skin health)

[0006] This invention provides a strain of *Lactobacillus plantarum* (… Lactiplantibacillus plantarum CCFM1354, taxonomically named Lactiplantibacillus plantarum It was deposited on October 25, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 63924, located at Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] The *Lactobacillus plantarum* CCFM1354 was isolated from the feces of healthy human beings. The 16S rRNA sequence of this strain is shown in SEQ ID NO.1 after sequencing analysis.

[0008] Under a microscope, the *Lactobacillus plantarum* CCFM1354 cells form colonies of medium diameter, raised, rough surface, and curled edges. After inoculation on MRS medium and cultured for 48 h, the colonies are generally pale yellow, smooth, raised, and round with a diameter of 3 mm.

[0009] The *Lactobacillus plantarum* CCFM1354 is a Gram-positive, facultative anaerobic bacterium that thrives in warm temperatures, with an optimal growth temperature of 35-40℃ and an optimal growth pH of 6.0-7.0.

[0010] The present invention also provides a metabiotic prepared using the aforementioned *Lactobacillus plantarum* CCFM1354.

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

[0012] In one embodiment, the inactivated or dead cells are prepared by culturing the *Lactobacillus plantarum* CCFM1354 in a culture medium for a period of time, collecting the bacterial cells in the cell culture medium, and obtaining inactivated bacterial cells by heat treatment or freeze-drying.

[0013] In one embodiment, the heat treatment conditions are: 60℃~70℃, 25~35 min.

[0014] In one embodiment, the method for preparing the bacterial lysate is as follows: the *Lactobacillus plantarum* CCFM1354 is cultured in a culture medium for a period of time, bacterial cells are collected, homogenized under high pressure, centrifuged, and the supernatant after centrifugation is collected to obtain the bacterial lysate.

[0015] In one embodiment, the fermentation supernatant is the supernatant obtained by culturing *Lactobacillus cereus* CCFM1354 in a culture medium for a period of time and then centrifuging it.

[0016] In one embodiment, drying includes, but is not limited to, preparation by spray drying, vacuum freeze drying, fluidized bed drying, and vacuum drying.

[0017] The present invention also provides compositions containing the aforementioned *Lactobacillus plantarum* CCFM1354 and / or its postgenes.

[0018] In one embodiment, the composition includes, but is not limited to, food, pharmaceuticals, health products, or cosmetics.

[0019] In one embodiment, the composition includes at least one of the following effects:

[0020] (1) Inhibit the formation of fluorescent AGEs;

[0021] (2) Prevent damage and functional decline of skin fibroblasts (HSF) caused by high glucose and AGE formation intermediate (acetone aldehyde);

[0022] (3) Reduce individual signs of aging, including but not limited to blood biochemical indicators and skin appearance.

[0023] (4) Prevent abnormal degradation of collagen in skin fibroblasts (HSF) under high glucose culture;

[0024] (5) Prevent abnormal expression of EGR2 mRNA, a key growth regulation target of skin fibroblasts (HSF) under high glucose culture;

[0025] (6) Reduce the AGE content in the serum, skin, liver, kidneys, and brain tissue of aging individuals;

[0026] (7) Reduce the levels of inflammatory markers IL-6 and TNF-α in the serum of aging individuals;

[0027] (8) Reduce the decrease in the moisture content of the stratum corneum of the back skin in aging individuals caused by glycation damage;

[0028] (9) Reduce the decrease in type III collagen content in the back skin of aging individuals caused by glycation damage.

[0029] This invention provides the application of *Lactobacillus plantarum* CCFM1354 and / or its metabiotics in the preparation of products for anti-glycation, anti-aging, and improvement of skin health problems caused by glycation.

[0030] In one embodiment, the anti-glycation and anti-aging measures include inhibiting the levels of glycated aging markers (AGEs) in blood, skin, liver, and / or kidneys; inhibiting the levels of inflammatory damage markers TNF-α and IL-6 in serum; inhibiting muscle strength decline during aging; and inhibiting skin aging characteristics.

[0031] In one embodiment, the improvement of skin health problems caused by glycation includes alleviating the reduction of skin stratum corneum moisture, alleviating the reduction of skin elasticity in multiple dimensions, and alleviating the loss of type III collagen in the skin.

[0032] In one embodiment, the product includes, but is not limited to, food or topical skincare ingredients.

[0033] In one embodiment, the product may be used in ways including, but not limited to, topical application or oral administration.

[0034] In one embodiment, the content of *Lactobacillus plantarum* CCFM1354 in the product is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0035] In one embodiment, the dosage of the postbiotic prepared from *Lactobacillus plantarum* CCFM1354 in the product is not less than 10 mg / kg body weight.

[0036] In one embodiment, the food contains *Lactobacillus plantarum* CCFM1354 and / or its post-generics, as well as conventional excipients.

[0037] In one embodiment, the conventional excipients include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

[0038] In one embodiment, the health product contains *Lactobacillus plantarum* CCFM1354 and / or its post-generics, as well as conventional excipients.

[0039] In one embodiment, the conventional excipients include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

[0040] In one embodiment, the pharmaceutical product contains *Lactobacillus plantarum* CCFM1354 and / or its postgenes, as well as a drug carrier and / or pharmaceutical excipients.

[0041] In one embodiment, the pharmaceutical excipient comprises excipients and additives.

[0042] In one embodiment, the pharmaceutical excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, and release inhibitors.

[0043] In one embodiment, the cosmetic contains a metagene of the *Lactobacillus plantarum* CCFM1354, as well as matrix ingredients and / or conventional excipients.

[0044] In one embodiment, the matrix raw materials include oil-based raw materials, wax-based raw materials, synthetic oil-based raw materials, powder-based raw materials, gel-based raw materials, coagulants, and surfactants.

[0045] In one embodiment, the conventional excipients include one or more of the following: moisturizers, whitening agents, flavoring agents, adhesives, lubricants, preservatives, film-forming agents, antioxidants, emulsifiers, and cosmetic nutritional additives.

[0046] This invention provides the use of the above composition in the preparation of products for preventing and / or alleviating symptoms related to skin aging.

[0047] In one embodiment, the symptoms associated with skin aging include dry skin, decreased elasticity, sagging, wrinkles, oxidative damage, and collagen loss.

[0048] The present invention also provides the application of the *Lactobacillus plantarum* CCFM1354 or the postbiotic in the preparation of food.

[0049] Beneficial effects:

[0050] This invention screened and obtained a strain of *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum CCFM1354, and its prepared metabiotics, both topical and oral, possess anti-glycation, anti-aging, and skin-health-improving abilities, specifically manifested in:

[0051] (1) Inhibits the generation of fluorescent AGE in the in vitro fructose-bovine serum albumin system;

[0052] (2) To prevent the decrease in cell viability in a model of acetone aldehyde-induced skin fibroblast (HSF) damage;

[0053] (3) Prevent abnormal degradation of collagen in skin fibroblasts (HSF) under high glucose culture;

[0054] (4) Prevent abnormal expression of EGR2 mRNA, a key growth regulation target of skin fibroblasts (HSF) under high glucose culture;

[0055] (5) Reduce the AGE content in the serum, skin, liver, kidneys, and brain tissue of aging individuals;

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

[0057] (7) Reduce the decrease in the moisture content of the stratum corneum of the back skin in aging individuals caused by glycation damage;

[0058] (8) Reduce the decrease in type III collagen content in the back skin of aging individuals caused by glycation damage.

[0059] Therefore, *Lactobacillus plantarum* ( Lactiplantibacillus plantarum CCFM1354 and its prepared post-biotics have great application potential in products that target anti-glycation and anti-aging and improve skin health.

[0060] Preservation of biological materials

[0061] A strain of Lactobacillus plantarum ( Lactiplantibacillus plantarum CCFM1354, taxonomically named Lactiplantibacillus plantarum It was deposited on October 25, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 63924, located at Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0062] Figure 1 Effects of different post-adrenergic glycosides on the inhibition of fluorescent AGE formation in the in vitro fructose-bovine serum albumin system;

[0063] Figure 2 Effects of different metageners on HSF cell proliferation;

[0064] Figure 3 Effects of different post-genetic intermediates, acetone aldehyde, on the viability of HSF cells;

[0065] Figure 4 The effect of different metabiotics on the expression of MMP-9 mRNA and EGR2 mRNA in HSF cells under high glucose culture;

[0066] Figure 5 Mouse experiment flowchart;

[0067] Figure 6 Effects of Lactobacillus plantarum CCFM1354 and its prepared postbiotic on the levels of AGE, IL-6, and TNF-α in mouse blood;

[0068] Figure 7 Effects of Lactobacillus plantarum CCFM1354 and its prepared postbiotic on AGE content in multiple organs (skin, liver, brain) of aging mice.

[0069] Figure 8 Effects of Lactobacillus plantarum CCFM1354 and its prepared metagenes on the water content of the stratum corneum of the skin;

[0070] Figure 9 The effects of Lactobacillus plantarum CCFM1354 and its prepared metagenerogen on the content of type III collagen in the skin;

[0071] Figure 10 Effects of *Lactobacillus plantarum* CCFM1354 and its prepared metagener on the expression of target genes (RAGE mRNA, DDOST mRNA, MMP-2 mRNA, COL3A1 mRNA) related to skin glycation loss;

[0072] Figure 11 The effect of fermentation of peanut skin by Lactobacillus plantarum CCFM1354 on enhancing the in vitro anti-glycation ability of peanut skin;

[0073] Figure 12 The effect of fermentation of peanut skin by Lactobacillus plantarum CCFM1354 on enhancing the ability of peanut skin to inhibit AGE accumulation after oral administration;

[0074] Figure 13The effect of fermentation of peanut skin by Lactobacillus plantarum CCFM1354 on enhancing the oral resistance of peanut skin to glycation damage.

[0075] "*" indicates a statistically significant difference from the Model group (P<0.05), "**" indicates a statistically significant difference from the Model group (P<0.01), "***" indicates an extremely statistically significant difference from the Model group (P<0.001), and "****" indicates an extremely statistically significant difference from the Model group (P<0.0001). Detailed Implementation

[0076] The present invention will be further described below with reference to specific embodiments.

[0077] The human skin fibroblasts (HSF) involved in the following examples were purchased from the Kunming Cell Bank.

[0078] The BALB / c mice used in the following examples were purchased from Vital Rivers.

[0079] The *Lactobacillus plantarum* CCFM1354, *Lactobacillus plantarum* FXJCJ22M3, *Lactobacillus plantarum* FSCDJY93L1, and *Lactobacillus plantarum* FXJCJ26M6 involved in the following examples were self-screened strains from the Food Biotechnology Center of Jiangnan University.

[0080] The culture media involved in the following examples are as follows:

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

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

[0083] MRS (Simplified) Liquid Culture Medium: Glucose 8 g / L, Yeast Extract 5 g / L, Calcium Carbonate 6 g / L, Anhydrous Sodium Acetate 2 g / L, Diammonium Citric Acid 2 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.

[0084] Peanut skin fermentation medium: 5 g / L of commercially available peanut skin extract, 4 g / L of glucose, 5 g / L of yeast powder, and 4 g / L of calcium carbonate were added. After adjusting the pH to 6.8-7.2, the medium was sterilized at 115 ℃ for 20 min to prepare the peanut skin fermentation medium (abbreviated as hsp). The commercially available peanut skin extract was purchased from Shaanxi Shengheng Biotechnology Co., Ltd., batch number SH20220328.

[0085] Cell culture medium: 89% (v / v) DMEM medium + 10% (v / v) fetal bovine serum + 1% (v / v) 100× penicillin and streptomycin mixed solution (the mixed solution contains 10,000 U / mL penicillin and 10 mg / mL streptomycin).

[0086] Example 1: Screening and Identification of Lactobacillus plantarum

[0087] 1. Screening

[0088] The samples were derived from feces of healthy individuals. After pretreatment, the samples were stored in 20% glycerol at -80 °C. After thawing, the samples were mixed and 0.5 mL was added to 4.5 mL of physiological saline. The samples were then serially diluted with physiological saline. The appropriate serial dilutions were spread on MRS solid medium and incubated at 37 °C for 48 h. Typical colonies of *Lactobacillus plantarum* were picked and streaked onto MRS solid medium for purification. Single colonies were then transferred to MRS liquid medium for enrichment and preserved in 30% glycerol to obtain the strain, which was named CCFM1354. The typical colonies of *Lactobacillus plantarum* were round, pale yellow, and smooth.

[0089] 2. Identification

[0090] The genome of strain CCFM1354 was extracted, and the 16S rDNA of strain CCFM1354 was amplified and sequenced (performed by Suzhou Genewiz Biotechnology Co., Ltd.; the nucleotide sequence of the amplified 16S rDNA of CCFM1354 is shown in SEQ ID NO.1). The sequence was then compared with the NCBI sequence, and the results showed that the strain was *Lactobacillus plantarum*, and it was named *Lactobacillus plantarum*. Lactiplantibacillus plantarum (CCFM1354)

[0091] Example 2: Cell resuscitation and culture

[0092] First, remove the frozen human skin fibroblasts (HSF), thaw them rapidly in a 37°C water bath, then centrifuge at 1000 r / min for 3 min, discard the supernatant, resuspend the cells in an appropriate volume of cell culture medium, place them in a culture dish, and incubate them in a 37°C incubator containing 5% CO2. When the cells regain viability and grow for 1-2 days and reach 70%-80% confluence, passage the cells.

[0093] Example 3: Preparation of postbiotic from *Lactobacillus plantarum* CCFM1354

[0094] (1) Use MRS solid medium to culture in a water-jacketed constant temperature incubator at 37 °C for 24-48 h to obtain single colonies; pick single colonies and inoculate them into MRS liquid medium, and culture at 37 °C for 12-18 h to obtain culture solution 1;

[0095] Culture medium 1 was inoculated into MRS liquid medium at an inoculation rate of 2% (v / v) and cultured at 37 °C for 12 h to obtain seed culture;

[0096] Seed culture was inoculated at 2-5% (v / v) into MRS liquid medium and MRS (simplified) liquid medium for expansion, and cultured at 37℃ for 18-24 h. The concentrations of the two cultures were then adjusted to equivalent levels (1.5×10⁻⁶). 9 Bacterial solution a and bacterial solution b were obtained by (CFU / mL).

[0097] The supernatant obtained by centrifuging bacterial culture a at 8000 r / min for 30 min was then heat-treated (65℃, 30 min) and freeze-dried to obtain powder for later use, thus preparing the fermentation supernatant of *Lactobacillus plantarum* CCFM1354 (denoted as CCFM1354_M).

[0098] The bacterial sludge obtained by centrifuging bacterial solution b at 8000 r / min for 30 min was resuspended in double-distilled water at 75% volume of the original bacterial solution. The resuspended solution was heat-treated (65 ℃, 30 min) and then homogenized under high pressure (1000~1200 MPa, 10 times). After homogenization, the supernatant was collected by centrifugation at 8000 r / min for 30 min. The bacterial cell lysate (denoted as CCFM1354_Z) was collected and freeze-dried to obtain postbiotic freeze-dried powder for later use.

[0099] The preparation method of live bacteria of *Lactobacillus plantarum* CCFM1354 is the same as that of post-biotic, except that the bacterial sludge obtained by centrifuging bacterial solution b at 8000 r / min for 30 min is resuspended in a ratio of 1 g: 2 mL freeze-drying protectant and then directly freeze-dried to obtain live bacterial powder of *Lactobacillus plantarum* CCFM1354, which is denoted as CCFM1354.

[0100] The following postbiotics of *Lactobacillus plantarum* CCFM1354 (cell lysate CCFM1354_Z and fermentation supernatant CCFM1354_M) were prepared by the above methods.

[0101] (2) The metagenes of *Lactobacillus plantarum* FXJCJ22M3, *Lactobacillus plantarum* FSCDJY93L1, and *Lactobacillus plantarum* FXJCJ26M6 were prepared according to the method in step (1).

[0102] Example 4: Preparation of peanut skin fermentation broth by fermentation of peanut skin with Lactobacillus plantarum CCFM1354

[0103] (1) Activation of Lactobacillus plantarum CCFM1354

[0104] Using an inoculation loop, streak a bacterial suspension of *Lactobacillus plantarum* CCFM1354 onto MRS solid medium and incubate upside down at 37 °C for 48 h. Take a single colony and transfer it to MRS liquid medium, incubate aerobically at 37 °C for 18 h, mix well, and then inoculate the bacterial suspension into a new MRS liquid medium at an inoculation rate of 2% (v / v). Repeat this process three times to obtain the final activated bacterial suspension.

[0105] (2) Preparation of culture medium for peanut skin fermentation

[0106] Add 5 g / L of commercially available peanut skin extract, 4 g / L of glucose, 5 g / L of yeast powder, and 4 g / L of calcium carbonate. After adjusting the pH to 6.8-7.2, sterilize at 115 ℃ for 20 min to prepare a culture medium for peanut skin fermentation (abbreviated as hsp).

[0107] (3) Fermentation of Lactobacillus plantarum CCFM1354

[0108] The activated bacterial culture obtained in step (1) was inoculated into the treated peanut skin fermentation culture obtained in step (2) at an inoculation rate of 2% (v / v) and cultured at 37 ℃ with shaking at 200 rpm for 72 h.

[0109] (4) Preparation of supernatant from peanut skin fermentation by Lactobacillus plantarum CCFM1354

[0110] The fermentation broth was collected after 72 h of culture. The supernatant obtained by centrifuging the bacterial broth at 8000 r / min for 30 min was then heat-treated (65 ℃ for 30 min) and freeze-dried to obtain powder for later use. The resulting supernatant of peanut skin fermentation by Lactobacillus plantarum CCFM1354 (denoted as CCFM1354_H) was prepared.

[0111] Example 5: Effect of postbiotic prepared from *Lactobacillus plantarum* CCFM1354 on the inhibition of fluorescent AGE formation in the in vitro fructose-bovine serum albumin system.

[0112] A final concentration of 10 mg / mL bovine serum albumin (BSA) and 0.5 M d-(+)-fructose were mixed in 0.1 M phosphate buffer (pH 7.4) and filtered through a 0.22 μm aqueous filter to obtain a sterile fructose-BSA saccharification system. 5% post-genetic precursor samples were prepared with PBS and incubated with the fructose-BSA saccharification system at 37 °C for 7 days. The incubated samples were used to detect the formation of fluorescent advanced photosynthetic processes (AGEs) 7 days after incubation.

[0113] The groups are as follows:

[0114] CCFM1354_Z: Used Lactobacillus plantarum CCFM1354 cell lysis buffer;

[0115] CCFM1354_M: Fermentation supernatant of Lactobacillus plantarum CCFM1354 was used.

[0116] FXJCJ22M3_Z: Used Lactobacillus plantarum FXJCJ22M3 cell lysis buffer;

[0117] FXJCJ22M3_M: Fermentation supernatant of Lactobacillus plantarum FXJCJ22M3;

[0118] FSCDJY93L1_Z: Used Lactobacillus plantarum FSCDJY93L1 cell lysis buffer;

[0119] FSCDJY93L1_M: Used supernatant of Lactobacillus plantarum FSCDJY93L1 cells;

[0120] FXJCJ26M6_Z: Use Lactobacillus plantarum FXJCJ26M6 cell lysis buffer;

[0121] FXJCJ26M6_M: Used supernatant of Lactobacillus plantarum FXJCJ26M6 cells.

[0122] After incubation, fluorescent AGEs in glucose-modified BSA were detected using an excitation wavelength of 340 nm and an emission wavelength of 440 nm. The percentage of inhibition of fluorescent AGE formation was calculated as 1 minus the difference in fluorescence intensity between the sample and the control (BSA+ / glucose+).

[0123] The effect of fructose-bovine serum albumin system on the inhibition rate of fluorescent AGE formation, such as Figure 1 As shown, the AGE inhibition rates of CCFM1354_Z and CCFM1354_M were 54.46% and 35.66%, respectively, which showed significant advantages compared with other Lactobacillus plantarum.

[0124] Example 6: Effect of metabiotic prepared from Lactobacillus plantarum CCFM1354 on HSF cell proliferation

[0125] The specific steps are as follows:

[0126] (1) Take 100 μL of HSF cells in the logarithmic growth phase and use 3×10 4 Cells per well were seeded at a concentration of 100 cells / well in 96-well plates, with the outermost ring filled with PBS solution to prevent edge effects. After culturing for 24 h to allow the cells to adhere, blank control, control and post-biotic treatment groups were set up.

[0127] Control group: Contains only cell culture medium and no HSF cells;

[0128] Control group: Contains cell culture medium and HSF cells, but does not contain post-genetics;

[0129] Treatment group: The metabiotic was resuspended in cell culture medium (the amount of resuspended metabiotic was the same as that fermented to a concentration of 5.0 × 10⁻⁶). 7 (The amount of metabiotic prepared from bacterial culture of CFU / mL is equivalent), and 100 μL of metabiotic prepared from *Lactobacillus plantarum* CCFM1283, *Lactobacillus plantarum* FXJCJ22M3, *Lactobacillus plantarum* FSCDJY93L1, or *Lactobacillus plantarum* FXJCJ26M6 is added respectively.

[0130] (2) The above well plates were incubated in an incubator at 37 °C for 24 h. After incubation, 10 μL of CCK8 solution was added to each well and incubated for 2 h. The absorbance (OD) at 450 nm was measured.

[0131] Cell viability is calculated using the following formula: Cell viability (%) = (OD value of treatment group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0132] Effects on cell proliferation, such as Figure 2As shown, compared with the control group (cell proliferation rate 100%), the addition of metagenes prepared from *Lactobacillus plantarum* CCFM1354 (CCFM1354_M and CCFM1354_Z), *Lactobacillus plantarum* FXJCJ22M3 (FXJCJ22M3_M and FXJCJ22M3_Z), *Lactobacillus plantarum* FSCDJY93L1 (FSCDJY93L1_M and FSCDJY93L1_Z), and *Lactobacillus plantarum* FXJCJ26M6 (FXJCJ26M6_M and FXJCJ26M6_Z) at an inactivated cell concentration of 5.0 × 10⁻⁶ cells / mL significantly increased the cell proliferation rate. 7 The cell proliferation rates at CFU / mL were 106.05%, 115.63%, 108.48%, 105.30%, 98.50%, 97.90%, 99.99%, 94.76%, and 107.73%, respectively.

[0133] According to the ISO 10993-5:2009 toxicity classification evaluation method, cells with a viability greater than 70% can be considered non-toxic. The above results indicate that an inactivated bacterial concentration of 5.0 × 10⁻⁶ is appropriate. 7 HSF cells at a postbiotic concentration of CFU / mL showed high viability (over 90%). Considering its non-cytotoxicity, an inactivated cell concentration of 5.0 × 10⁻⁶ cells was selected. 7 CFU / mL is a suitable postbiotic concentration for subsequent cell experiments.

[0134] Example 7: Effect of metabiotic prepared from *Lactobacillus plantarum* CCFM1354 on preventing acetone-induced glycation damage in HSF cells.

[0135] The specific steps are as follows:

[0136] (1) Take 100 μL of HSF cells in the logarithmic growth phase and use 3×10 4 Cells per well were seeded at a concentration of 100 cells / well in a 96-well plate, with the outermost ring filled with PBS solution to prevent edge effects. After culturing for 24 h to allow the cells to adhere, a blank group, a control group 1, and a treatment group 1 were set up.

[0137] Control group: Contains only cell culture medium and no HSF cells;

[0138] Control group 1: Contains cell culture medium and HSF cells, but does not contain post-biotics;

[0139] Treatment group 1: Contains cell culture medium and HSF cells, and also contains metagenes.

[0140] Metabiotics included *Lactobacillus plantarum* CCFM1354, *Lactobacillus plantarum* FXJCJ22M3, *Lactobacillus plantarum* FSCDJY93L1, and a metabiotic prepared from *Lactobacillus plantarum* FXJCJ26M6. The metabiotics were resuspended in cell culture medium (the amount of resuspended metabiotics was the same as the amount fermented to a concentration of 5.0 × 10⁻⁶). 7 The amount of metabiotics prepared from bacterial culture at CFU / mL is equivalent.

[0141] (2) The above-mentioned well plates were incubated in an incubator at 37 ℃ for 24 h. After incubation, the old culture medium of the control group and the modeling agent group was discarded, and the plates were washed three times with PBS. The control group, model group and treatment group were set up.

[0142] Control group: After changing the medium in step (1), control group 1 contained cell culture medium and HSF cells, without post-genetic treatment, and without acetone aldehyde modeling agent;

[0143] Model group: The control group 1 in step (1) was replaced with a cell culture medium containing acetone aldehyde modeling agent, containing the original HSF cells, without post-genetic treatment.

[0144] Cell culture medium containing acetone aldehyde modeling agent: acetone aldehyde is mixed evenly in ordinary cell culture medium and sterilized by passing through a 0.22 μm aqueous filter membrane. The final concentration of acetone aldehyde in the cell culture medium is 400 μmol / L.

[0145] Treatment group: The treatment group 1 in step (1) was changed to a cell culture medium containing acetone aldehyde modeling agent, containing the original HSF cells, and then treated with post-biotics.

[0146] (3) The well plates prepared in step (2) were incubated in an incubator at a temperature of 37 °C for 24 h. After incubation, 10 μL of CCK8 solution was added to each well and incubated for 2 h to measure the absorbance (OD) at 450 nm.

[0147] Cell viability was calculated using the following formulas: Model group cell viability (%) = (Model group OD value - Blank group OD value) / (Control group OD value - Blank group OD value) × 100%; Treatment group cell viability (%) = (Treatment group OD value - Blank group OD value) / (Control group OD value - Blank group OD value) × 100%.

[0148] Results of preventing damage to HSF cells caused by acetone aldehyde: Figure 3 Compared with the control group (100% cell viability), the cell viability of the model group was 54.93%, and acetone aldehyde modeling caused significant damage to HSF cells.

[0149] After adding CCFM1354_M and CCFM1354_Z to the treatment groups, the cell viability was 68.80% and 79.54%, respectively. Compared with the model group, CCFM1354_Z significantly improved the HSF cell viability, indicating that the metagenetic agent of Lactobacillus plantarum CCFM1354 can effectively prevent glycosylation damage to HSF cells caused by acetone aldehyde.

[0150] After treatment with other treatment groups of *Lactobacillus plantarum* FXJCJ22M3 (FXJCJ22M3_M and FXJCJ22M3_Z), *Lactobacillus plantarum* FSCDJY93L1 (FSCDJY93L1_M and FSCDJY93L1_Z), and *Lactobacillus plantarum* FXJCJ26M6 (FXJCJ26M6_M and FXJCJ26M6_Z), the HSF cell viability was 37.99%, 43.54%, 42.92%, 35.04%, 49.72%, and 36.02%, respectively. This indicates that the other *Lactobacillus plantarum* metageners did not possess the ability to prevent acetone aldehyde damage to HSF cells exhibited by the *Lactobacillus plantarum* CCFM1354 metagener.

[0151] Example 8: The effect of metabiotics prepared from Lactobacillus plantarum CCFM1354 on the expression of MMP-9 mRNA and EGR2 mRNA in HSF cells under high glucose culture.

[0152] The specific steps are as follows:

[0153] (1) HSF cells were loaded with 1×10 5 Cells were seeded at a density of 100 cells / mL in 6-well plates and cultured overnight until cell attachment was achieved. The old culture medium was discarded, and the cells were washed three times with PBS. A control group and a treatment group were set up.

[0154] Control group 1: The group that did not receive post-genetic agents;

[0155] The treatment groups consisted of *Lactobacillus plantarum* CCFM1354 lysis buffer (CCFM1354_Z), *Lactobacillus plantarum* FXJCJ22M3 lysis buffer (FXJCJ22M3_Z), *Lactobacillus plantarum* FSCDJY93L1 lysis buffer (FSCDJY93L1_Z), and *Lactobacillus plantarum* FXJCJ26M6 lysis buffer (FXJCJ26M6_Z). After treatment, the metabiotics were resuspended in cell culture medium (the amount of resuspended metabiotics was the same as that fermented to a concentration of 5.0 × 10⁻⁶). 7 The amount of metabiotics prepared from bacterial culture at CFU / mL is equivalent.

[0156] Two mL of each of the following lysates were added to a 6-well plate: CCFM1354 lysate (CCFM1354_Z), FXJCJ22M3 lysate (FXJCJ22M3_Z), FSCDJY93L1 lysate (FSCDJY93L1_Z), and FXJCJ26M6 lysate (FXJCJ26M6_Z). The plates were incubated for 24 h, with three replicates for each sample.

[0157] (2) The above-mentioned well plates were incubated in an incubator at 37°C for 24 h. After incubation, the old culture medium of the control group and the modeling agent group was discarded, and the plates were washed three times with PBS. The control group, model group and treatment group were set up:

[0158] Control group: Step (1) After changing the medium, the original HSF cells, without post-genetic treatment, were added to 2 mL of ordinary cell culture medium;

[0159] Model group: Step (1) Control group 1 was changed to cell culture medium containing 35 mmol / L glucose, containing the original HSF cells, without post-genetic treatment;

[0160] Treatment group: Step (1) Control group 1 changed the medium to a cell culture medium containing 35 mmol / L glucose, containing the original HSF cells, and then treated with post-biotics.

[0161] (3) The above-mentioned well plate from step (2) was incubated in an incubator at 37 ℃ for 24 h. The culture supernatant was discarded, and each well was washed three times rapidly with PBS. 1 mL of cell lysis buffer was added to each well, and the cells were repeatedly pipetted. RNA was extracted from the cell lysis buffer and reverse transcribed into cDNA using an RT-PCR reverse transcription kit. The expression of genes in HSF cells was detected by real-time quantitative PCR. -△△Ct The expression levels of MMP-9 mRNA and EGR2 mRNA were calculated using formulas, with β-actin as the internal reference. The primers used are described in Table 1 below, and the results are as follows: Figure 4 As shown.

[0162] Table 1 Primer Sequences

[0163]

[0164] The results are as follows Figure 4As shown, the expression level of MMP-9 mRNA in the control group was approximately 1, while the expression level in the model group increased to 2.54 after intervention with high glucose medium (cell culture medium containing 35 mmol / L glucose). The metageneric agent prepared by *Lactobacillus plantarum* CCFM1354 (CCFM1354_Z) significantly reduced the expression level of MMP-9 mRNA in HSF cells to 0.75. The expression levels of MMP-9 mRNA after treatment with other *Lactobacillus plantarum* metageneric agents (FXJCJ22M3_Z, FSCDJY93L1_Z, FXJCJ26M6_Z) were approximately 5.19, 2.30, and 1.22, respectively, and none of them showed a significant downregulation effect on MMP-9 mRNA expression compared with the model group. The expression level of EGR2 mRNA in the control group was approximately 1. After intervention with high glucose medium, the expression level decreased to 0.75. The post-biotic prepared by Lactobacillus plantarum CCFM1354 (CCFM1354_Z) significantly increased the expression level of EGR2 mRNA in HSF cells to 2.59, which was significantly improved. Other treatments did not achieve similar regulatory effects on EGR2 mRNA in cells damaged by glycation.

[0165] Matrix metalloproteinase 9 (MMP-9) is an enzyme belonging to the zinc-metalloproteinase family. It is an enzyme that mainly degrades type IV collagen and elastin, participating in the degradation of the extracellular matrix in normal physiological and pathological processes. Under high glucose conditions, MMP-9 expression increases, leading to slowed proliferation, decreased viability, reduced migration, and decreased collagen secretion capacity of skin fibroblasts. Early growth response protein 2 (EGR2), induced by T cell receptors, is essential for inducing T cell dysfunction and participates in the regulation of inflammatory pathways. Under hyperglycemic conditions, EGR2 expression decreases, inhibiting anti-inflammatory Socs1 and increasing pro-inflammatory genes. These results indicate that the metabiotic (cell lysate) prepared from *Lactobacillus plantarum* CCFM1354 can downregulate MMP-9 mRNA expression and upregulate EGR2 mRNA expression under high glucose culture conditions, preventing glucose damage to HSF cells caused by high glucose culture conditions.

[0166] Example 9: Effects of *Lactobacillus plantarum* CCFM1354 and its prepared metabiotic on the levels of blood biochemical indicators (AGE, TNF-α, IL-6) in aging mice.

[0167] The preparation methods of the post-biotics (CCFM1354_M and CCFM1354_Z) of Lactobacillus paracasei CCFM1354 involved in the following examples are the same as in Example 3; wherein, the CCFM1354_Z group is obtained by centrifuging the obtained bacterial solution b at 8000 r / min for 30 min to obtain bacterial sludge, resuspending the bacterial sludge in physiological saline at 75% volume of the original bacterial solution, heat-treating the resuspended solution (65 ℃, 30 min), without high-pressure homogenization, to obtain inactivated bacterial cells (denoted as CCFM1354_Z), and freeze-drying to obtain post-biotic freeze-dried powder for later use.

[0168] The specific steps are as follows:

[0169] (1) Forty-five healthy male BALB / c mice aged 8 weeks were randomly divided into 9 cages, with 5 mice in each cage. The 9 cages were as follows: except for the model group (2 cages), the blank group and the other groups (1 cage each), as follows:

[0170] Control group: Using physiological saline as a control;

[0171] Model group:

[0172] CCFM1354 group: Live Lactobacillus plantarum CCFM1354 was used at a dose of 5 × 10⁻⁶. 9 CFU / kg mouse body weight;

[0173] CCFM1354_Z group: The mice were given a postbiotic (inactivated bacterial cells) of Lactobacillus plantarum CCFM1354 at a dose of 500 mg / kg of mouse body weight.

[0174] CCFM1354_M group: The mice were given a metabolite of Lactobacillus plantarum CCFM1354 (fermentation supernatant) at a dose of 500 mg / kg body weight.

[0175] Among them, the fermentation supernatant or inactivated bacterial cells in each of the above groups are inactivated bacterial cells or metabolites prepared from bacterial broth after fermentation with an equal amount of live bacteria.

[0176] The experiment lasted 7 weeks: After one week of acclimatization, all groups except the control group received a subcutaneous injection of D-galactose (1000 mg / kg) at a dose of 0.1 mL / mouse / day. Starting from the second week, each intervention group received lyophilized powder of the corresponding strain or post-biotic lyophilized powder (lysis buffer and fermentation broth) prepared by the strain dissolved in physiological saline at the appropriate dose, administered to mice by gavage at a dose of 0.1 mL / mouse / day. The control group and the model group were administered an equal volume of physiological saline by gavage as controls until the end of the experiment. All groups had free access to water and food, and the experimental procedure was as follows. Figure 5 As shown.

[0177] After the experiment, mice were sacrificed and blood was collected from the eyeballs. After standing for 40 minutes, the blood was centrifuged at 3000 r / min for 20 minutes to collect serum. The levels of AGE, IL-6, and TNF-α in the skin were detected using an ELISA kit. Figure 6 As shown:

[0178] (1) AGE content: Compared with the control group (262.70 ng / L), the AGE content in the serum of the model group increased significantly to 406.22 ng / L. Oral administration of postbiotics prepared by *Lactobacillus plantarum* CCFM1354 significantly reduced the content of AGE, a marker of glycosylated aging, in the serum of mice. The CCFM1354_Z group and the CCFM1354_M group reduced the serum AGE content to 305.51 ng / L and 286.87 ng / L, respectively (a reduction of 24.8% and 29.4% compared with the model group). Oral administration of live *Lactobacillus plantarum* CCFM1354 had the best effect in reducing the AGE content in the serum of aging mice, reducing the AGE content to 178.67 ng / L (a reduction of 56% compared with the model group).

[0179] (2) IL-6 content: Compared with the control group (80.20 ng / L), the IL-6 content in the serum of the model group increased significantly to 126.79 ng / L. Oral administration of postbiotic prepared by Lactobacillus plantarum CCFM1354 significantly reduced the IL-6 content in the serum of mice. The IL-6 content in the serum of the CCFM1354_Z group, CCFM1354_M group and CCFM1354 live bacteria group was 89.18 ng / L, 114.167 ng / L and 97.19 ng / L, respectively. Among them, CCFM1354_Z reduced the IL-6 content of inflammatory factor in the model group by 29.66%, which was the most significant effect.

[0180] (3) TNF-α content: Compared with the control group (297.24 ng / L), the serum TNF-α content in the model group increased significantly to 382.15 ng / L. Oral administration of postbiotic prepared by Lactobacillus plantarum CCFM1354 significantly reduced the serum TNF-α content in mice. The CCFM1354_Z group, CCFM1354_M group and CCFM1354 live bacteria group reduced the serum TNF-α content to 324.40 ng / L, 379.53 ng / L and 374.67 ng / L, respectively. Only CCFM1354_Z significantly reduced the inflammatory factor TNF-α content in the model group by 15.1%.

[0181] Based on the results of relevant biochemical indicators in animal serum, it can be concluded that Lactobacillus plantarum CCFM1354 and its prepared metabiotic (inactivated bacterial cells) can combat the overall aging of the host by significantly reducing AGE content and reducing the content of inflammatory factors IL-6 and TNF-α, thereby resisting glycation and alleviating inflammation.

[0182] Example 10: Effects of Lactobacillus plantarum CCFM1354 and its prepared postbiotic on AGE content in the skin of aging mice

[0183] The method for establishing the animal model involved in the following examples is the same as in Example 9. The difference is that after the mice were sacrificed after the experiment, the skin tissue on the back was cut off and ground into a homogenate at a weight-to-volume ratio of 1:10 with PBS. The homogenate was centrifuged at 3000 r / min for 20 min and the supernatant was taken to detect the AGE content in the skin using an ELISA kit.

[0184] The results of detecting the levels of AGE (glycation end products) markers in various organs are as follows: Figure 7 As shown:

[0185] Skin: Skin is the largest organ in the body. The accumulation of AGEs in the skin affects skin condition, and increased AGE content leads to significant changes in skin aging characteristics. Compared with the control group (336.76 ng / L), the AGE content in the model group's skin significantly increased to 463.21 ng / L. Oral administration of postbiotics prepared from Lactobacillus plantarum CCFM1354 reduced the content of AGE markers in mouse skin. The CCFM1354_Z group and CCFM1354_M group reduced the AGE content in the skin to 406.50 ng / L and 421.13 ng / L, respectively (a reduction of 12.2% and 9.1% compared with the model group). Oral administration of live CCFM1354 reduced the AGE content in the skin to 350.13 ng / L, a reduction of 24.4% compared with the model group, showing significant effects.

[0186] The results above indicate that *Lactobacillus plantarum* CCFM1354 and its prepared metabiotics (inactivated cells and fermentation supernatant) can not only significantly reduce the AGE content in serum, but also alleviate the accumulation of AGE in skin tissue, thereby achieving the goal of host anti-glycation, anti-aging, and improved skin health.

[0187] Example 11: Effects of Lactobacillus plantarum CCFM1354 and its prepared postbiotic on the skin's resistance to glycation damage in aging mice.

[0188] The animal models used in the following examples were established using the same method as in Example 9. At the end of the experiment, the stratum corneum water content on the backs of each mouse was measured using a skin elasticity tester MPA580 from CK GmbH (Germany) equipped with a stratum corneum moisture measurement probe (Corneometer CM825). The results are shown below. Figure 8 As shown.

[0189] The preparation method for mouse skin tissue homogenates used for ELISA kit detection was the same as in Example 10. The type III collagen content in the skin homogenate was detected using a skin type III collagen ELISA kit, and the results are as follows: Figure 9 As shown.

[0190] RNA was extracted from skin using the Trizol method and reverse transcribed into cDNA to detect gene expression at relevant target sites during glycation loss. Genes directly related to glycation damage in the skin included COL3A1, MMP-2, DDOST, and RAGE. The primers used are described in Table 2 below, and the gene expression detection results are as follows: Figure 10 As shown.

[0191] Table 2 Primer Sequences

[0192]

[0193] (1) The moisture content of the stratum corneum is from Figure 8 It can be seen that, compared with the blank group (66.78%), the water content of the model group was significantly reduced to 47.95%. The water content of the stratum corneum in the *Lactobacillus plantarum* CCFM1354_Z group (62.75%) was about 30.87% higher than that in the model group. The water content of the *Lactobacillus plantarum* CCFM1354_M group (55.62%) was about 15.60% higher than that in the model group. The water content of the *Lactobacillus plantarum* CCFM1354 group (62.34%) was 29.97% higher than that in the model group. In other words, the experimental results show that the postbiotic prepared from *Lactobacillus plantarum* CCFM1354, especially its inactivated cells (*Lactobacillus plantarum* CCFM1354_Z), can increase the water content of the back of aged mice after glycation damage.

[0194] During aging, AGE concentration gradually increases, cross-links with surrounding longevity proteins, or enhances the expression of metalloproteinases through the AGE-RAGE pathway, leading to loosening of skin structure and decreased water-holding capacity of the stratum corneum. Exogenous supplementation with post-glycation endogenous bacteria with anti-glycation functions can alleviate skin moisture loss during aging. *Lactobacillus plantarum* CCFM1354 and its prepared post-glycation endogenous bacteria, when taken orally, have the effect of preventing skin dryness during aging. Among them, the post-glycation endogenous bacteria prepared from *Lactobacillus plantarum* CCFM1354 (CCFM1354_Z) significantly alleviated the decrease in stratum corneum moisture content caused by glycation damage compared to the pure fermentation supernatant CCFM1354_M group, while live CCFM1354 bacteria had the strongest upregulating effect on stratum corneum moisture content.

[0195] (2) The content of type III collagen in the skin on the back of mice is as follows Figure 9 As shown, compared with the blank group (7.17 μg / L), the skin elasticity and firmness of the model group were significantly reduced to 6.09 μg / L. The skin elasticity of the *Lactobacillus plantarum* CCFM1354_Z group (8.16 μg / L) increased by 33.93% compared with the model group, and the skin elasticity of the CCFM1354_M group (7.60 μg / L) increased by 24.78% compared with the model group. The post-biotic prepared from *Lactobacillus plantarum* CCFM1354 significantly increased the collagen content in the back of aging mice.

[0196] (3) To investigate the specific mechanism by which post-genetic agents inhibit glycation damage from affecting collagen synthesis, skin gene expression was detected. The results are as follows: Figure 10As shown, ① the post-biotic CCFM1354_Z prepared from *Lactobacillus plantarum* CCFM1354 significantly downregulated the expression of AGE receptor RAGE mRNA to a relative expression level of 0.93 (68.4% lower than the model group's 2.95); simultaneously upregulated the expression of the RAGE competitive receptor AGER1, i.e., DDOST mRNA, to 0.95 (129.3% higher than the model group's 0.41), thus inhibiting AGE-RAGE binding from two aspects. Live *Lactobacillus plantarum* CCFM1354 reduced RAGE mRNA to 1.01 (65.6% lower than the model group), a slightly lower effect than CCFM1354_Z; however, the live bacteria did not significantly increase the expression of the RAGE competitive receptor DDOST mRNA, with an expression level of only 0.45 (model group 0.41). ② The post-biotic CCFM1354_Z, prepared from *Lactobacillus plantarum* CCFM1354, significantly downregulated the expression of the metalloproteinase MMP-2 mRNA, reducing its relative expression level to 0.95 (a 67.6% decrease compared to the model group of 2.77). Live *Lactobacillus plantarum* CCFM1354 reduced MMP-2 mRNA expression to 0.48 (an 83.8% decrease compared to the model group). This indicates that both *Lactobacillus plantarum* CCFM1354 and its prepared post-biotic can inhibit the adverse effects of glycation on protein function. ③ Both *Lactobacillus plantarum* CCFM1354 and its prepared post-biotic CCFM1354_Z significantly upregulated the expression of type III collagen synthase COL3A1 mRNA, with expression levels of 2.04 and 1.89 after administration (an increase of 362.3% and 314.2% respectively compared to the model group of 0.44), alleviating the abnormal decline in type III collagen synthesis caused by glycation loss and maintaining normal collagen function.

[0197] This study demonstrates that *Lactobacillus plantarum* CCFM1354 and its prepared metabiotics can inhibit AGE-RAGE binding by downregulating RAGE mRNA expression and upregulating DDOST mRNA expression, thereby reducing the continuous downstream glycation reaction and targeting anti-glycation and anti-aging. Furthermore, by downregulating MMP-2 mRNA expression and upregulating COL3A1 mRNA expression, it can alleviate the weakening of protein function caused by glycation damage during aging and increase collagen synthesis, thereby improving the host's skin health.

[0198] Example 12: Effect of *Lactobacillus plantarum* CCFM1354 fermentation of peanut skin on improving the anti-saccharification ability of peanut skin

[0199] (1) Fermentation of peanut skin supernatant by Lactobacillus plantarum CCFM1354 enhances the in vitro anti-glycation ability of peanut skin:

[0200] The preparation of peanut skin fermentation culture medium (hsp) and peanut skin fermentation supernatant of Lactobacillus plantarum CCFM1354 (CCFM1354_H) is as shown in Example 4; the construction of the in vitro fructose-bovine serum albumin system and the generation and detection of fluorescent AGE are as shown in Example 5; the detection of the prevention of glycosylation damage caused by acetone aldehyde to HSF cells is as shown in Example 7.

[0201] Peanut skin extract mainly contains flavonoids such as proanthocyanidins, resveratrol, and quercetin. Oligomeric proanthocyanidins possess bioactivities including free radical scavenging, antioxidant activity, cardiovascular protection, anti-inflammation, and tumor inhibition. Since 2017, numerous papers have reported the in vitro inhibitory effect of peanut skin on AGE formation, and its anti-glycation damage ability has also been verified in cell experiments. However, the content of anti-glycation active substances in peanut skin is low, and its bioavailability is not high. Furthermore, high-concentration peanut skin extract is dark red to black in color. To better utilize the anti-glycation properties of peanut skin, we attempted to ferment peanut skin using *Lactobacillus plantarum* CCFM1354 to prepare *Lactobacillus plantarum* CCFM1354 fermented peanut skin supernatant (CCFM1354_H). We then used a peanut skin fermentation culture medium of 100 μg / mL and the concentration of the fermented peanut skin supernatant to verify the effect of *Lactobacillus plantarum* CCFM1354 on the anti-glycation ability of peanut skin.

[0202] Depend on Figure 11 It was found that the original inhibition rate of the fluorescent AGE formation in the fructose-bovine serum albumin system was 61.70%, while the inhibition rate of the supernatant after fermentation with *Lactobacillus plantarum* CCFM1354 reached 69.77%, an increase of 13.1% compared to the unfermented group, indicating a significant increase in the ability to inhibit AGE formation. Furthermore, after administration of peanut skin to prevent acetone aldehyde damage, the cell viability of HSF cells was only 56.16%, a 5.5% improvement compared to the model group (53.23%). After fermentation with *Lactobacillus plantarum* CCFM1354, the cell viability of HSF cells increased to 76.07%, significantly improving the cell viability of HSF cells after acetone aldehyde damage (an increase of 42.9% compared to the model group). Therefore, *Lactobacillus plantarum* CCFM1354 can enhance its original in vitro anti-glycation ability by fermenting peanut skin.

[0203] (2) Effect of *Lactobacillus plantarum* CCFM1354 fermentation supernatant on increasing serum AGE levels in mice with aging after oral administration of peanut skin:

[0204] For the experimental construction of aging mice and the methods for serum and skin AGE detection, as in Examples 9 and 10, the group that was gavaged with a synthetic peanut skin preparation included: live bacteria *Lactobacillus plantarum* CCFM1354 and peanut skin fermentation broth (denoted as CCFM1354+hsp).

[0205] Depend on Figure 12 It was found that although peanut skin itself could reduce the AGE content in the serum of aging mice to 246.95 ng / L (control group 262.70 ng / L, model group 406.22 ng / L), a significant reduction of 39.2% compared with the model group; the peanut skin fermentation broth treated with Lactobacillus plantarum CCFM1354 was more effective, with the AGE content reaching 208.42 ng / L, a reduction of 48.7% compared with the model group; the effect of the peanut skin synthetic preparation group was not as good as the other two groups, and it could only reduce the AGE content by 27.1% of the model group.

[0206] The above experimental results, combined with in vitro experimental results, demonstrate that fermentation of peanut skin by *Lactobacillus plantarum* CCFM1354 not only enhances the anti-glycation function of unfermented peanut skin in inhibiting AGE production in vitro, but also exerts a synergistic effect in vivo after oral administration. In other words, the anti-glycation ability of peanut skin is further enhanced by *Lactobacillus plantarum* CCFM1354.

[0207] (3) The effect of oral administration of peanut skin supernatant fermented with Lactobacillus plantarum CCFM1354 on alleviating skin glycation damage in aging mice:

[0208] Skin collagen content is also a direct biochemical indicator for detecting the skin condition of aging mice. Aging modeling significantly reduces the content of type III collagen in mouse skin, with a level of 6.09 μg / mL in the model group (compared to 7.17 μg / mL in the control group). The HSP group significantly increased the content of type III collagen to 7.43 μg / mL (22.0% higher than the model), and CCFM1354_H significantly increased the content of type III collagen to 8.42 μg / mL (38.3% higher than the model). The synthetic preparation group increased the content of type III collagen but only reached 6.73 μg / mL (10.5% higher than the model).

[0209] The above results collectively demonstrate that the supernatant of peanut skin fermented by *Lactobacillus plantarum* CCFM1354 alleviates the effects of glycation damage by increasing the content of type III collagen in the skin of aging mice after oral administration of peanut skin.

[0210] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of *Lactobacillus plantarum* ( Lactiplantibacillus plantarum CCFM1354 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 25, 2023, with accession number GDMCC No: 63924.

2. A metabiotic prepared using *Lactobacillus plantarum* CCFM1354 according to claim 1, wherein the metabiotic comprises inactivated cells, fermentation supernatant, and / or cell lysate; the method for preparing the inactivated cells is as follows: culturing *Lactobacillus plantarum* CCFM1354 in a culture medium for a period of time, collecting the cell cells in the cell culture medium, and obtaining inactivated cell cells after heat treatment; the method for preparing the cell lysate is as follows: culturing *Lactobacillus plantarum* CCFM1354 in a culture medium for a period of time, collecting the cell cells, homogenizing under high pressure, centrifuging, collecting the supernatant after centrifugation, and obtaining the cell lysate; the method for preparing the fermentation supernatant is as follows: culturing *Lactobacillus plantarum* CCFM1354 in a culture medium for a period of time, and obtaining the supernatant after centrifugation of the culture.

3. A composition containing *Lactobacillus plantarum* CCFM1354 as described in claim 1 and / or the metabiotic as described in claim 2.

4. The composition according to claim 3, characterized in that, The composition includes food, medicine, or health products.

5. The composition according to claim 4, wherein the composition is the supernatant obtained by fermenting *Lactobacillus plantarum* CCFM1354 in a culture medium containing peanut skin.

6. The application of the post-genetic agent as described in claim 2 in the preparation of cosmetics.

7. The application according to claim 6, characterized in that, Includes at least one of the following functions: (1) Inhibit the formation of fluorescent AGEs; (2) Prevent damage and functional decline of skin fibroblasts caused by high glucose and AGE formation intermediates; (3) Reduce individual signs of aging; (4) Prevent abnormal degradation of collagen in skin fibroblasts under high glucose culture; (5) Prevent abnormal expression of EGR2 mRNA, a key growth regulator of skin fibroblasts under high glucose culture. (6) Reduce the AGE content in the serum, skin, liver, kidneys, and brain tissue of aging individuals; (7) Reduce the levels of inflammatory markers IL-6 and TNF-α in the serum of aging individuals; (8) Reduce the decrease in the moisture content of the stratum corneum of the back skin in aging individuals caused by glycation damage; (9) Reduce the decrease in type III collagen content in the back skin of aging individuals caused by glycation damage.

8. The use of *Lactobacillus plantarum* CCFM1354 as described in claim 1 or the metabiotic as described in claim 2 in the preparation of food.

Images