Lactobacillus muci genus fermentation based on multi-target repair skin barrier CCFM1401 and its postbiotic

Abstract

The application discloses a strain of fermented Lactobacillus muciadis CCFM1401 based on multi-target skin barrier repair and a postbiotic thereof, and belongs to the technical fields of microorganisms and medicines. The bacterial lysate of the fermented Lactobacillus muciadis CCFM1401 provided by the application can improve the cell activity of damaged keratinocytes and the expression levels of FLG, LOR, IVL, ZO-1, Occludin and CLDN genes in the aspect of external use. The live bacteria and the postbiotic (including the bacterial lysate and the fermentation supernatant) of the fermented Lactobacillus muciadis CCFM1401 can reduce TEWL, up-regulate the water content of the stratum corneum, reduce inflammatory reactions (such as skin histamine and serum IgE), and also up-regulate the expression of skin barrier function and structural proteins. Therefore, the fermented Lactobacillus muciadis has a great application prospect in the preparation of external or oral drugs for repairing skin barrier, and in the preparation of food, health products or cosmetics.

Description

TECHNICAL FIELD

[0001] The present application relates to a strain of Limosilactobacillus fermentum CCFM1401 based on multi-target repair of skin barrier and its postbiotic, belonging to the fields of microbial technology and medicine. BACKGROUND

[0002] Skin is the largest organ of the human body and an important tissue for defending against various physical, chemical, and pathogenic microorganism invasions from the outside world. The skin barrier is crucial for the skin to resist the outside world and protect the internal environment of the body. Weak skin barrier damage leads to functional decline and decreased defense function, endangering the health of the body. At the same time, the microecology has an important influence on skin health, and probiotics, as a means to regulate the balance of skin microecology, play a role through antioxidant, reduction of extracellular matrix degradation, and inhibition of expression of inflammatory factors.

[0003] Among the inventions for repairing the skin barrier, most of them produce excellent effects when used externally. For example, CN118304225A discloses a composition comprising L-linalool, olive oil, jojoba seed oil, complex plant essential oil, phospholipid, glycerol, polyglyceryl monolaurate, and deionized water, and the complex plant essential oil is composed of essential oil compositions of perilla, acanthopanax seed, Citrus aurantium, Gynura japonica, chamomile, Blumea orientalis, and grapefruit peel, which has significant effects of acne removal, sensitive skin soothing, and skin repair promotion. CN118477030A discloses a high-moisture repair mask containing Dendrobium huoshanense and a preparation method thereof, which has excellent antioxidant capacity and can provide more comprehensive protection for the skin and accelerate skin repair. Although these external products have certain effects in repairing damaged skin barrier, there is still room for improvement. First, their mechanism is relatively single, mainly by applying on the skin surface to play a role. Second, external products usually have significant effects in the short term, but are insufficient in repairing chronic or long-term skin barrier damage. In addition, external products can only act on the outer layer of the skin and cannot regulate skin health from the inside, while the integrity of the skin barrier is closely related to the health of the body. Therefore, it is particularly necessary to provide a probiotic repair product that can be taken internally and externally, and a probiotic product that combines internal and external synergistic effects can more comprehensively and effectively repair the skin barrier and provide more comprehensive repair effects. SUMMARY

[0004] The present application provides a fermented Limosilactobacillus fermentum CCFM1401 and its postbiotic for use in the preparation of a product for repairing the skin barrier.

[0005] The application provides a strain of Limosilactobacillus fermentum CCFM1401, which is preserved in the Guangdong Microbial Culture Collection Center, has a preservation number of GDMCC No: 64895 and a preservation date of July 22, 2024.

[0006] The Limosilactobacillus fermentum CCFM1401 is derived from the feces of a healthy population, and the strain is sequenced and analyzed, and the obtained sequence is subjected to nucleic acid sequence alignment in NCBI, and the result shows that the strain is Limosilactobacillus fermentum of the genus Lactobacillus, and is named Limosilactobacillus fermentum CCFM1401.

[0007] The Limosilactobacillus fermentum CCFM1401 has a white, smooth and round colony on a MRS solid culture medium.

[0008] The application also provides a postbiotic prepared from the Limosilactobacillus fermentum CCFM1401.

[0009] In an embodiment, the postbiotic comprises a cell lysate, inactivated or inactive cells, a fermentation supernatant, or a powder prepared by drying any of the above.

[0010] In an embodiment, the inactivated or inactive cells are prepared by culturing the Limosilactobacillus fermentum CCFM1401 in a culture medium for a period of time, collecting the cell bodies in the cell culture solution, and obtaining inactivated cell bodies after heat treatment.

[0011] In an embodiment, the heat treatment is performed at 65 DEG C for 30 min.

[0012] In an embodiment, the cell lysate is prepared by culturing the Limosilactobacillus fermentum CCFM1401 in a culture medium for a period of time, collecting the cell bodies, high-pressure homogenizing, and centrifuging the supernatant to obtain the cell lysate.

[0013] In an embodiment, the fermentation supernatant is the supernatant after centrifugation of the Limosilactobacillus fermentum CCFM1401 cultured in a culture medium for a period of time.

[0014] The application also provides a composition containing the Limosilactobacillus fermentum CCFM1401 and / or the postbiotic thereof.

[0015] In an embodiment, the composition includes, but is not limited to, food, medicine, health care products or cosmetics.

[0016] In an embodiment, the food includes the above-mentioned composition and conventional auxiliary materials.

[0017] In an embodiment, the conventional excipient comprises one or more of a filler, a flavoring agent, a binder, a disintegrant, a lubricant, an antacid, and a nutritional fortifier.

[0018] In an embodiment, the health product comprises the above composition and a conventional excipient.

[0019] In an embodiment, the conventional excipient comprises one or more of a filler, a flavoring agent, a binder, a disintegrant, a lubricant, an antacid, and a nutritional fortifier.

[0020] In an embodiment, the composition is a probiotic powder.

[0021] In an embodiment, the probiotic powder is a solid-state powder postbiotic prepared by drying a prepared liquid postbiotic of L. m. subs. jensenii CCFM1401.

[0022] In an embodiment, drying comprises, but is not limited to, spray drying, vacuum freeze-drying, fluidized bed drying, vacuum drying.

[0023] The present application also provides use of the composition in the preparation of a product for repairing skin barrier.

[0024] In an embodiment, the product comprises at least one of the following effects:

[0025] (1) improving cell viability of skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage in vitro;

[0026] (2) improving barrier function and structural protein expression of skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage in vitro;

[0027] (3) reducing skin barrier damage indicators (blood biochemical indicators, skin appearance) of an individual;

[0028] (4) increasing barrier function and structural proteins of skin tissue of an individual;

[0029] (5) increasing basement membrane proteins of skin tissue of an individual;

[0030] (6) reducing inflammatory response of an individual.

[0031] In an embodiment, the skin barrier damage related symptoms comprise increased skin appearance, skin proteins, and inflammatory factors in blood.

[0032] In an embodiment, the skin damage comprises dry skin, reduced elasticity, sagging, wrinkle formation, oxidative damage, protein loss.

[0033] In an embodiment, the application mode is topical external use and oral use.

[0034] In an embodiment, the product contains fermented L. mukhgosei CCFM1401 in an amount of not less than 1×10 7 CFU / mL.

[0035] In an embodiment, the product contains fermented L. mukhgosei CCFM1401 in an amount of not less than 430 μg / kg body weight.

[0036] In an embodiment, the product is a pharmaceutical product or a cosmetic product.

[0037] In an embodiment, the pharmaceutical product contains the fermented L. mukhgosei CCFM1401, a pharmaceutical carrier and / or a pharmaceutical excipient.

[0038] In an embodiment, the pharmaceutical excipient contains an excipient and an additional agent.

[0039] In an embodiment, the pharmaceutical excipient contains a solvent, a propellant, a solubilizer, a co-solvent, an emulsifier, a coloring agent, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending agent, a coating material, a fragrance, an anti-adhesion agent, an integrating agent, a penetration enhancer, a pH regulator, a buffer, a plasticizer, a surfactant, a foaming agent, an antifoaming agent, a thickening agent, a complexing agent, a humectant, an absorbent, a diluent, a flocculating agent and a deflocculating agent, a filter aid, and a release retardant.

[0040] In an embodiment, the cosmetic product contains the fermented L. mukhgosei CCFM1401, a base material, and / or a conventional excipient.

[0041] In an embodiment, the base material includes an oil-based material, a wax-based material, a synthetic oil-based material, a powder-based material, a gum-based material, a coagulant, and a surfactant.

[0042] In an embodiment, the conventional excipient includes one or more of a humectant, a whitening agent, a flavoring agent, a binder, a lubricant, a preservative, a film agent, an antioxidant, an emulsifier, and a cosmetic nutrient additive.

[0043] The present application also provides use of the fermented L. mukhgosei CCFM1401 in food production.

[0044] The present application also provides use of the fermented L. mukhgosei CCFM1401 and / or a postbiotic thereof in the preparation of a product for repairing skin barrier or repairing skin damage.

[0045] The application also provides the use of the fermented Limosilactobacillus fermentum CCFM1401 and / or the postbiotic thereof in the preparation of a product for improving skin hydration.

[0046] Beneficial effects:

[0047] The fermented Limosilactobacillus fermentum CCFM1401 of the application and the postbiotic prepared therefrom have the ability to relieve skin barrier damage and improve the expression of related proteins, which is specifically embodied in:

[0048] (1) improving the cell activity of skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage;

[0049] (2) improving the mRNA expression of FLG, IVL, LOR, ZO-1, Occludin and CLDN of skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage in vitro;

[0050] (3) improving the content of skin barrier function proteins (FLG and LOR) of skin barrier damage individuals;

[0051] (4) improving the content of skin barrier structure proteins (ZO-1, OCCLUDIN and CLDN) of skin barrier damage individuals;

[0052] (5) enhancing the basement membrane barrier effect of skin barrier damage individuals;

[0053] (6) repairing the skin appearance of skin barrier damage individuals;

[0054] (7) reducing the inflammatory response caused by skin barrier damage individuals.

[0055] Therefore, the postbiotic prepared from the fermented Limosilactobacillus fermentum CCFM1401 has great application prospects in products for relieving skin barrier damage of hosts.

[0056] Biological material preservation

[0057] The fermented Limosilactobacillus fermentum CCFM1401, taxonomically named Limosilactobacillus fermentum, has been deposited in the Guangdong Microbial Culture Collection Center on July 22, 2024, with the accession number GDMCC No: 64895, and the address of the deposit is No. 59, Building 5, Guangzhou, Guangdong, China. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 Evaluation of the effect of different concentrations of SDS on HaCaT cell damage

[0059] Figure 2 Effect of different postbiotics on HaCaT cell proliferation

[0060] Figure 3 Effect of different postbiotics on the vitality of HaCaT cells after SDS damage

[0061] Figure 4 Effect of different postbiotics on the barrier function of HaCaT cells and the expression of structural protein genes after SDS damage

[0062] Figure 5 Receptors of the pathway for the repair effect of fermented L. muci- dalis CCFM1401 postbiotic

[0063] Figure 6 Animal experiment process

[0064] Figure 7 Effect of fermented L. mucilaginis CCFM1401 and the postbiotic prepared therefrom on the barrier function proteins in the skin tissue of mice

[0065] Figure 8 Effect of fermented L. mucilaginis CCFM1401 and the postbiotic prepared therefrom on the barrier structural proteins in the skin tissue of mice

[0066] Figure 9 Effect of fermented L. mucilaginis CCFM1401 and the postbiotic prepared therefrom on the expression of barrier protein genes in the basal layer of the skin tissue of mice

[0067] Figure 10 Effect of fermented L. mucilaginis CCFM1401 and the postbiotic prepared therefrom on the TEWL and the water content of the stratum corneum of the skin tissue of mice

[0068] Figure 11 Effect of fermented L. mucilaginis CCFM1401 and the postbiotic prepared therefrom on inflammation in the skin tissue and serum of mice

[0069] Figure 12 Pathway receptors for the repair effect of fermented L. mucilaginis CCFM1401 and the postbiotic prepared therefrom on the skin barrier

[0070] " " indicates a statistically significant difference (P < 0.05) from the Model group, " " indicates a significantly statistically significant difference (P < 0.01) from the Model group, " " indicates an extremely statistically significant difference (P < 0.001) from the Model group, and " " indicates an extremely statistically significant difference (P < 0.0001) from the Model group. DETAILED DESCRIPTION

[0071] The present application will be further described below in conjunction with specific examples.

[0072] The human keratinocytes (HaCaT) involved in the following examples were purchased from Shanghai Cell Bank.

[0073] The BALB / c mice involved in the following examples were purchased from Vantian Li Hua Company.

[0074] The L. fermentum FJSNTL31, L. fermentum FJSNTL41, and L. fermentum CCFM1401 involved in the following examples were obtained from the Food Biotechnology Center of Jiangnan University.

[0075] The culture medium involved in the following examples is as follows:

[0076] Modified MRS liquid medium: 5.0 g / L of yeast powder, 10.0 g / L of protein peptone, 20.0 g / L of glucose, 2.0 g / L of anhydrous sodium acetate, 2.0 g / L of citric acid diamine, 2.6 g / L of potassium phosphate dibasic, 0.25 g / L of manganese sulfate monohydrate, 0.5 g / L of magnesium sulfate heptahydrate, and 1 mL / L of Tween-80, pH 6.2-6.4.

[0077] Modified MRS solid medium: 5.0 g / L of yeast powder, 10.0 g / L of protein peptone, 20.0 g / L of glucose, 2.0 g / L of anhydrous sodium acetate, 2.0 g / L of citric acid diamine, 2.6 g / L of potassium phosphate dibasic, 0.25 g / L of manganese sulfate monohydrate, 0.5 g / L of magnesium sulfate heptahydrate, 1 mL / L of Tween-80, and 20.0 g / L of agar, pH 6.2-6.4.

[0078] Cell culture medium: 89% (v / v) DMEM medium + 10% (v / v) fetal bovine serum + 1% (v / v) 100 x penicillin and streptomycin mixed solution (penicillin content 10000 U / mL, streptomycin concentration 10 mg / mL in the mixed solution).

[0079] The cell resuscitation and culture method involved in the following examples is as follows:

[0080] Firstly, take out the frozen human keratinocyte strain (HaCaT) from the -80°C refrigerator, quickly melt in the 37°C water bath, then add 5mL cell culture medium and centrifuge at 1000r / min for 3min, discard the supernatant. Add 1mL of cell culture medium to resuspend the cells, then place them in a culture dish and put them in a 37°C incubator containing 5% CO2 for culture. When the cells grow and recover vitality for 1-2d to reach 70%-80% confluence, the cells are subcultured.

[0081] Example 1: Preparation of postbiotic by fermenting Limosilactobacillus fermentum CCFM1401

[0082] 1. Screening and identification of fermenting Limosilactobacillus fermentum CCFM1401

[0083] The strain sample was derived from the feces of a healthy person, and after pretreatment, it was stored in 20% glycerol at -80°C in the refrigerator. After thawing, mix and take 0.5mL sample and add it to 4.5mL normal saline. Gradient dilution was performed with normal saline, and the appropriate gradient dilution was coated on MRS solid medium and cultured at 37°C for 48h. Typical colonies of fermenting Limosilactobacillus fermentum were picked and streaked on MRS solid medium for purification, and single colonies were transferred to MRS liquid medium for enrichment. The strain was obtained and stored in 30% glycerol. The strain genome was extracted for 16S rDNA amplification and sequencing (performed by Suzhou Jinyuzhi Biological Technology Co., Ltd.). The results were determined by NCBI sequence alignment to be fermenting Limosilactobacillus fermentum, named Limosilactobacillus fermentum CCFM1401, which was deposited at the Guangdong Microbial Culture Collection Center on July 22, 2024, with the accession number GDMCC No: 64895, and the address is No. 59, Building 5, 100, Martyrs' Road, Guangzhou.

[0084] 2. Preparation of postbiotic of fermenting Limosilactobacillus fermentum CCFM1401

[0085] (1) The fermenting Limosilactobacillus fermentum CCFM1401 was streaked from the preservation tube and cultured on modified MRS solid medium at 37°C in a water-jacketed incubator for 48h to obtain single colonies; the single colonies were inoculated into modified MRS liquid medium and cultured at 37°C for 12-18h to obtain culture 1;

[0086] Culture 1 was inoculated into modified MRS liquid medium at a 2% (v / v) inoculation amount and cultured at 37°C for 12h to obtain seed liquid;

[0087] The seed liquid was inoculated into modified MRS liquid medium at a 2% (v / v) inoculation amount for expansion culture, and the viable cell count was recorded after 18h of culture at 37°C to obtain bacterial liquid a.

[0088] The bacterial liquid a was centrifuged at 8000 r / min for 30 min, and the supernatant and bacterial slurry were collected. The supernatant was heat-treated (65°C, 30 min), and freeze-dried to obtain a powder for standby. The fermented L. mesenteroides CCFM1401 supernatant freeze-dried powder (denoted as CCFM1401-Q) was prepared. The bacterial slurry was resuspended with double-distilled water at 75% of the original bacterial liquid volume, and the resuspension was heat-treated (65°C, 30 min) and then homogenized in a high-pressure homogenizer (1000 MPa, 10 P times). After homogenization, the supernatant was obtained by centrifugation at 8000 r / min for 30 min to obtain the bacterial lysate (denoted as CCFM1401-L).

[0089] The preparation method of the live L. mesenteroides CCFM1401 is as follows: the bacterial liquid is prepared by the same method as above, and the concentration of the bacterial liquid is adjusted to the same level as that of the bacterial liquid a. The bacterial slurry obtained by centrifugation at 8000 r / min for 30 min is resuspended with glycerol at a ratio of 1 g:2 mL to obtain the live L. mesenteroides CCFM1401 glycerol tube, denoted as CCFM1401-H.

[0090] The postbiotics of L. mesenteroides CCFM1401 (bacterial lysate CCFM1401-L and fermented supernatant CCFM1401-Q) are prepared by the above-mentioned means.

[0091] (2) The postbiotics of L. mesenteroides FJSNTL31 and L. mesenteroides FJSNTL41 are simultaneously prepared according to the method of step (1).

[0092] Example 2: Establishment of an in vitro sodium dodecyl sulfate (SDS) damaged keratinocyte (HaCaT) model

[0093] (1) The HaCaT cells in the logarithmic growth phase were inoculated in a 96-well plate at a concentration of 1.2×10 4 cells / 100 μL / well, and the outermost circle of the well plate was filled with PBS solution to prevent edge effects. After 36 h of culture, the cells were attached, and a blank group, a control group, and an SDS treatment group were set up.

[0094] The blank group only contained cell culture medium without HaCaT cells.

[0095] The control group contained cell culture medium and HaCaT cells, but no SDS.

[0096] The treatment group contained cell culture medium, HaCaT cells, and SDS (concentrations of 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, and 50 μg / mL).

[0097] (2) Respectively, the above-mentioned hole plate is incubated in the incubator with the temperature of 37℃ for 6h, 12h and 24h, 10μL CCK8 solution is added to each hole after the incubation is finished, and the incubation is carried out for 1.5h to measure the absorbance (OD) at 450nm.

[0098] The cell viability is calculated according to the following formula: cell viability (%) = (treatment group OD value-blank group OD value) / (control group OD value-blank group OD value) x 100%.

[0099] (3) After the non-linear regression fitting, the influence on the cell viability is as shown in Figure 1 Compared with the control group (cell proliferation rate 100%), the cell viability of the model group treated by adding 15μg / mL SDS for 6h is 60.6%, and the SDS modeling causes significant damage to HaCaT cells.

[0100] Example 3: Effect of metachemion prepared by fermenting Lactobacillus muciadis CCFM1401 on HaCaT cell viability

[0101] (1) The HaCaT cells in the logarithmic growth phase are inoculated in the 96-hole plate at the concentration of 1.2×10 4 cells / 100μL / hole, wherein the outermost circle of the hole plate is filled with PBS solution to prevent edge effect, and the cells are cultured for 36h to be attached, and then the blank group, the control group and the metachemion treatment group are set;

[0102] The blank group only contains the cell culture medium without HaCaT cells;

[0103] The control group contains the cell culture medium and HaCaT cells without metachemion;

[0104] The metachemion treatment group contains the cell culture medium and HaCaT cells, and contains metachemion.

[0105] The metachemion is resuspended with the cell culture medium (the amount of resuspended metachemion is equivalent to the amount of metachemion prepared by fermenting the bacteria solution to the concentration of 5.0×10 7 CFU / ml), and 100μL of metachemion prepared by fermenting Lactobacillus muciadis FJSNTL31, Lactobacillus muciadis FJSNTL41 and Lactobacillus muciadis CCFM1401 is added respectively.

[0106] (2) Respectively, the above-mentioned hole plate is incubated in the incubator with the temperature of 37℃ for 6h, 12h and 24h, 10μL CCK8 solution is added to each hole after the incubation is finished, and the incubation is carried out for 1.5h to measure the absorbance (OD) at 450nm.

[0107] The cell viability is calculated according to the following formula: cell viability (%) = (treatment group OD value-blank group OD value) / (control group OD value-blank group OD value) x 100%.

[0108] The effect on cell activity was compared with the control group (cell proliferation rate 100%) as shown in Table 1. Figure 2 As shown in Table 1, the cell proliferation rates of HaCaT cells treated with the postbiotics prepared from L. fermentum CCFM1401 (CCFM1401-Q and CCFM1401-L), L. fermentum FJSNTL31 (FJSNTL31-Q and FJSNTL31-L), and L. fermentum FJSNTL41 (FJSNTL41-Q and FJSNTL41-L) at an inactivated cell concentration of 5.0 x 10 7 CFU / ml were 99.92%, 100%, 99.91%, 100.13%, and 99.72%, respectively.

[0109] According to the toxicity grading evaluation method in ISO 10993-5:2009, cell viability greater than 70% can be considered non-toxic. The above results show that the cell viability of HaCaT cells at a postbiotic concentration of 5.0 x 10 7 CFU / ml is higher than 95%, and considering its non-cytotoxicity, an inactivated cell concentration of 5.0 x 10 7 CFU / ml is a suitable postbiotic concentration for subsequent cell experiments.

[0110] Example 4: Effect of postbiotics prepared from L. fermentum CCFM1401 on HaCaT cell damage caused by SDS

[0111] 1. Postbiotics prepared from L. fermentum CCFM1401 maintain the cell activity of SDS-damaged HaCaT cells

[0112] (1) HaCaT cells in the logarithmic growth phase were inoculated in a 96-well plate at a concentration of 1.2 x 10 4 cells / 100 μL / well, and the outermost circle of the well plate was filled with PBS solution to prevent edge effects, and cultured for 36 h until the cells adhered;

[0113] (2) After the cells adhered, different groups were treated with different medium changes, and incubated for 6 h.

[0114] Control group: After the medium change in step (1), the cell culture medium containing HaCaT cells was not treated with postbiotics and did not contain SDS modeling agent.

[0115] Model group: The medium in step (1) was changed to a cell culture medium containing 15 μg / mL SDS, which contained the original HaCaT cells and was not treated with postbiotics; the cell culture medium containing SDS modeling agent was prepared by mixing SDS uniformly in the culture medium and filtering through a 0.22 μm water system filter to sterilize, so that the final concentration of SDS in the cell culture medium was 15 μg / mL.

[0116] Postbiotic treatment group: the treatment method is the same as the model group.

[0117] (3) After the incubation is completed, the original culture medium is discarded, and PBS is used for cleaning once. In the postbiotic treatment group, the corresponding postbiotic sample is added, and in the control group and the model group, the cell culture medium is added. Incubate for 24 hours again.

[0118] Among them, the postbiotic treatment group uses cell culture medium to resuspend the postbiotic (the amount of resuspended postbiotic is equivalent to the amount of postbiotic prepared from bacteria solution with a concentration of 5.0×10 7 CFU / ml), and 100 μL of postbiotic prepared from fermented Lactobacillus mucosus FJSNTL31, Lactobacillus mucosus FJSNTL41 and Lactobacillus mucosus CCFM1401 is added respectively.

[0119] (4) Add 10 μL of CCK8 solution to each well and incubate for 1.5 hours to measure the absorbance value (OD) at 450 nm.

[0120] The cell viability is calculated according to the following formula: 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%.

[0121] The results of the effect of postbiotics on SDS-induced HaCaT cell damage are as follows Figure 3 Compared with the control group (cell viability 100%), the cell viability of the model group is 68.91%, and SDS modeling causes significant damage to HaCaT cells.

[0122] After adding CCFM1401-Q and CCFM1401-L to the treatment group, the cell viability is 56.10% and 81.15% respectively, and among them, compared with the model group 68.91%, CCFM1401-L significantly improves the viability of HaCaT cells, indicating that the postbiotic of fermented Lactobacillus mucosus CCFM1401 can effectively alleviate the damage of SDS to HaCaT cells;

[0123] After the postbiotics of fermented Lactobacillus mucosus FJSNTL31 (FJSNTL31-Q and FJSNTL31-L) and Lactobacillus mucosus FJSNTL41 (FJSNTL41-Q and FJSNTL41-L) are treated, the viability of HaCaT cells is 65.77%, 67.82%, 74.75% and 63.53% respectively, that is, the postbiotics of other fermented Lactobacillus mucosus do not have the excellent ability to alleviate the damage of SDS to HaCaT cells as the postbiotic of fermented Lactobacillus mucosus CCFM1401.

[0124] 2. Effect of Postbiotic Prepared by Lactobacillus Mucosus CCFM1401 on Repairing SDS Damaged HaCaT Cell Barrier Function and Structural Protein Gene Expression

[0125] (1) HaCaT cells were seeded in a 6-well plate at 1.8 x 10 4 cells / well, and the cells were cultured for 36 h to adhere.

[0126] (2) After the cells adhered, different groups were treated with different medium changes for 6 h.

[0127] Control group: After step (1), the medium containing HaCaT cells was not treated with postbiotics and did not contain SDS modeling agent.

[0128] Model group: The medium in step (1) was changed to a medium containing 15 μg / mL SDS, which contained the original HaCaT cells and was not treated with postbiotics. The preparation method of the cell culture medium containing SDS modeling agent: the SDS was mixed uniformly in the medium and sterilized through a 0.22 μm water filter membrane, so that the final concentration of SDS in the cell culture medium was 15 μg / mL.

[0129] Postbiotic treatment group: The treatment method was the same as the model group.

[0130] (3) After incubation, the original medium was discarded and washed once with PBS. In the postbiotic treatment group, the corresponding postbiotic sample was added, and in the control group and model group, the cell culture medium was added. Incubate for another 24 h.

[0131] Among them, the postbiotic treatment group used cell culture medium to resuspend the postbiotic (the amount of resuspended postbiotic was equivalent to the amount of postbiotic prepared by fermenting bacteria to a concentration of 5.0 x 10 7 CFU / mL), and 2 mL of postbiotic prepared by Lactobacillus Mucosus CCFM1401-L was added.

[0132] (4) After incubation, the culture supernatant was discarded, each well was washed with PBS for 3 times, 1 mL of cell lysis solution was added to each well, and the cell lysis solution was extracted by repeated blowing and sucking. RNA was extracted and reverse transcribed into cDNA using an RT-PCR reverse transcription kit. The expression of genes in HaCaT cells was detected by real-time fluorescence quantitative method, and the mRNA expression of FLG, IVL, LOR, ZO-1, OCCLUDIN and CLDN was calculated using the 2 -△△Ct formula, where the internal reference was GAPDH. The results are shown in Table 1. Figure 4

[0133] Table 1 Primer Sequences

[0134] ​

[0135] According to the data results of step 1, only Lactobacillus fermentum CCFM1401-L has the effect of up-regulating cell activity, so the subsequent qPCR test of cell barrier and functional proteins selects this group for experiment.

[0136] Filaggrin (FLG) is essential for skin barrier function and is an important molecule that connects keratin fibers in the stratum corneum of human skin. With the help of FLG monomers, keratin fibers regularly aggregate to form a solid physical barrier in the outermost layer of the epidermis, which can prevent the loss of epidermal water and the invasion of external allergens. The absence of FLG is associated with many skin diseases, such as eczema (atopic dermatitis) and ichthyosis. Defects or absence of FLG protein in these diseases can lead to weakened skin barrier function, thereby increasing skin sensitivity to external stimuli. Therefore, the goal is to alleviate the damage of SDS to keratinocytes by increasing the expression of FLG. The expression results of FLG are shown in Table 4. Figure 4 As can be seen, the expression of FLG mRNA in the control group is 1, and after SDS intervention, the expression of the model group decreases to 0.29; the postbiotic CCFM1401-L prepared by fermenting Mucilaginibacter sp. CCFM1401 significantly increases the expression of FLG mRNA in HaCaT cells to 3.09, which is 10.6 times that of the model group.

[0137] Involucrin (IVL) is cross-linked with loricrin under the catalysis of transglutaminase in keratinocytes, forming an insoluble cornified envelope that constitutes the unique stratum corneum barrier structure of the epidermis. It also interacts with other keratinocyte differentiation proteins such as filaggrin (FLG) to maintain the structure and function of the skin barrier. The expression results of IVL are shown in Table 5. Figure 4 As can be seen, the expression of IVL mRNA in the control group is about 1, and after SDS intervention, the expression of the model group decreases to 0.18; the postbiotic CCFM1401-L prepared by fermenting Mucilaginibacter sp. CCFM1401 significantly increases the expression of IVL mRNA in HaCaT cells to 5.3, which is 29 times higher than the model group.

[0138] Loricrin (LOR) is involved in the formation of the cornified envelope during keratinization, which is a tough, non-water-soluble outer membrane that is essential for maintaining the integrity of the skin barrier. In addition, it helps maintain the hydration status of the skin and, through its interaction with other keratinocyte differentiation proteins, maintains the softness and elasticity of the skin. Defects or absence of LOR protein in these diseases can lead to weakened skin barrier function, thereby increasing skin sensitivity to external stimuli. Its abnormal expression or loss of function is associated with certain skin diseases, such as ichthyosis and certain types of keratosis. The expression results of LOR are shown in Table 6. Figure 4It can be seen that the expression of LOR mRNA in the control group is about 1, and the expression in the model group after SDS intervention decreases to 0.24. The postbiotic CCFM1401-L prepared from fermented L. m. subspecies johnsonii CCFM1401 significantly increases the expression of LOR mRNA in HaCaT cells to 10.47.

[0139] Zonula occludens-1 (ZO-1), occludin (OCCLUDIN) and claudin (CLDN) are key components of tight junctions (TJs) and are structural proteins of the skin barrier. They are involved in controlling the transport of substances between cells and are essential for maintaining the selective permeability of the skin barrier. In addition, they not only participate in the physical connection between cells, but also participate in the intracellular signal transduction process, affecting cell proliferation, differentiation and migration. When the skin is damaged, these proteins participate in the reconstruction of tight junctions and help repair the skin barrier. The results of the expression of the three structural proteins are shown in Table 2. Figure 4 It can be seen that CCFM1401-L has a significant up-regulating effect on ZO-1, CLDN and OCCLUDIN compared with the model group, and can increase the mRNA to 1.99, 1.52 and 2.25, respectively.

[0140] From the above results, it can be seen that the postbiotic (bacterial lysate) prepared from fermented L. m. subspecies johnsonii can up-regulate the expression of functional proteins (FLG, IVL and LOR) and structural proteins (ZO-1, OCCLUDIN and CLDN) mRNA when HaCaT cells are damaged by SDS, and can repair the damage of SDS to HaCaT cells by reducing abnormal cell protein function and relieving apoptosis.

[0141] Example 5: Effect of postbiotic prepared from fermented L. m. subspecies johnsonii CCFM1401 on cell pathway receptors

[0142] (1) HaCaT cells were seeded at 1.8 x 10 4 cells / well in a 6-well plate, and the cells were cultured for 36 h to adhere.

[0143] (2) After adhesion, the original culture medium was discarded, and the corresponding postbiotic sample was added to the postbiotic treatment group, and the cell culture medium was added to the control group. Then, the cells were incubated for another 24 h.

[0144] Among them, the postbiotic treatment group used cell culture medium to resuspend the postbiotic (the amount of resuspended postbiotic was equivalent to the amount of postbiotic prepared from bacteria with a fermentation concentration of 5.0 x 10 7 CFU / ml), and 2 mL of postbiotic prepared from fermented L. m. subspecies johnsonii CCFM1401-L was added.

[0145] (3) Incubation is over, discard the culture supernatant, wash each well with PBS quickly for 3 times, add 1 mL cell lysis solution to each well, repeatedly blow and suck, extract RNA from the cell lysis solution, and reverse transcribe into cDNA using an RT-PCR reverse transcription kit, detect the expression of genes in HaCaT cells by real-time fluorescence quantitative method, and use 2 -△△Ct The mRNA expression of STAT3 is calculated by the formula, wherein the internal reference is GAPDH, the primers are described in Table 2 below, and the results are shown in Figure 5

[0146] Table 2: Primer sequences

[0147]

[0148] JAK / STAT is also a classic pathway for maintaining skin barrier and inhibiting inflammation, and the content of STAT3 can be used as a characteristic target in this pathway. According to the results shown in Figure 5 The expression of STAT3 can be down-regulated to 0.43 by the CCFM1401 bacterial lysate.

[0149] In summary, the CCFM1401 bacterial lysate can regulate protein expression through the JAK / STAT pathway, thereby achieving the effect of repairing the barrier of HaCaT cells.

[0150] Example 6: Fermented Lactobacillus muciaparius CCFM1401 and postbiotic prepared therefrom for relieving skin barrier damage

[0151] 1. Effect of fermented Lactobacillus muciaparius CCFM1401 and postbiotic prepared therefrom on the level of barrier function in the skin of mice with skin barrier damage

[0152] The preparation method of the postbiotic (CCFM1401-Q and CCFM1401-S) of fermented Lactobacillus muciaparius CCFM1401 involved in the following examples is the same as that in Example 1, except that the bacterial slurry obtained by centrifuging the bacterial liquid at 8000 r / min for 30 min is resuspended in double distilled water at 75% of the original bacterial liquid volume, the resuspended solution is heat-treated at 65°C for 30 min, and high-pressure homogenization is not performed.

[0153] (1) 45 healthy female BALB / c mice aged 7 weeks were randomly divided into 9 cages, 5 mice in each cage, and the 9 cages were respectively:

[0154] Control group (Control): physiological saline was used as a control;

[0155] Model group (Model): physiological saline was used as a control;

[0156] ​CCFM1401-H group: live Lactobacillus mucosae CCFM1401 was used, with a dosage of 1 x 10 9 CFU;

[0157] CCFM1401-S group: Lactobacillus mucosae CCFM1401 postbiotic (inactivated bacteria) was used, with a dosage of 1 x 10 9 CFU;

[0158] CCFM1401-Q group: Lactobacillus mucosae CCFM1401 metabolite (fermentation supernatant) was used, with a dosage of 43 mg / kg mouse body weight.

[0159] In the above groups, the postbiotic (inactivated bacteria) or metabolite was prepared from the inactivated bacteria or metabolite of the corresponding amount of bacteria as the bacteria.

[0160] The experiment lasted for four weeks, and gavage was performed on days 7-28. Before modeling, the mouse back skin was shaved with a razor and depilatory cream, with a size of 2 cm long x 1.5 cm wide. A base solution was prepared with acetone and olive oil (4:1 v / v), and then 0.5% and 0.2% DNFB sensitizing drugs were prepared by mixing DNFB and the base solution. Modeling started from week 3, and on the first day of modeling, 25 μL of 0.5% DNFB drug was applied to the mouse back skin. Then, 25L of 0.2% DNFB drug was used on days 18, 21, 24, and 27. The blank group mice used the same amount of base solution. The specific experimental method is shown in Figure 6 .

[0161] After the experiment, the mice were sacrificed and the eyeball blood was taken. After standing for 40 min, the blood supernatant was centrifuged at 3000 r / min for 20 min for ELISA detection. The back skin tissue was cut and ground to prepare a homogenate with a weight-volume ratio of 1:10 with PBS, centrifuged at 3000 r / min for 20 min, and the skin supernatant was detected by an ELISA kit.

[0162] The content of filaggrin (FLG) and loricrin (LOR) in the mouse skin was detected by an Elisa kit as shown in Figure 7 .

[0163] Compared with the control group (3888 pg / 100 mg), the content of FLG in the skin of the model group decreased significantly to 2724.17 pg / 100 mg, and the content of FLG in the oral CCFM1401-S and CCFM1401-Q groups was significantly higher than that in the model group, restoring the content of FLG in the skin to 3938 pg / 100 mg and 3751.33 pg / 100 mg, respectively. The content of FLG in the oral CCFM1401-H group (2829.72 pg / 100 mg) was not significantly higher than that in the model group. The content of LOR in the control group was 514.42 pg / 100 mg, which was 1.17 times the content of LOR (439.5 pg / 100 mg) in the model group. Oral CCFM1401-S significantly increased the content of LOR to 505.92 pg / 100 mg compared with the model group, while CCFM1401-H (458.5 pg / 100 mg) and CCFM1401-Q (420.25 pg / 100 mg) groups had no significant difference compared with the model group.

[0164] In summary, oral CCFM1401-S has a good up-regulation effect on barrier function proteins (LOR and FLG), while oral CCFM1401-H and CCFM1401-Q have poor up-regulation effects. Therefore, oral CCFM1401-S has a good skin barrier repair effect.

[0165] 2. Effect of Lactobacillus fermentum CCFM1401 and its prepared probiotics on the level of skin barrier structure proteins in skin barrier damaged mice

[0166] The animal experiment design, gavage group, and skin homogenate sample preparation and steps 1 used in the following examples are the same. The content of skin barrier structure proteins ZO-1, CLDN, and Occludin in the skin of skin barrier damaged mice is shown in Figure 8 .

[0167] (1) Skin ZO-1 content: The content of ZO-1 in the control group was 58.13 pg / 100 mg, and the content in the model group was 47.78 pg / 100 mg. Oral CCFM1401-S, CCFM1401-H, and CCFM1401-Q groups significantly increased the content of ZO-1 compared with the model group, which was 58.51 pg / 100 mg, 61.7 pg / 100 mg, and 52.9 pg / 100 mg, respectively. Among them, the content of ZO-1 in the oral CCFM1401-H group was the highest.

[0168] (2) The CLDN content of the skin: the CLDN content of the control group was 97.05 pg / 100 mg, and the content of the model group was 67.59 pg / 100 mg. The oral administration of CCFM1401-S, CCFM1401-H and CCFM1401-Q significantly increased the CLDN content compared with the model group, which was 98.90 pg / 100 mg, 92.26 pg / 100 mg and 78.22 pg / 100 mg, respectively.

[0169] (3) The Occludin content of the skin: the Occludin content of the control group was 87.78 pg / 100 mg, and the content of the model group was 69.15 pg / 100 mg. The oral administration of CCFM1401-S, CCFM1401-H and CCFM1401-Q significantly increased the Occludin content compared with the model group, which was 83.34 pg / 100 mg, 88.23 pg / 100 mg and 78.47 pg / 100 mg, respectively.

[0170] In summary, according to the comprehensive indicators of skin structural proteins, the fermented L. m. CCFM1401 and the metaplasma prepared therefrom can increase the content of skin structural proteins, and the dead bacteria and live bacteria groups have better effects.

[0171] 3. Effect of fermented L. m. CCFM1401 and the metaplasma prepared therefrom on the expression of skin basement membrane barrier protein genes in skin barrier damage mice

[0172] The animal experiment design and the intragastric administration group in the following examples were the same as step 1. The skin RNA was extracted by the Nuozhan kit, and the gene expression of fibronectin, perlecan and laminin in the skin was detected by reverse transcription into cDNA. The primer sequences are described in Table 3 below. The relative content of fibronectin and laminin mRNA is shown in Table 4. Figure 9

[0173] Table 3: Primer sequences

[0174]

[0175] ​Fibronectin and laminin are proteins essential to the skin barrier health in the skin basement layer. By detecting the mRNA content of fibronectin and laminin, it is known that the relative expression of fibronectin mRNA of oral CCFM1401-S, CCFM1401-H and CCFM1401-Q is 3.96, 1.98 and 1.38, all of which have a significant up-regulation effect compared with the model group (0.46), in which the expression of oral CCFM1401-S is 8.62 times that of the model group. As for laminin, oral CCFM1401-S, CCFM1401-H and CCFM1401-Q mRNA relative expression is up-regulated to 1.7, 2.63 and 1.40, all of which have significant differences compared with the model group (0.28).

[0176] According to the above results, oral CCFM1401-S and CCFM1401-Q groups have better effects on the target points of basement membrane barrier repair.

[0177] 4、Fermented Lactobacillus muciaparius CCFM1401 and its prepared probiotics affect the TEWL and stratum corneum water content of skin barrier damaged mice

[0178] The animal experiment design and intragastric group synchronization steps in the following examples are the same as step 1. The skin tester Cutometer DUAL MPA580 is used with TEWL probe and stratum corneum water content CM825 probe to test the skin appearance. The skin TEWL and water content of oral CCFM1401-S, CCFM1401-H and CCFM1401-Q are shown in Figure 10 The TEWL of oral CCFM1401-S group decreased to 14.55 g / m 2 / h (equivalent to the control group), which is 3.19 times lower than the model group (46.48 g / m 2 / h). At the same time, this group has a significant up-regulation in the stratum corneum water content compared with the model group, and the water content of oral CCFM1401-S is 26.26, which is 1.49 times that of the model group (17.53). However, oral CCFM1401-H and CCFM-S have no significant difference in TEWL and stratum corneum water content compared with the model group.

[0179] In summary, oral CCFM1401-S has a good repair effect on the skin appearance.

[0180] 5、Fermented Lactobacillus muciaparius CCFM1401 and its prepared probiotics affect the TEWL and stratum corneum water content of skin barrier damaged mice

[0181] The animal experiment design, gavage group and skin homogenate sample preparation and steps 1 of the Elisa kit used in the following examples are the same. The three indicators of serum IgE, skin histamine and spleen index to some extent reflect the inflammation of the skin and the whole body, and the contents are shown in Table 2. Figure 11

[0182] Oral CCFM1401-S can significantly down-regulate the content of IgE compared with the model group, the IgE content of the model group is 1.46 μg / mL, and the IgE content of the CCFM1401-S group is 0.98 μg / mL. The contents of oral CCFM1401-H and CCFM1401-Q are 1.31 μg / mL and 1.32 μg / mL respectively, and there is no significant difference compared with the model group.

[0183] In the index of skin histamine, compared with the control group (11.00 ng / 100 mg), the histamine content of the skin of the model group increased significantly to 14.74 ng / 100 mg. Oral CCFM1401-S and CCFM1401-H can significantly reduce the content of skin histamine to 11.76 ng / 100 mg and 10.98 ng / 100 mg compared with the model group. Oral CCFM1401-Q cannot significantly reduce the content of skin histamine, and the content is 13.25 ng / 100 mg.

[0184] Spleen index is the reaction of systemic inflammation, in which the spleen index of the blank group is 0.38, and the model group is 0.59, indicating that DNFB damaged skin can cause systemic inflammatory response. Oral CCFM1401-S can significantly reduce the spleen index to 0.49, while oral CCFM1401-H and CCFM1401-Q cannot reduce the spleen index, and the values are both 0.6.

[0185] In summary, oral CCFM1401-S can alleviate the inflammatory condition in the DNFB damaged mouse skin barrier model to different extents, and has better anti-inflammatory effect.

[0186] 6、Fermented lactobacillus muciiparus CCFM1401 fermentation supernatant repairs skin barrier damage in mice through JAK / STAT pathway and OX40 receptor

[0187] The animal experiment design and gavage group in the following examples are the same as step 1. The skin RNA is extracted by Novozyme kit, and the gene expression of STAT3 and OX40 in the skin is detected by reverse transcription into cDNA, wherein the primer sequence is described in Table 4 below.

[0188] According to the results obtained in the above examples, oral CCFM1401-S has better skin barrier repair effect, so oral CCFM1401-S group is selected to analyze the repair mechanism. From the results of the above examples, oral CCFM1401-S can significantly down-regulate the content of IgE, reduce the content of skin histamine, and reduce the spleen index.​Figure 12 It can be seen that oral administration of CCFM1401-S can significantly down-regulate the mRNA expression of STAT3 and OX40 to 0.77 and 0.8, which is 5.69 and 2.39 times lower than the model group, respectively.

[0189] Based on this, it can be inferred that CCFM1401-Q can repair the skin barrier in the DNFB damaged mouse skin model by down-regulating the expression of STAT3 and OX40, two different pathway receptors.

[0190] Table 4: Primer sequences

[0191]

[0192] Although the present application has been disclosed in the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.

Claims

1. A strain of Lactobacillus mucosae (L. mucosae) CCFM1401, characterized in that, Limosilactobacillus fermentum The fermenting Limosilactobacillus CCFM1401 has been deposited in Guangdong Microbial Culture Collection Center on July 22, 2024, and the deposit number is GDMCC No: 64895. ​ 2. A microbial preparation containing the fermenting Limosilactobacillus CCFM1401 of claim 1.

3. The microbial preparation according to claim 2, characterized in that, The content of L. fermentum CCFM1401 in the microbial preparation is ≥ 1 x 10 6 CFU / mL or 1 x 10 6 CFU / g.

4. The postbiotic prepared from the L. m. fermentum CCFM1401 of claim 1, characterized by, The postbiotic comprises a cell lysate, a fermentation supernatant of the fermenting Limosilactobacillus CCFM1401, or a powder prepared by drying any of the cell lysate, the fermentation supernatant.

5. A composition containing the fermenting Limosilactobacillus CCFM1401 of claim 1, or the microbial preparation of claim 2 or 3, or the postbiotic of claim 4.

6. The composition of claim 5, wherein, The composition comprises a food, a drug, a health product, or a daily use product.

7. The composition of claim 6, wherein, The daily use product comprises a skin care product or a hair care product; the drug comprises a topical drug or an oral drug.

8. The composition of claim 6, wherein, The daily use product comprises a cosmetic product.

9. Use of the fermenting Limosilactobacillus CCFM1401 of claim 1 and / or a postbiotic thereof in the preparation of a product for repairing skin barrier or repairing skin damage.

10. Use of the fermenting Limosilactobacillus CCFM1401 of claim 1 and / or a postbiotic thereof in the preparation of a product for improving skin hydration.

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