Inactivated culture and product of lactobacillus plantarum bgi-n6 with anti-wrinkle and anti-aging effects

Abstract

The present application belongs to the field of microbial technology, and more specifically, the present application relates to an inactivated culture of plant lactobacillus BGI-N6, its use and products containing the inactivated culture. The present application provides an inactivated culture product of probiotics, which can break through the condition limitation of application of active probiotics, effectively eliminate free radicals, inhibit collagenase and elastase, inhibit skin pathogenic bacteria, so as to achieve better effect of slowing down skin aging.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and more specifically, this invention relates to an inactivated culture of Lactiplantibacillus plantarum BGI-N6, its uses, and products containing said inactivated culture. Background Technology

[0002] The skin consists of three layers: the epidermis, dermis, and subcutaneous tissue. As the skin ages, these three components undergo degenerative changes, with the dermis showing the most significant alterations. The external characteristics of skin aging mainly manifest as sagging skin, decreased elasticity, wrinkles, dull skin tone, dryness, and roughness. The skin aging process involves both endogenous factors (genetics, excess free radicals, DNA damage, etc.) and exogenous factors (UV radiation, exposure to chemicals, smoking, lack of sleep, etc.). Regardless of whether it's endogenous or exogenous aging, wrinkle formation and decreased elasticity are the most common and typical symptoms of skin aging. One of the main mechanisms of dermal atrophy is considered to be the reduction in the content of the extracellular matrix (collagen and elastin). In aging skin, collagen production decreases while degradation increases, leading to an overall reduction in collagen quantity. Oxidative stress induces elevated levels of matrix metalloproteinases in the body, further reducing collagen synthesis and accelerating collagen degradation, resulting in deeper wrinkles. Furthermore, aging skin gradually weakens, becoming more susceptible to environmental aggressors, leading to impaired skin barrier function. Therefore, it is necessary to develop anti-wrinkle products to delay skin aging, and antioxidants and protease inhibitors are effective ingredients in anti-wrinkle products.

[0003] Probiotics have been proven to possess a variety of beneficial properties for the skin, such as antioxidant activity, regulation of skin inflammation, treatment of various skin diseases, prevention of allergic contact dermatitis, and inhibition of skin pathogens. Probiotics can also replenish beneficial bacteria in the skin, strengthen the skin barrier, promote better moisture absorption, and help delay signs of skin aging. *Lactobacillus plantarum* is one of the most widely studied probiotics, with broad sources, safety, non-toxicity, and significant probiotic effects. Current research indicates that *Lactobacillus plantarum* has been assessed to have skin-targeting beneficial effects when administered topically or orally with live or dead cells, cell lysates, or fermentation broth (supernatant).

[0004] Currently, existing technologies mainly focus on the wrinkle-improving effects of probiotic cultures, lacking analysis of their specific active ingredients. This greatly limits the process development and functional application of probiotics and their cultures.

[0005] Therefore, it is necessary to study the specific active ingredients in inactivated probiotic cultures to clarify their efficacy and enhance their application value and market potential. Furthermore, there is an urgent need in this field for an inactivated probiotic culture product that can overcome the limitations of live probiotic applications, effectively scavenging free radicals, inhibiting collagenase and elastase, suppressing skin pathogens, reducing wrinkles, and strengthening the skin barrier, thereby achieving a better effect in slowing down skin aging. Summary of the Invention

[0006] In view of this, in a first aspect, the present invention provides an inactivated culture of Lactiplantibacillus plantarum BGI-N6, which was deposited at the China Center for Type Culture Collection on June 5, 2023, with accession number CCTCC NO: M2023948.

[0007] In a second aspect, the present invention provides the use of the inactivated culture of the first aspect of the present invention in enhancing the antioxidant capacity of the skin.

[0008] In a third aspect, the present invention provides the use of the inactivated culture of the first aspect of the invention in inhibiting collagenase in the skin.

[0009] In a fourth aspect, the present invention provides the use of the inactivated culture of the first aspect of the present invention in the inhibition of elastase in the skin.

[0010] In a fifth aspect, the present invention provides the use of the inactivated culture of the first aspect of the present invention in preventing or slowing down skin aging.

[0011] In a sixth aspect, the present invention provides a product for preventing or slowing down skin aging, said product comprising an inactivated culture of the first aspect of the present invention.

[0012] The beneficial effects of the present invention include one or more of the following, but are not limited thereto:

[0013] 1) The inactivated culture of BGI-N6 of Lactobacillus plantarum provided by the present invention includes inactivated bacterial cells and metabolites, and belongs to postbiotic products. It contains bioactive substances with probiotic functions, which can overcome the condition limitations of active probiotics.

[0014] 2) The inactivated culture of BGI-N6 of Lactobacillus plantarum provided by the present invention is rich in active ingredients such as lactic acid, phenyllactic acid and hydroxymethylcoumarin, and can be used to prepare anti-wrinkle and anti-aging products;

[0015] 3) The inactivated culture of BGI-N6 of Lactobacillus plantarum provided by the present invention can effectively scavenge free radicals, inhibit collagenase and elastase, inhibit skin pathogens, reduce wrinkles, and enhance the skin barrier, thereby achieving the effect of slowing down skin aging.

[0016] 4) Compared with existing anti-wrinkle and anti-aging microbial or post-biotic products, the inactivated culture of Lactobacillus plantarum BGI-N6 provided by the present invention has excellent, or even better, effects in slowing down skin aging. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other implementation schemes can be obtained based on these drawings without creative effort.

[0018] Figure 1 The results of antioxidant capacity assays for Lactobacillus plantarum BGI-N6 postbiotics were presented, specifically including the DPPH radical scavenging rate, hydroxyl radical scavenging rate, and reducing capacity of Lactobacillus plantarum BGI-N6 postbiotic (N6), with vitamin C as a positive control.

[0019] Figure 2 The inhibition rate of *Lactobacillus plantarum* BGI-N6 postgenerant against collagenase was shown, specifically including the test results of *Lactobacillus plantarum* BGI-N6 postgenerant (N6) diluted by one time (i.e., 50% concentration postgenerant), *Lactobacillus plantarum* 299v postgenerant (299v), and *Lactobacillus rhamnosus* GG (LGG), with 0.1 mg / mL epigallocatechin gallate (EGCG) as a positive control.

[0020] Figure 3 The inhibition rate of elastase by Lactobacillus plantarum BGI-N6 postbiotic was shown, specifically including the test results of Lactobacillus plantarum BGI-N6 postbiotic (N6), Lactobacillus plantarum 299v postbiotic (299v) and Lactobacillus rhamnosus GG postbiotic (LGG), with 0.1 mg / mL EGCG as a positive control.

[0021] Figure 4 This is the LC / MS spectrum of MRS medium and Lactobacillus plantarum BGI-N6 postbiotic.

[0022] Figure 5 The inhibition rates of six components in the postbiotic of *Lactobacillus plantarum* BGI-N6 on collagenase were shown.

[0023] Figure 6The inhibition rates of six components in the postbiotic of *Lactobacillus plantarum* BGI-N6 against elastase were shown. Detailed Implementation

[0024] The present invention will now be clearly and completely described in conjunction with its embodiments and accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments that can be obtained by those skilled in the art based on the embodiments of the present invention are within the scope of protection of the present invention.

[0025] As mentioned above, existing technologies lack an analysis of the specific active ingredients of Lactobacillus plantarum for anti-wrinkle and anti-aging effects, and are limited by the strict application conditions of live probiotics.

[0026] Therefore, the purpose of this invention is to provide an inactivated culture of Lactobacillus plantarum BGI-N6, which can overcome the limitations of the application of active probiotics and produce a better effect in slowing down skin aging.

[0027] Therefore, in a first aspect, the present invention provides an inactivated culture of *Lactobacillus plantarum* BGI-N6, which was deposited on June 5, 2023, at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, with accession number CCTCC NO: M2023948.

[0028] In this invention, the inactivated culture of *Lactobacillus plantarum* BGI-N6 is a mixture obtained by fermenting and inactivating *Lactobacillus plantarum* BGI-N6. Therefore, the main components of the inactivated culture include microbial cells, residual culture medium, nutrients not fully utilized by the microorganisms, various metabolites of the microorganisms, and small molecules released after microbial inactivation. The inactivated culture described in this invention conforms to the definition of postbiotics by the International Society for the Study of Probiotics and Prebiotics (ISAPP). Therefore, "inactivated culture" and "postbiotic" are used interchangeably and have equivalent meanings herein.

[0029] In this invention, "fermentation" refers to inoculating *Lactobacillus plantarum* BGI-N6 into a culture medium for expansion culture, and then allowing it to stand at a suitable temperature for a period of time to obtain a fermentation mixture of *Lactobacillus plantarum* BGI-N6. The culture medium can be MRS medium, M17 medium, or TPY medium, preferably MRS medium; the fermentation temperature can be 35°C to 38°C, preferably 37°C; and the fermentation time can be 16h to 48h, preferably 24h.

[0030] In some implementations, *Lactobacillus plantarum* BGI-N6 is inoculated into MRS liquid medium for expansion culture and then incubated statically at 37°C for 24 hours.

[0031] In some embodiments, the OD of the fermentation broth of *Lactobacillus plantarum* BGI-N6 is... 600 =1.6±0.2.

[0032] In some embodiments, the cell count in the fermentation broth of *Lactobacillus plantarum* BGI-N6 is 1-3 × 10⁻⁶. 9 CFU / mL.

[0033] In this invention, the fermentation product of *Lactobacillus plantarum* BGI-N6 is inactivated to obtain an inactivated culture. The inactivation treatment can be carried out using various microbial inactivation methods known to those skilled in the art, as long as the inactivation method does not destroy the active ingredients in the *Lactobacillus plantarum* BGI-N6 fermentation product. The inactivation treatment includes, but is not limited to, heat treatment, physical treatment (e.g., high-pressure treatment, ultrasonic treatment), and chemical treatment (e.g., enzymatic hydrolysis, solvent extraction), among which heat treatment is simple and easy to operate and suitable for production. Therefore, in some embodiments, the inactivation treatment method is heat treatment, which can be carried out under the following conditions: placing the *Lactobacillus plantarum* BGI-N6 fermentation mixture in a water bath at 55°C to 65°C for 15 to 60 minutes; preferably, placing the *Lactobacillus plantarum* BGI-N6 fermentation mixture in a water bath at 60°C for 30 minutes.

[0034] In some embodiments of the present invention, due to the limitations of the experimental methods, only the supernatant of the inactivated culture of *Lactobacillus plantarum* BGI-N6 was used for effect verification. However, it is understood that the technical effect obtained by using the supernatant for verification is also applicable to the inactivated culture of *Lactobacillus plantarum* BGI-N6.

[0035] In some embodiments, the inactivated culture comprises inactivated cells and metabolites of Lactobacillus plantarum BGI-N6.

[0036] Through experiments, the inventors discovered that in the inactivated culture of *Lactobacillus plantarum* BGI-N6, the levels of lactic acid, 6-hydroxyhexanoic acid, phenyllactic acid, hydroxymethylcoumarin, hydroxyphenyllactic acid, and 2-hydroxy-3-methylbutyric acid were significantly increased. Furthermore, they found that lactic acid, phenyllactic acid, and hydroxymethylcoumarin exhibited significant collagenase and elastase inhibitory abilities, making them effective anti-wrinkle and anti-aging active ingredients in the inactivated culture of BGI-N6.

[0037] In some embodiments, the inactivated culture contains lactic acid, phenyllactic acid, and / or hydroxycoumarin as active ingredients.

[0038] In a second aspect, the present invention provides the use of the inactivated culture of the first aspect of the present invention in enhancing the antioxidant capacity of the skin.

[0039] As the body's first line of defense, the skin is constantly exposed to the external environment and is easily attacked by exogenous factors. This leads to the production of a large number of free radicals in skin cells, which in turn triggers a series of oxidation reactions, accelerating skin aging. "Antioxidant" refers to the skin cells using their own defense system or externally provided antioxidants to combat and eliminate free radicals, reducing their damage to the skin and maintaining its health and vitality. "Free radicals" are atoms or molecules with unpaired electrons produced during the body's metabolism. They are highly reactive and easily react with other molecules, thereby damaging cell structure and function. Long-term accumulation can easily lead to skin aging, age spots, wrinkles, and other problems.

[0040] In some implementations, enhancing the skin's antioxidant capacity includes scavenging free radicals in the skin, including DPPH free radicals and hydroxyl free radicals.

[0041] In a third aspect, the present invention provides the use of the inactivated culture of the first aspect of the invention in inhibiting collagenase in the skin.

[0042] Collagenase is an enzyme in the matrix metalloproteinase family that degrades collagen in the skin, leading to skin aging. A higher collagenase inhibition rate indicates stronger anti-wrinkle ability. In this invention, the inventors discovered that an inactivated culture of *Lactobacillus plantarum* BGI-N6 can effectively inhibit the activity of collagenase in the skin, thus delaying skin aging.

[0043] In a fourth aspect, the present invention provides the use of the inactivated culture of the first aspect of the present invention in the inhibition of elastase in the skin.

[0044] Elastase is an enzyme in the matrix metalloproteinase family that degrades elastin in the skin, causing it to lose elasticity. A higher elastase inhibition rate indicates stronger anti-wrinkle ability. In this invention, the inventors discovered that an inactivated culture of *Lactobacillus plantarum* BGI-N6 can effectively inhibit the activity of elastase in the skin, maintaining skin elasticity.

[0045] In a fifth aspect, the present invention provides the use of the inactivated culture of the first aspect of the present invention in preventing or slowing down skin aging.

[0046] As described above, the inventors have discovered that an inactivated culture of *Lactobacillus plantarum* BGI-N6 can effectively inhibit the activity of collagenase and elastase in the skin. Therefore, this inactivated culture can prevent or slow down skin aging.

[0047] In a sixth aspect, the present invention provides a product for preventing or slowing down skin aging, said product comprising an inactivated culture of the first aspect of the present invention.

[0048] In some implementations, the products include skincare products, cosmetics, and food.

[0049] In some implementations, the product is administered topically or orally.

[0050] In some embodiments, the main anti-wrinkle ingredients of the product are lactic acid, phenyllactic acid, and / or hydroxycoumarin.

[0051] Those skilled in the art will understand that the descriptions relating to inactivated cultures above apply to the sixth aspect of the present invention, and will not be repeated here.

[0052] The present invention will be further described below through the following embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0053] Example

[0054] Example 1. Preparation of *Lactobacillus plantarum* BGI-N6 postbiotic (N6)

[0055] Strain activation: Under aseptic conditions, cryopreserved Lactiplantibacillus plantarum BGI-N6 (preservation number: CCTCC NO: M2023948) was inoculated into MRS liquid medium (purchased from Guangdong Huankai Microbial Technology Co., Ltd.) at a ratio of 2% (v / v) and cultured at 37℃ for 24 h to obtain Lactiplantibacillus plantarum seed culture.

[0056] Inoculation and fermentation: The seed culture was transferred to an appropriate amount of MRS liquid medium at a ratio of 2% (v / v) for scale-up culture, and then incubated statically at 37°C for 24 hours to obtain the fermentation broth (OD). 600 =1.6±0.2, bacterial count is 1-3×10 9 (CFU / mL)

[0057] Inactivation: The fermentation broth was placed in a 60°C water bath for 30 minutes to obtain an inactivated fermentation broth, which is the *Lactobacillus plantarum* BGI-N6 postbiotic.

[0058] Comparative Example 1. Preparation of *Lactobacillus plantarum* 299v postbiotic (299v)

[0059] Using the same preparation method as in Example 1, *Lactobacillus plantarum* 299v (a commercially available control strain preserved in the laboratory) was fermented and inactivated to obtain *Lactobacillus plantarum* 299v post-biotic.

[0060] Comparative Example 2. Preparation of Lactobacillus rhamnosus GG postbiotic (LGG)

[0061] Using the same preparation method as in Example 1, Lactobacillus rhamnosus GG (a commercially available control strain preserved in the laboratory) was fermented and inactivated to obtain Lactobacillus rhamnosus GG post-genetic precursor (LGG).

[0062] Example 2. Antioxidant Capacity Test

[0063] The following three antioxidant capacity tests were performed on the postbiotic (N6) of *Lactobacillus plantarum* BGI-N6:

[0064] Determination of the free radical scavenging ability of 1,2,2-biphenyl-1-picrylhydrazine (DPPH)

[0065] Prepare a 0.2 mmol / L DPPH solution using anhydrous ethanol. Add 0.1 mL of N6 to 3 mL of DPPH solution, mix well, and react in the dark at room temperature for 30 min. Then centrifuge and measure the absorbance (A) of 200 μL of the reaction solution at a wavelength of 517 nm. 样 Vitamin C (Vc) was used to replace the postbiotic from *Lactobacillus lactis* as a control, and a sample control well containing the postbiotic to be tested + anhydrous ethanol was also set up (A). 样空白 (A0) blank wells containing anhydrous ethanol and distilled water and (A1) control wells containing DPPH solution and distilled water.

[0066] The calculation formula is: DPPH free radical scavenging rate = [1 - (A 样 -A 样空白 [(A1-A0)]×100%

[0067] The higher the DPPH free radical scavenging rate of a sample, the stronger its antioxidant capacity. The results are as follows: Figure 1 As shown, the DPPH scavenging rate of N6 was 68.52%, which was higher than that of Vc at 0.2 mg / mL, showing a statistically significant difference. This indicates that Lactobacillus plantarum BGI-N6 postbiotic has a strong DPPH free radical scavenging ability.

[0068] 2. Determination of hydroxyl radical scavenging ability

[0069] Prepare 0.01 mol / L phosphate buffer, 2.5 mmol / L o-phenanthroline solution, 2.5 mmol / L ferrous sulfate solution, and 0.1% hydrogen peroxide solution, respectively. Take 1 mL of phosphate buffer, 1 mL of o-phenanthroline solution, 1 mL of deionized distilled water, and 1 mL of ferrous sulfate solution, mix well, then add 0.5 mL of N6 and mix well. Finally, add 1 mL of hydrogen peroxide solution and mix well. Incubate at 37℃ for 1 h. Take 200 μL of the reaction solution and measure the absorbance at a wavelength of 536 nm (A). 样 Vc was used instead of N6 as a control, and control wells (A1) without hydrogen peroxide and blank wells (A0) without N6 were also set up.

[0070] The calculation formula is: Hydroxyl radical scavenging rate = (A 样 -A0) / (A1-A0)×100%

[0071] The higher the hydroxyl radical scavenging rate of a sample, the stronger its antioxidant capacity. Results are as follows... Figure 1 As shown, the hydroxyl radical scavenging rate of N6 was 50.26%, which was higher than that of Vc at 1 mg / mL, indicating that Lactobacillus plantarum BGI-N6 postbiotic has a strong hydroxyl radical scavenging ability.

[0072] 3. Reducing power measurement

[0073] Prepare 0.2 mol / L phosphate buffer (pH 6.6), 1% potassium ferricyanide solution, 10% trichloroacetic acid solution, and 0.1% ferric chloride solution, respectively. Take 0.5 mL of phosphate buffer, add 0.1 mL of N6 or control (Vc) solution and 0.5 mL of potassium ferricyanide solution, mix well, incubate at 50 °C for 30 minutes, then add 0.5 mL of trichloroacetic acid and mix well. Centrifuge at 3000 r / min for 10 minutes, take 100 μL of supernatant, then add 100 μL of distilled water and 25 μL of ferric chloride solution and mix well. React at room temperature for 10 minutes, and measure the absorbance at a wavelength of 700 nm.

[0074] In this test, the sample's reducing power reduced the ferric iron in potassium ferricyanide to ferrous iron. The ferrous iron then reacted with ferric chloride to form Prussian blue, which has maximum absorbance at 700 nm. Therefore, the higher the absorbance of the sample, the stronger its reducing power. The results are as follows... Figure 1 As shown, the reducing power of N6 was 0.665, which was higher than that of Vc at 0.1 mg / mL, indicating a statistically significant difference. This suggests that the postbiotic of Lactobacillus plantarum BGI-N6 has a strong reducing ability.

[0075] The results of the above three tests indicate that *Lactobacillus plantarum* BGI-N6 postbiotic has a strong antioxidant capacity.

[0076] Example 3. Anti-wrinkle ability test

[0077] The anti-wrinkle ability of the supernatants obtained after centrifugation of *Lactobacillus plantarum* BGI-N6 postgenerant (N6), *Lactobacillus plantarum* 299v postgenerant (299v), and *Lactobacillus rhamnosus* GG postgenerant (LGG) was tested as follows:

[0078] 1. Determination of collagenase inhibition ability

[0079] Prepare a tris(hydroxymethyl)methylglycine (Tricine) buffer (50 mmol / L, pH 7.5) containing 400 mmol / L NaCl and 10 mmol / L CaCl2. Prepare a 0.5 mg / mL collagenase solution and a 1 mmol / L N-[3-(2-furanyl)acryloyl]-leucine-glycine-proline-alanine (FALGPA) solution using the Tricine buffer. Mix 20 μL of a 1-fold diluted supernatant with 20 μL of collagenase solution, then add 120 μL of Tricine buffer. Incubate at 37 °C for 15 min, then add 40 μL of FALGPA solution. After 20 min, measure the absorbance at 335 nm (A). 样 0.1 mg / mL epigallocatechin gallate (EGCG) was used as a control instead of the metagenic agent. Control wells (A1) without the metagenic agent, blank wells (A0) without the metagenic agent and enzyme, and blank sample wells (A1) without the enzyme were also included. 样空白 ).

[0080] The calculation formula is: Collagenase inhibition rate = [1 - (A)] 样 -A 样空白 [(A1-A0)]×100%

[0081] The higher the collagenase inhibition rate of a sample, the stronger its anti-wrinkle ability. Results are as follows... Figure 2 As shown, the collagenase inhibition rate of 50% N6 reached 71.76%, which was significantly higher than that of 299v (69.26%) and LGG (63.08%), and comparable to that of the positive control EGCG (72.04%), indicating that Lactobacillus plantarum BGI-N6 postbiotic has excellent collagenase inhibition ability.

[0082] 2. Elastase Inhibition Capacity Assay

[0083] Prepare 0.2 mol / L Tris-HCl buffer (pH 8.0). Use Tris-HCl buffer to prepare 0.5 U / mL elastase solution and 1.6 mmol / L N-succinyl-alanine-alanine-p-nitroaniline (AAAPAN) solution. Mix 40 μL of Tris-HCl buffer (pH 8.0) with 50 μL of postbiotic solution, then add 30 μL of elastase solution. Incubate at 25 °C for 15 min, then add 80 μL of AAAPAN solution. After 30 min, measure the absorbance at 405 nm (A). 样 0.1 mg / mL epigallocatechin gallate (EGCG) was used as a control instead of the metagenic agent. Control wells (A1) without the metagenic agent, blank wells (A0) without the metagenic agent and enzyme, and blank sample wells (A1) without the enzyme were also included. 样空白 ).

[0084] The calculation formula is: elastase inhibition rate = [1 - (A 样 -A 样空白 [(A1-A0)]×100%

[0085] The higher the elastase inhibition rate of a sample, the stronger its anti-wrinkle ability. The results are as follows... Figure 3 As shown, the elastase inhibition rate of N6 reached 41.97%, which was significantly higher than that of 299v (25.8%), LGG (27.57%) and the positive control EGCG (20.7%), indicating that Lactobacillus plantarum BGI-N6 postbiotic has excellent elastase inhibition ability.

[0086] The above test results indicate that *Lactobacillus plantarum* BGI-N6 postbiotic has excellent anti-wrinkle ability.

[0087] Example 4. Test on the ability to inhibit skin pathogens

[0088] The following pathogenicity tests were performed on the supernatants after centrifugation and filtration of *Lactobacillus plantarum* BGI-N6 postgenerant (N6), *Lactobacillus plantarum* 299v postgenerant (299v), and *Lactobacillus rhamnosus* GG postgenerant (LGG):

[0089] Common skin pathogens such as Staphylococcus aureus, Staphylococcus epidermidis, Propionibacterium acnes, and Candida albicans were selected as indicator bacteria, inoculated into BHI medium, and cultured overnight at 37°C for later use.

[0090] Prepare a double-concentration BHI medium, sterilize it, and inoculate Staphylococcus aureus, Staphylococcus epidermidis, Propionibacterium acnes, and Candida albicans into the double-concentration BHI medium at a 2% (v / v) inoculation rate. Mix well, and take 100 μL of the inoculated double-concentration BHI culture, then add 100 μL of the aforementioned metabiotic. A blank control was set up: 100 μL of the inoculated double-concentration BHI culture + 100 μL of sterile water. OD was then measured. 600 The values ​​are denoted as OD. 实验1 and OD 空白1 After incubation at 37℃ for 20 hours, OD was measured again. 600 The values ​​are denoted as OD. 实验2 and OD 空白2 .

[0091] Calculation formula: Pathogen inhibition rate = 1 - (OD) 实验2 -OD 实验1 ) / (OD 空白2 -OD 空白1 )×100%

[0092] The higher the pathogen inhibition rate of a sample, the stronger its antibacterial ability. Table 1 shows the results: N6 showed an inhibition rate of 97.37% against Staphylococcus aureus, 98.72% against Staphylococcus epidermidis, 94.54% against Propionibacterium acnes, and 76.52% against Candida albicans, indicating that *Lactobacillus plantarum* BGI-N6 postbiotic can effectively inhibit common skin pathogens.

[0093] Table 1 Results of antibacterial ability test

[0094] Post-natal Staphylococcus aureus Staphylococcus epidermidis Propionibacterium acnes Candida albicans 299v 96.57％ 84.75％ 88.92％ 39.60％ LGG 96.15％ 89.37％ 89.36％ 85.02％ N6 97.37％ 98.72％ 94.54％ 76.52％

[0095] Example 5: Detection of active ingredients in *Lactobacillus plantarum* BGI-N6 postbiotic.

[0096] Take 100 μL of centrifuged and filtered MRS liquid culture medium and *Lactobacillus plantarum* BGI-N6 postbiotic, add 1 mL of extraction buffer (methanol:acetonitrile:water = 2:2:1, v / v), vortex to mix, sonicate in an ice-water bath for 10 min, and flash freeze in liquid nitrogen for 1 min, repeating three times. After incubation at -20℃ for 1 h, centrifuge at 13000 r / min and 4℃ for 15 min. Take the supernatant, dry it with nitrogen evaporation apparatus, add 100 μL of acetonitrile:water = 1:1 (v / v) to reconstitute, vortex for 30 s, sonicate in an ice-water bath for 10 min, and centrifuge at 13000 r / min and 4℃ for 15 min. Take the supernatant and perform LC / MS analysis to detect the components of the fermentation broth. The results are shown in Table 2. Figure 4 As shown in a-4b.

[0097] Table 2. Substances that were significantly increased after fermentation with Lactobacillus plantarum BGI-N6

[0098]

[0099] The results showed that lactic acid, 6-hydroxyhexanoic acid, phenyllactic acid, hydroxymethylcoumarin, hydroxyphenyllactic acid, and 2-hydroxy-3-methylbutyric acid were the substances that were significantly increased after inoculation of *Lactobacillus plantarum* BGI-N6 as a metagener.

[0100] Example 6: Verification of the anti-wrinkle ability of the post-biotic active ingredient of Lactobacillus plantarum BGI-N6

[0101] Each active component of *Lactobacillus plantarum* BGI-N6 postbiotic detected in Example 5 was prepared into a 1 mg / mL solution. Following the method provided in Example 3, its collagenase and elastase inhibitory abilities were determined. The results are as follows: Figure 5 , Figure 6 As shown.

[0102] The results showed that lactic acid, phenyllactic acid, and hydroxycoumarin all exhibited significant collagenase and elastase inhibitory abilities, indicating that lactic acid, phenyllactic acid, and hydroxycoumarin in the post-biotic of *Lactobacillus plantarum* BGI-N6 are closely related to its anti-wrinkle effect.

Claims

1. An inactivated culture of Lactiplantibacillus plantarum BGI-N6, wherein Lactiplantibacillus plantarum BGI-N6 was deposited at the China Center for Type Culture Collection on June 5, 2023, with accession number CCTCC NO: M2023948。 2. The inactivated culture according to claim 1, wherein the inactivated culture comprises inactivated cells and metabolites of Lactobacillus plantarum BGI-N6.

3. The inactivated culture according to claim 1 or 2, wherein the inactivated culture comprises lactic acid, phenyllactic acid and / or hydroxycoumarin as active ingredients.

4. Use of the inactivated culture according to any one of claims 1-3 in enhancing the antioxidant capacity of the skin.

5. Use of the inactivated culture according to any one of claims 1-3 in inhibiting collagenase in the skin.

6. Use of the inactivated culture according to any one of claims 1-3 in inhibiting elastase in the skin.

7. Use of the inactivated culture according to any one of claims 1-3 in preventing or slowing down skin aging.

8. A product for preventing or slowing down skin aging, said product comprising the inactivated culture according to any one of claims 1-3.

9. The product according to claim 8, wherein the product includes skin care products, cosmetics, and food.

10. The product of claim 8, wherein the product is administered by topical application or oral administration.

11. The product according to claim 8, wherein the main anti-wrinkle components of the product are lactic acid, phenyllactic acid and / or hydroxymethylcoumarin.

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