Lactobacillus acidophilus ccfm1460 and postbiotic for promoting skin barrier repair

Abstract

The application discloses a lactic acid bacteria CCFM1460 which can produce multiple bioactive substances to promote skin barrier repair and a postbiotic of the lactic acid bacteria CCFM1460, and belongs to the technical field of microorganisms and medical technology.The lactic acid bacteria CCFM1460 provided in the application can produce 3,4-dihydroxyphenyl glycol, N-tetradecyl diethanolamine and 3-phenyl lactic acid through fermentation.The postbiotic of the lactic acid bacteria CCFM1460 has a good skin barrier repair effect.The cell lysate and fermentation supernatant of the lactic acid bacteria CCFM1460 can improve the cell activity of damaged keratinocytes and the expression levels of FLG, IVL and LOR genes, and the cell lysate of the CCFM1460 can improve the expression levels of ZO-1, OCCLUDIN and CLDN genes.Therefore, the lactic acid bacteria has a great application prospect in the preparation of external use or oral medicine or cosmetics for repairing skin barrier.

Description

Technical Field

[0001] This invention relates to a strain of Lactobacillus acidophilus CCFM1460 that produces a variety of bioactive substances to promote skin barrier repair and its post-biotics, belonging to the fields of microbial technology and pharmaceutical technology. Background Technology

[0002] One of the key functions of the skin is to form an effective barrier between the organism and its environment. The outer epidermis is composed of stratified squamous epithelial cells. Keratinocytes constitute the main part of the human skin's epidermis (accounting for approximately 90% of all epidermal cells) and form a barrier against damage from external organisms, heat, ultraviolet radiation, pathogenic bacteria, fungi, parasites, and viruses. Keratinocytes can produce a large number of antimicrobial molecules, further contributing to the formation of a strong protective barrier. By regulating cell signaling pathways, they promote the proliferation of fibroblasts and keratinocytes, enhance the skin's repair capabilities, and accelerate wound healing and tissue remodeling.

[0003] Regarding skin barrier repair, both topical and oral technologies have demonstrated certain effectiveness. For example, patent CN117618306A discloses a soothing and repairing composition and its preparation method, comprising ginseng, coptis chinensis, salvia miltiorrhiza, Dunaliella salina extract, hydrolyzed lupin protein, argan oil, and modified filaggrin peptides, primarily used topically to comprehensively repair the skin barrier. CN118516436A discloses an enzymatic hydrolysis product of sea cucumber collagen peptides with uniform molecular weight (250–1000 Da) and high purity (96%), which exhibits certain skin barrier repair effects when taken orally.

[0004] While these technologies have potential applications, they also have significant limitations: First, the large molecular components in topical compositions (such as filaggrin peptides and some plant extracts) have limited permeability, making it difficult to penetrate deep into the skin barrier and thus limiting their repair effects. Plant extracts also suffer from poor stability, easily becoming ineffective due to environmental influences, and may cause allergic reactions in different individuals, especially those with sensitive skin. Second, although oral forms of sea cucumber collagen peptides have small molecular weights and high purity, their bioavailability still decreases during digestion and absorption, affecting the final repair effect. More importantly, these topical and oral repair methods have limited effectiveness in improving the expression of core regulatory proteins related to the skin barrier (such as filaggrin, endoplasmin, and tight junction proteins), often failing to achieve ideal levels of regulation and lacking long-term, significant regulatory effects.

[0005] Furthermore, from the perspective of metabolites, current research on probiotics mainly focuses on conventional short-chain fatty acids and lactic acid metabolites. However, existing strains generally struggle to produce certain active small molecules closely related to skin health, such as 3,4-dihydroxyphenylethylene glycol (with antioxidant and anti-inflammatory activities), N-tetradecyl diethanolamine (with cell membrane stabilizing and moisturizing effects), and 3-phenyllactic acid (with antibacterial and anti-inflammatory potential), resulting in significant gaps in related research and applications. Therefore, screening and obtaining strains capable of producing these specific active metabolites while significantly improving the expression of core regulatory proteins of the skin barrier remains a pressing technical challenge in this field. Summary of the Invention

[0006] This invention provides the application of Lactobacillus acidophilus CCFM1460 and its postbiotics in the preparation of products for repairing the skin barrier.

[0007] This invention provides a strain of Lactobacillus acidophilus, CCFM1460, which is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 65750.

[0008] In one embodiment, the Lactobacillus acidophilus CCFM1460 is derived from the feces of healthy individuals. The strain is sequenced and analyzed, and the obtained sequence is compared with the nucleic acid sequence in NCBI. The result shows that it belongs to Lactobacillus acidophilus of the Lactobacillus genus, and it is named Lactobacillus acidophilus CCFM1460.

[0009] In one embodiment, the Lactobacillus acidophilus CCFM1460 colonies on MRS solid medium are raised, semi-opaque white, with an uneven surface and rough edges.

[0010] The present invention also provides a metabiotic prepared using the aforementioned Lactobacillus acidophilus CCFM1460.

[0011] In one embodiment, the metabiotic includes cell lysate, inactivated or dead cells, fermentation supernatant, 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 acidophilus CCFM1460 in a culture medium for a period of time, collecting the bacterial cells in the cell culture medium, and obtaining inactivated bacterial cells after heat treatment.

[0013] In one embodiment, the heat treatment conditions are: 65°C for 30 minutes.

[0014] In one embodiment, the method for preparing the bacterial lysate is as follows: Lactobacillus acidophilus CCFM1460 is cultured in a culture medium for a period of time, bacterial cells are collected, homogenized under high pressure, and the supernatant obtained by centrifugation is used to obtain the bacterial lysate.

[0015] In one embodiment, the fermentation supernatant is the supernatant obtained by centrifuging Lactobacillus acidophilus CCFM1460 after culturing it in a culture medium for a period of time.

[0016] The present invention also provides products containing the Lactobacillus acidophilus CCFM1460 and / or its postbiotic.

[0017] In one embodiment, the product includes, but is not limited to, food, medicine, health products, or daily chemical products.

[0018] In one embodiment, the food product includes the above-described composition and conventional excipients.

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

[0020] In one embodiment, the health product includes the above-described composition and conventional excipients.

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

[0022] In one embodiment, the dosage form of the product includes at least one of creams, lotions, oils, liquids, gels, powders, and lyophilized products.

[0023] In one embodiment, the product is a probiotic powder.

[0024] In one embodiment, the bacterial powder is a solid powder of Lactobacillus acidophilus CCFM1460 prepared by drying the liquid postbiotic.

[0025] In one embodiment, drying includes, but is not limited to, preparation by spray drying, vacuum freeze drying, fluidized bed drying, and vacuum drying.

[0026] The present invention also provides the use of the composition in the preparation of products that repair the skin barrier.

[0027] In one embodiment, the product includes at least one of the following functions:

[0028] (1) Increase barrier function proteins in individual skin tissue;

[0029] (2) Increase the barrier function structural proteins of individual skin tissue.

[0030] In one embodiment, the skin barrier disruption-related symptoms include an increase in skin appearance, skin proteins, and inflammatory factors in the blood.

[0031] In one embodiment, the skin damage includes dry skin, decreased elasticity, sagging, wrinkle formation, oxidative damage, and protein loss.

[0032] In one embodiment, the application can be either topical or oral.

[0033] In one embodiment, the product contains at least 1 × 10⁻⁶ Lactobacillus acidophilus CCFM1460. 7 CFU / mL.

[0034] In one embodiment, the dose of the postbiotic prepared from Lactobacillus acidophilus CCFM1460 in the product is not less than 250 μg / kg.

[0035] In one embodiment, the product is a pharmaceutical or cosmetic product.

[0036] In one embodiment, the pharmaceutical product comprises the Lactobacillus acidophilus CCFM1460, a drug carrier, and / or pharmaceutical excipients.

[0037] In one embodiment, the pharmaceutical excipient comprises excipients and additives.

[0038] 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.

[0039] In one embodiment, the cosmetic contains Lactobacillus acidophilus CCFM1460, matrix ingredients, and / or conventional excipients.

[0040] 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.

[0041] 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.

[0042] The present invention also provides the application of the Lactobacillus acidophilus CCFM1460 in food production.

[0043] The present invention also provides the use of the Lactobacillus acidophilus CCFM1460 or its microbial preparation in the preparation of 3,4-dihydroxyphenylethylene glycol, N-tetradecyl diethanolamine and / or 3-phenyllactic acid.

[0044] In one embodiment, the Lactobacillus acidophilus CCFM1460 or the microbial preparation is fermented in a culture medium.

[0045] Beneficial effects:

[0046] (1) The Lactobacillus acidophilus CCFM1460 of the present invention can ferment to produce 3,4-dihydroxyphenyl glycol, N-tetradecyl diethanolamine and 3-phenyllactic acid.

[0047] (2) The Lactobacillus acidophilus CCFM1460 of the present invention and its prepared metabiotics, whether applied topically or orally, have the ability to alleviate skin barrier damage and improve the expression of related proteins, specifically manifested in:

[0048] (1) Increase the levels of skin barrier functional proteins (FLG, LOR, and IVL) in individuals with skin barrier damage;

[0049] (2) Increase the content of skin barrier structural proteins (ZO-1, OCCLUDIN, CLDN) in individuals with skin barrier damage.

[0050] Therefore, metabiotics prepared from Lactobacillus acidophilus CCFM1460 have great application potential in products that alleviate damage to the host skin barrier.

[0051] Preservation of biological materials

[0052] Lactobacillus acidophilus (CCFM1460), taxonomically named Lactobacillus acidophilus, was deposited on January 10, 2025, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No.: 65750), located at Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0053] Figure 1 : The number of viable Lactobacillus acidophilus bacteria per mL;

[0054] Figure 2 : The freeze-dried survival rate of different Lactobacillus acidophilus strains;

[0055] Figure 3 Effects of Lactobacillus acidophilus CCFM1460 and its prepared metabiotic on the barrier function protein FLG in mouse skin tissue;

[0056] Figure 4 Effects of Lactobacillus acidophilus CCFM1460 and its prepared metagener on barrier function protein IVL in mouse skin tissue;

[0057] Figure 5 Effects of Lactobacillus acidophilus CCFM1460 and its prepared metagener on the barrier function protein LOR in mouse skin tissue;

[0058] Figure 6 Effects of Lactobacillus acidophilus CCFM1460 and its prepared metagener on the barrier structural protein ZO-1 in mouse skin tissue;

[0059] Figure 7 Effects of Lactobacillus acidophilus CCFM1460 and its prepared metabiotic on OCCLUDIN, a barrier structural protein in mouse skin tissue;

[0060] Figure 8 Effects of Lactobacillus acidophilus CCFM1460 and its prepared metabiotic on the barrier structural protein CLDN in mouse skin tissue;

[0061] Figure 9 Liquid chromatography-mass spectrometry (LC-MS) of 3,4-dihydroxyphenyl ethylene glycol standard.

[0062] Figure 10 Liquid chromatography-mass spectrometry (LC-MS) plot of 3,4-dihydroxyphenyl ethylene glycol content in fermentation supernatant sample.

[0063] Figure 11 Liquid chromatography-mass spectrometry (LC-MS) plot of 3,4-dihydroxyphenylethylene glycol content in bacterial samples.

[0064] Figure 12 A comparison chart of retention times for 3,4-dihydroxyphenyl ethylene glycol standards, fermentation supernatant, and cell samples.

[0065] Figure 13 : Comparison of retention times of sphingosine and the sample.

[0066] Figure 14 Liquid chromatography-mass spectrometry (LC-MS) of tetradecyldiethanolamine (Tetradecyldiethanolamine) standard.

[0067] Figure 15 Liquid chromatography-mass spectrometry (LC-MS) plot showing the tetradecyl diethanolamine content in the fermentation supernatant sample.

[0068] Figure 16 Liquid chromatography-mass spectrometry (LC-MS) of tetradecyl diethanolamine content in fermentation supernatant sample (mass spectrum obtained by low-energy bombardment).

[0069] Figure 17 : Liquid chromatography-mass spectra of tetradecyl diethanolamine content in bacterial samples (mass spectra after energy bombardment of the same group as fermentation supernatant).

[0070] Figure 18 A comparison chart of retention times for standard tetradecyl diethanolamine, fermentation supernatant, and cell samples.

[0071] Figure 19 : Comparison of retention times of dihydrocoumarin and the sample.

[0072] Figure 20 3-Phenylonic acid standard LC-MS chromatogram.

[0073] Figure 21 Liquid chromatography-mass spectrometry (LC-MS) plot of 3-phenyllactic acid content in fermentation supernatant sample.

[0074] Figure 22 Liquid chromatography-mass spectrometry (LC-MS) plot of 3-phenyllactic acid content in bacterial samples.

[0075] Figure 23 A comparison chart of retention times for 3-phenyllactic acid standards, fermentation supernatant, and cell samples.

[0076] "*" 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

[0077] The present invention will be further described below with reference to specific embodiments.

[0078] The human keratinocytes (HaCaT) involved in the following examples were purchased from the Shanghai Cell Bank.

[0079] The Lactobacillus acidophilus CCFM1460 involved in the following examples was a self-screened strain from the Food Biotechnology Center of Jiangnan University.

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

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

[0082] Modified MRS solid medium: yeast extract 5.0 g / L, peptone 10.0 g / L, glucose 20.0 g / L, anhydrous sodium acetate 2.0 g / L, diammonium citrate 2.0 g / L, dipotassium hydrogen phosphate 2.6 g / L, manganese sulfate monohydrate 0.25 g / L, magnesium sulfate heptahydrate 0.5 g / L, Tween-80 1 mL / L, cysteine ​​1 g / L, and agar 16.0 g / L, pH 6.2–6.4.

[0083] Cell culture medium: 89% (v / v) DMEM medium + 10% (v / v) fetal bovine serum + 1% (v / v) 100× penicillin and streptomycin mixed solution (penicillin content 10000U / mL, streptomycin concentration 10mg / mL).

[0084] Example 1: Screening of Lactobacillus acidophilus CCFM1460

[0085] 1. Screening and identification of Lactobacillus acidophilus

[0086] The bacterial strain samples were obtained from the 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 of each sample was added to 4.5 mL of physiological saline. The samples were then serially diluted with physiological saline. The appropriate serial dilutions were plated onto modified MRS solid medium and incubated at 37°C for 48 h. Typical colonies of *Lactobacillus acidophilus* were picked and streaked onto MRS solid medium for purification. Single colonies were then transferred to modified MRS liquid medium for enrichment and preserved in 30% glycerol to obtain the bacterial strain. The genome of the strain was extracted and amplified using 16S rDNA (performed by Suzhou Genewiz Biotechnology Co., Ltd.). The results, confirmed by NCBI sequence alignment, identified the strain as *Lactobacillus acidophilus*. The target strain was named *Lactobacillus acidophilus* CCFM1460 and was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 10, 2025, with accession number GDMCC No: 65750.

[0087] 2. Screening of Lactobacillus acidophilus based on viable count in fermentation broth and lyophilization survival rate

[0088] After purifying the screened Lactobacillus acidophilus on modified MRS solid medium, single colonies were picked and cultured in modified MRS liquid medium at 37°C for 12 h. The culture was then inoculated into modified MRS liquid medium at an inoculum of 2% (v / v) to complete one subculture. After three subcultures, viable cell count and lyophilization survival rate were determined.

[0089] The viable cell count experiment method is as follows: Take 0.5 mL of Lactobacillus acidophilus suspension after pH adjustment and mix it evenly with 4.5 mL of sterile physiological saline to obtain a 10-fold diluted bacterial suspension. Repeat the above steps to dilute it sequentially. After diluting the Lactobacillus acidophilus suspension to an appropriate concentration, take 1 mL of the diluted bacterial suspension and place it in a sterile Petri dish. Set up three parallels for each concentration gradient. Then pour in solid culture medium cooled to 42℃ and mix well. After it is completely solidified, place it in a 37℃ constant temperature incubator for 48 hours and then take it out to count.

[0090] The specific method for determining the freeze-dried survival rate is as follows: Pre-freeze-drying counting of *Lactobacillus acidophilus*: Take 0.5 mL of pH-adjusted *Lactobacillus acidophilus* suspension and mix it thoroughly with 4.5 mL of sterile physiological saline to obtain a tenfold dilution. Repeat the above steps to dilute the suspension sequentially until a suitable concentration is reached. Take 1 mL of the diluted suspension and place it in a sterile Petri dish, setting up three replicates for each concentration gradient. Pour in solid culture medium cooled to 42℃ and mix well. After complete solidification, incubate at 37℃ for 48 hours, then remove and count. Post-freeze-drying counting of *Lactobacillus acidophilus* (using a freeze-drying protectant: 13% skim milk powder, mixed with bacterial sludge at a ratio of 1 (g bacterial sludge): 2 (g freeze-drying protectant)). Take 1 mL of sterile physiological saline and add it to the freeze-dried *Lactobacillus acidophilus* sample, mix thoroughly, and allow it to fully rehydrate. Then, perform post-freeze-drying counting according to the above steps.

[0091] Calculation of freeze-dried Lactobacillus survival rate: The freeze-dried survival rate is calculated using the following formula:

[0092]

[0093] The viable cell count of the CCFM1460 fermentation broth was 1.14 x 10⁻⁶ after screening. 10 The CFU / mL and freeze-dried survival rate (67.31%) were significantly higher than those of other Lactobacillus acidophilus strains.

[0094] Example 2: Preparation of postbiotics from Lactobacillus acidophilus CCFM1460

[0095] First, the postbiotic preparation of Lactobacillus acidophilus CCFM1460 was carried out:

[0096] (1) Lactobacillus acidophilus CCFM1460 was streaked from the preservation tube and revived. It was then cultured in a water-jacketed incubator at 37°C for 48 hours on modified MRS solid medium to obtain single colonies. Single colonies were picked and inoculated into modified MRS liquid medium and cultured at 37°C for 12-18 hours to obtain culture solution 1.

[0097] Culture medium 1 was inoculated into modified MRS liquid medium at an inoculation rate of 2% (v / v) and cultured at 37°C for 12 h to obtain seed culture;

[0098] The seed culture was inoculated at 2% (v / v) into modified MRS liquid medium for expansion culture, and cultured at 37℃ for 18h. The number of viable bacteria was recorded and bacterial culture a was obtained.

[0099] The bacterial culture a was centrifuged at 8000 r / min for 10 min, and the supernatant and bacterial sludge were collected. The supernatant was heat-treated (65℃, 30 min) and freeze-dried to obtain powder for later use, thus preparing the freeze-dried powder of Lactobacillus acidophilus CCFM1460 fermentation supernatant (denoted as CCFM1460-Q). The bacterial sludge was resuspended in 75% of the original bacterial culture volume of double-distilled water, and the resuspended liquid was heat-treated (65℃, 30 min), and then homogenized under high pressure (1000 MPa, 10 times) using a high-pressure homogenizer. After homogenization, the supernatant was collected by centrifugation at 8000 r / min for 30 min to obtain the bacterial cell lysate (denoted as CCFM1460-L).

[0100] The preparation method of live Lactobacillus acidophilus CCFM1460 is as follows: prepare bacterial solution according to the same method as above, adjust the concentration of bacterial solution to the same level as bacterial solution a, centrifuge at 8000 r / min for 30 min to obtain bacterial sludge, and then reselect it with glycerol at a ratio of 1 g: 2 mL to obtain live Lactobacillus acidophilus CCFM1460 glycerol tube, which is denoted as CCFM1460-H.

[0101] The following postbiotics of Lactobacillus acidophilus CCFM1460 (cell lysate CCFM1460-L and fermentation supernatant CCFM1460-Q) were prepared by the above methods.

[0102] Example 3: Effects of postbiotic strain prepared from Lactobacillus acidophilus CCFM1460 on the expression of barrier function protein genes in SDS-damaged HaCaT cells.

[0103] (1) HaCaT cells were fed with 1.8 × 10⁻⁶ cells. 4 One cell per well was seeded into a 6-well plate and cultured for 36 hours until the cells adhered.

[0104] (2) After the cells adhered to the wall, different groups underwent different medium changes and were incubated for 6 hours.

[0105] Control group (blank group): After changing the medium in step (1), the medium contained cell culture medium and HaCaT cells, without post-genetic treatment and without SDS modeling agent;

[0106] Model group: The culture medium in step (1) was changed to a cell culture medium containing 15 μg / mL SDS, containing the original HaCaT cells, without post-genetic treatment; the cell culture medium containing SDS modeling agent was prepared by mixing SDS evenly in the culture medium and passing it through a 0.22 μm aqueous filter membrane for sterilization, so that the final concentration of SDS in the cell culture medium was 15 μg / mL.

[0107] Postgenetic treatment group: The treatment method is the same as that of the model group.

[0108] (3) After incubation, discard the original culture medium and wash once with PBS. Add the corresponding metagenic sample to the metagenic treatment group, and add cell culture medium to the control group and model group. Incubate again for 24 hours.

[0109] In the metabiotic treatment group, 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 postbiotic prepared from the bacterial culture at CFU / mL was equivalent, and 2 mL of postbiotic prepared from Lactobacillus acidophilus CCFM1460 (CCFM1460-Q and CCFM1460-L) was added respectively.

[0110] (4) After incubation, discard the culture supernatant, wash each well three times rapidly with PBS, add 1 mL of cell lysis buffer to each well, repeatedly pipette, extract RNA from the cell lysis buffer, and reverse transcribe it into cDNA using an RT-PCR reverse transcription kit. Detect gene expression in HaCaT cells using real-time quantitative PCR. -△△Ct The formula calculates the mRNA expression levels of FLG, IVL, and LOR, with GAPDH as the internal reference. The primers are described in Table 1 below, and the results are as follows: Figure 3 , 4 As shown in Figure 5.

[0111] Table 1 Primer sequences

[0112]

[0113]

[0114] Filamentin (FLG) is crucial for skin barrier function. It is a key molecule in the stratum corneum of human skin, connecting keratin fibers. With the assistance of FLG monomers, keratin fibers aggregate regularly, forming a robust physical barrier on the outermost layer of the epidermis, preventing moisture loss and the invasion of external allergens. FLG deficiency is associated with numerous skin diseases, such as eczema (atopic dermatitis) and ichthyosis. In these diseases, FLG protein deficiency or absence leads to weakened skin barrier function, thereby increasing skin sensitivity to external stimuli. Therefore, the goal is to alleviate SDS-induced damage to keratinocytes by increasing FLG gene expression. FLG expression results were obtained from… Figure 3 It can be seen that the expression level of FLG mRNA in the control group was about 1, while the expression level in the model group decreased to 0.46 after SDS intervention. The post-biotics (CCFM1460-L and CCFM1460-Q) prepared by Lactobacillus acidophilus CCFM1460 significantly increased the expression level of FLG mRNA in HaCaT cells to 6.44 and 5.45, respectively. In this indicator, both the fermentation supernatant and cell lysate of CCFM1460 had a significant upregulation effect on FLG mRNA expression, but the upregulation effect of CCFM1460-L was better than that of CCFM1460-Q.

[0115] Inner lamina protein (IVL) is formed by the cross-linking of inner lamina protein and laminarin under the catalysis of keratinocyte transglutaminase, creating an insoluble keratinized capsule that constitutes the unique stratum corneum barrier structure of the epidermis. It also interacts with other keratinocyte differentiation proteins such as filaggrin (FLG) to jointly maintain the structure and function of the skin barrier. IVL expression results are derived from... Figure 4 It can be seen that the expression level of IVL mRNA in the control group was about 1, while the expression level in the model group decreased to 0.20 after SDS intervention. The post-biotics (CCFM1460-L and CCFM1460-Q) prepared by Lactobacillus acidophilus CCFM1460 significantly increased the expression level of IVL mRNA in HaCaT cells to 20.046 and 17.73, respectively. In this indicator, the fermentation supernatant and cell lysate of CCFM1460 also had a significant upregulation effect on FLG mRNA expression.

[0116] LOR (Leg-like protein) participates in the formation of the keratinized capsule during keratinization, a tough, water-insoluble outer membrane crucial for maintaining the integrity of the skin barrier. Furthermore, it helps maintain skin hydration and, through its interaction with other keratinocyte differentiation proteins, preserves skin softness and elasticity. Defects or absence of LOR in these diseases lead to weakened skin barrier function, thereby increasing skin sensitivity to external stimuli. Abnormal expression or loss of function is associated with certain skin diseases, such as ichthyosis and certain types of keratosis. LOR expression results are derived from… Figure 5It was found that the expression level of LOR mRNA in the control group was approximately 1, while the expression level in the model group decreased to 0.58 after SDS intervention. The postbiotics (CCFM1460-L and CCFM1460-Q) prepared from Lactobacillus acidophilus CCFM1460 significantly increased the expression level of LOR mRNA in HaCaT cells to 4.21 and 2.00, respectively.

[0117] The results above indicate that the metabiotics (cell lysate and fermentation supernatant) prepared from Lactobacillus acidophilus can upregulate the expression of functional protein (FLG, IVL, and LOR) mRNA in HaCaT cells when SDS damages them. CCFM1460-L has a better repair effect than CCFM1460-Q, as it repairs SDS-induced damage to HaCaT cells by reducing abnormal cell protein function and alleviating apoptosis.

[0118] Example 4: Effect of postbiotic strain prepared from Lactobacillus acidophilus CCFM1460 on the expression of barrier structural protein genes in SDS-damaged HaCaT cells.

[0119] (1) HaCaT cells were fed with 1.8 × 10⁻⁶ cells. 4 One cell per well was seeded into a 6-well plate and cultured for 36 hours until the cells adhered.

[0120] (2) After the cells adhered to the wall, different groups underwent different medium changes and were incubated for 6 hours.

[0121] Control group: After changing the medium in step (1), it contains cell culture medium and HaCaT cells, without post-genetic treatment, and without SDS modeling agent;

[0122] Model group: The culture medium in step (1) was changed to a cell culture medium containing 15 μg / mL SDS, containing the original HaCaT cells, without post-genetic treatment; the cell culture medium containing SDS modeling agent was prepared by mixing SDS evenly in the culture medium and passing it through a 0.22 μm aqueous filter membrane for sterilization, so that the final concentration of SDS in the cell culture medium was 15 μg / mL.

[0123] Postgenetic treatment group: The treatment method is the same as that of the model group.

[0124] (3) After incubation, discard the original culture medium and wash once with PBS. Add the corresponding metagenic sample to the metagenic treatment group, and add cell culture medium to the control group and model group. Incubate again for 24 hours.

[0125] In the metabiotic treatment group, 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). 7The amount of postbiotic prepared from the bacterial culture at CFU / ml was equivalent, and 2 mL of postbiotic prepared from Lactobacillus acidophilus CCFM1460 (CCFM1460-Q and CCFM1460-L) was added respectively.

[0126] (4) After incubation, discard the culture supernatant, wash each well three times rapidly with PBS, add 1 mL of cell lysis buffer to each well, repeatedly pipette, extract RNA from the cell lysis buffer, and reverse transcribe it into cDNA using an RT-PCR reverse transcription kit. Detect gene expression in HaCaT cells using real-time quantitative PCR. -△△Ct The formula calculates the mRNA expression levels of ZO-1, OCCLUDIN, and CLDN, with GAPDH as the internal reference. The primers are described in Table 2 below, and the results are as follows: Figure 6 , 7 As shown in Figure 8.

[0127] Table 2 Primer sequences

[0128]

[0129]

[0130] ZO-1 (Zonula Occludens-1), a core component of the tight junction protein complex, plays a crucial role in maintaining skin barrier function. It helps the skin resist external stimuli and maintain its healthy function by strengthening intercellular tight junctions, regulating hydration, participating in skin immune responses and repair processes. ZO-1 deficiency can lead to various skin problems, such as eczema (atopic dermatitis), allergic skin diseases (such as contact dermatitis), and psoriasis. Reduced ZO-1 expression leads to skin barrier damage, thereby increasing the risk of various skin diseases. ZO-1 expression results are determined by… Figure 6 It can be seen that the expression level of mRNA in the control group was about 1, while the expression level in the model group decreased to 0.79 after SDS intervention. The post-biotic CCFM1460-L prepared by Lactobacillus acidophilus CCFM1460 can increase the mRNA expression level of ZO-1 in HaCaT cells to 1.80, but the post-biotic CCFM1460-Q has no significant upregulation effect.

[0131] OCCLUDIN is a key member of the tight junction protein family, primarily distributed between epithelial and endothelial cells. It plays a crucial role in maintaining skin barrier function, particularly in preserving selective skin permeability, regulating hydration and skin immune responses, and protecting the skin from external pathogens. OCCLUDIN expression results are provided by... Figure 7It can be seen that the mRNA expression level of OCCLUDIN in the control group was about 1, while the expression level in the model group decreased to 0.79 after SDS intervention. The post-biotic CCFM1460-L prepared by Lactobacillus acidophilus CCFM1460 can increase the mRNA expression level of ZO-1 in HaCaT cells to 1.80, while the post-biotic CCFM1460-Q can increase it to 1.10.

[0132] Claudins (CLDNs) are an important class of tight junction proteins widely distributed in various epithelial cell types. They play a crucial role in the skin barrier function, maintaining selective skin permeability, preventing the entry of harmful substances, preserving hydration, and protecting the skin from external stimuli. CLDNs work in conjunction with other tight junction proteins (such as OCCLUDIN and ZO-1) to establish and maintain the tight junction structure of the skin. CLDN expression results are... Figure 8 It can be seen that the mRNA expression level in the control group was about 1, while the expression level in the model group decreased to 0.52 after SDS intervention. The post-biotic CCFM1460-L prepared by Lactobacillus acidophilus CCFM1460 can increase the mRNA expression level of ZO-1 in HaCaT cells to 1.17, but the post-biotic CCFM1460-Q has no significant upregulation effect.

[0133] The results above indicate that the bacterial lysate prepared from Lactobacillus acidophilus can upregulate the expression of structural proteins (ZO-1, OCCLUDIN, and CLDN) mRNA in HaCaT cells damaged by SDS. CCFM1460-L can alleviate SDS-induced damage to HaCaT cells through the protective effect of skin structural proteins and by promoting cell repair and regeneration. However, the repair effect of the fermentation supernatant was not significant.

[0134] Example 5: Analysis of the effective substances of Lactobacillus acidophilus CCFM1460 in repairing SDS-damaged HaCaT cells based on non-target metabolomics analysis

[0135] 1. Sample preparation and detection before CCFM1460 fermentation supernatant metabolomics analysis:

[0136] (1) Following the method for preparing seed culture in Example 2, after culturing the strain for 12 hours, take 1 mL of bacterial culture, centrifuge at 4°C, 10000 g, for 5 min to collect the supernatant 1; use the same blank liquid culture medium as liquid 2.

[0137] (2) Transfer 100 μL of supernatant 1 and 2 into a 1.5 mL centrifuge tube;

[0138] (3) Add 400 μL of methanol:acetonitrile = (1:1, v / v) (pre-cool at -20℃ in advance) to precipitate the protein;

[0139] (4) Vortex for 30 seconds, then sonicate in an ice bath for 10 minutes;

[0140] (5) Place the sample in a -20℃ refrigerator for 1 hour to improve the protein precipitation rate (secondary precipitation removes protein);

[0141] (6) Centrifuge at 15000 rpm for 15 min at 4℃.

[0142] (7) Take the supernatant and concentrate it under vacuum;

[0143] (8) Redissolve by adding 100 μL of acetonitrile:water (1:1) and vortexing for 30 s;

[0144] (9) Centrifuge at 15000rpm for 15 minutes at 4℃, take the supernatant, transfer the appropriate volume into a sample vial for testing;

[0145] (10) For polar metabolites, this project used a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph (UPLC) with a Waters ACQUITY UPLC BEH Amide (2.1 mm × 50 mm, 1.7 μm) column for chromatographic separation of the target compounds. Phase A of the HPLC was aqueous, containing 25 mmol / L ammonium acetate and 25 mmol / L ammonia, and phase B was acetonitrile. Sample tray temperature: 4 °C, injection volume: 2 μL. The Orbitrap Exploris 120 mass spectrometer was able to acquire primary and secondary mass spectrometry data under the control of the software (Xcalibur, version 4.4, Thermo). Detailed parameters are as follows: Sheath gas flow rate: 50 Arb, Aux gas flow rate: 15 Arb, Capillary temperature: 320℃, Fullms resolution: 60000, MS / MS resolution: 15000, Collision energy: SNCE 20 / 30 / 40, Spray voltage: 3.8kV (positive) or -3.4kV (negative).

[0146] 2. Sample preparation and detection before CCFM1460 bacterial cell structure metabolomics analysis:

[0147] After culturing for 12 hours, take 1 mL of bacterial solution, centrifuge at 4℃, 10000g, for 5 minutes to collect the bacterial sludge;

[0148] (1) Wash once with pre-cooled 0.9% physiological saline to remove residual culture medium;

[0149] (2) After centrifuging to remove physiological saline, 1.5 mL centrifuge tubes containing bacterial sludge are placed into liquid nitrogen for quenching to stop metabolic activity;

[0150] (3) Remove the sample from the liquid nitrogen and add 500 μL of methanol:acetonitrile = (1:1, v / v) (pre-cool at -20℃ in advance);

[0151] (4) After vortexing for 30 seconds, the cells were repeatedly frozen and thawed three times with liquid nitrogen to lyse the cells and release intracellular metabolites.

[0152] (5) Place the sample in a -20℃ refrigerator for 1 hour to improve the protein precipitation rate (secondary precipitation removes protein);

[0153] (6) Centrifuge at 15,000 rpm for 15 min at 4℃;

[0154] (7) Take the supernatant and evaporate it to dryness using a rotary evaporator;

[0155] (8) Redissolve by adding 100 μL of acetonitrile:water (1:1) and vortexing for 30 s;

[0156] (9) Centrifuge at 15000rpm for 15 minutes at 4℃, take the supernatant, transfer an appropriate volume into a vial for testing;

[0157] (10) For polar metabolites, this project used a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph (UPLC) with a Waters ACQUITY UPLC BEH Amide (2.1 mm × 50 mm, 1.7 μm) column for chromatographic separation of the target compounds. Phase A of the HPLC was aqueous, containing 25 mmol / L ammonium acetate and 25 mmol / L ammonia, and phase B was acetonitrile. Sample tray temperature: 4 °C, injection volume: 2 μL. The Orbitrap Exploris 120 mass spectrometer was able to acquire primary and secondary mass spectrometry data under the control of the software (Xcalibur, version 4.4, Thermo). Detailed parameters are as follows: Sheath gas flow rate: 50 Arb, Aux gas flow rate: 15 Arb, Capillary temperature: 320℃, Fullms resolution: 60000, MS / MS resolution: 15000, Collision energy: SNCE 20 / 30 / 40, Spray voltage: 3.8kV (positive) or -3.4kV (negative).

[0158] 3. Analysis of active ingredients in CCFM1460:

[0159] After the raw data was converted into mzXML format using ProteoWizard software, metabolite identification was performed using a collaboratively developed R package with BiotreeDB (V3.0) as the database. Then, visualization analysis was performed using a self-developed R package.

[0160] We consulted relevant literature and collected the chemical formulas, molecular formulas, and molecular weights of potential effective substances in CCFM1403. Combining non-target metabolomics, we screened potential effective substances based on P-value < 0.05 and differences compared to pre-fermentation substances, as well as research related to skin barrier repair. The substances are shown in Table 3.

[0161] Table 3. Potentially effective substances related to the skin barrier in CCFM1460

[0162]

[0163] Example 6: Quantitative detection of potential active substances in Lactobacillus acidophilus CCFM1460 based on non-target metabolomics results

[0164] 1. Sample preparation and detection before liquid chromatography-mass spectrometry (LC-MS) in CCFM1460:

[0165] Same as Example 5.

[0166] 2. Content detection results

[0167] Based on the results of non-targeted metabolomics, targeted quantitative detection revealed three substances in the sample: 3,4-dihydroxyphenylglycol, N-tetradecyldiethanolamine (a D-erythrosine disphingosine isomer), and 3-phenyllactic acid (dihydrocoumarin with an additional H2O structure, (+ / -)-3-Phenyllactic Acid). The specific contents and spectral information are as follows.

[0168] (1) 3,4-Dihydroxyphenylethylene glycol

[0169] like Figure 9-12As shown, this substance was only present in the fermentation supernatant, at a concentration of 0.68 ppm. This substance possesses clear antioxidant and anti-inflammatory potential. Oxidative stress and inflammation are key external factors disrupting lipid homeostasis and keratin differentiation (such as FLG, LOR, and IVL) in the stratum corneum; therefore, antioxidant / anti-inflammatory effects can be considered a supporting mechanism for skin barrier repair. In vitro studies evaluating olive oil extract and its representative molecules (including 3,4-dihydroxyphenylethylene glycol and oleuropein) in human skin keratinocytes showed that olive oil polyphenols as a whole can promote keratinocyte proliferation and inhibit the NF-κB inflammatory pathway.

[0170] (2) N-Tetradecyldiethanolamine (an isomer of sphingosine)

[0171] like Figure 13-18 As shown, the retention time of the sphingosine standard is significantly different from that in the sample, so it is suspected that the real substance in the sample is an isomer of sphingosine. After re-comparing the secondary mass spectra, it was found that the substance in the sample is most similar to the mass spectrum of "Tetradecyldiethanolamine". Subsequently, the target verification of Tetradecyldiethanolamine was carried out again.

[0172] N-Tetradecyldiethanolamine was detected in both the fermentation supernatant and bacterial cells, at concentrations of 0.05 ppm and 0.38 ppm, respectively. This substance is a homologue of N-alkyldiethanolamine (ECHA registered name: 2,2'-(tetradecylimino)bisethanol), exhibiting surface-active / amphiphilic chemical properties and is found in industrial and formulation chemistry. It is an important alkanolamine used in cosmetic formulations as an emulsifier, thickener, wetting agent, detergent, and alkalizing agent. Studies have shown that the transdermal absorption of diethanolamine (DEA) in cosmetics such as shampoos, hair dyes, and lotions is extremely low, with less than 1% entering the systemic circulation, and no significant difference between living and inactivated skin. This indicates that the systemic exposure risk of DEA is extremely low under normal use conditions and falls within the safe range for use. Furthermore, DEA can accumulate in the skin and increase the transdermal absorption potential of other substances, but it itself hardly enters the bloodstream, making it suitable for use in cosmetics requiring improved penetration of active ingredients.

[0173] (3)(±)-3-Phenylonic acid (with one more H2O than dihydrocoumarin analogs)

[0174] like Figure 19-23As shown, the retention time of the standard dihydrocoumarin is significantly different from that in the sample. Therefore, it is suspected that the real substance in the sample is an isomer (analogue) of dihydrocoumarin. After re-comparing the secondary mass spectra, it was found that the substance in the sample is most similar to the mass spectrum of "(+ / -)-3-Phenyllactic Acid". Subsequently, the target verification of (+ / -)-3-Phenyllactic Acid was performed again.

[0175] Triethanolamine was detected in both the fermentation supernatant and bacterial cells, at concentrations of 1.52 ppm and 1.33 ppm, respectively. This substance is an aromatic organic acid with broad-spectrum inhibitory and anti-biofilm activity against Staphylococcus aureus and various fungi. In skin barrier diseases (such as AD / eczema, chronic wounds), pathogenic bacterial load and biofilm can significantly exacerbate inflammation, disrupt tight junctions / keratin differentiation, and delay barrier recovery.

[0176] Therefore, the Lactobacillus acidophilus CCFM1460 of the present invention produces new active metabolites such as 3,4-dihydroxyphenylethylene glycol, N-tetradecyl diethanolamine and (±)-3-phenyllactic acid after fermentation.

[0177] 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 acidophilus ( Lactobacillus acidophilus CCFM1460, characterized in that, The Lactobacillus acidophilus CCFM1460 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 65750.

2. A microbial preparation containing the Lactobacillus acidophilus CCFM1460 of claim 1.

3. A metabiotic prepared from Lactobacillus acidophilus CCFM1460 as described in claim 1.

4. The epigenetic agent as described in claim 3, characterized in that, The metabiotics include bacterial lysates, inactivated or dead cells, and fermentation supernatants.

5. A medicine containing Lactobacillus acidophilus CCFM1460 as described in claim 1 and / or its postbiotic.

6. The use of Lactobacillus acidophilus CCFM1460 or its post-biotic as described in claim 1 in the preparation of a medicament for repairing the skin barrier, characterized in that, The skin barrier repair includes at least one of the following: (1) Enhance the activity of skin keratinocytes; (2) Regulate the expression of barrier-related proteins and genes in skin keratinocytes.

7. The use of Lactobacillus acidophilus CCFM1460 of claim 1 or the microbial preparation of claim 2 in the preparation of 3,4-dihydroxyphenylethylene glycol, N-tetradecyl diethanolamine and / or 3-phenyllactic acid.

8. The application as described in claim 7, characterized in that, Ferment the Lactobacillus acidophilus CCFM1460 of claim 1 or the microbial preparation of claim 2 in a culture medium.

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