Fermented lactobacillus mucus CCFM1401 for repairing skin barrier based on multiple target spots and metaplast of fermented lactobacillus mucus CCFM1401

Abstract

The invention discloses a lactobacillus mucus CCFM1401 for repairing a skin barrier based on multiple target spots and a metagen of the lactobacillus mucus CCFM1401, and belongs to the technical field of microorganisms and medicines. The thallus lysate of the fermented lactobacillus mucus CCFM1401, provided by the invention, can be used for improving the cell viability of damaged keratinocytes and the expression of FLG, LOR, IVL, ZO-1, Occludin and CLDN gene levels in the aspect of external application; and viable bacteria and metabiotics (including thallus lysate and fermentation supernatant) of the fermented lactobacillus mucus CCFM1401 are orally taken, so that TEWL can be reduced, the water content of cuticle can be up-regulated, inflammatory reactions (such as skin histamine and serum IgE) can be reduced, and the skin barrier function and expression of structural protein can be up-regulated. Therefore, the fermented lactobacillus mucilaginosus has a huge application prospect in preparation of external or oral medicines for repairing the skin barrier and preparation of food, health care products or cosmetics.

Description

Technical Field

[0001] The invention relates to a fermented mucus lactobacillus CCFM1401 for repairing skin barrier based on multiple targets and a postbiotic thereof, and belongs to the technical field of microorganisms and the technical field of medicine. Background Art

[0002] The skin is the largest organ in the human body and an important tissue for the body to defend against various physical, chemical and pathogenic microorganisms. The skin barrier is crucial for the skin to resist the outside world and protect the body's internal environment. A weak skin barrier will lead to its functional decline and defense function decline, which will endanger the health of the body. At the same time, the microecology has an important impact on skin health, and probiotics, as a means of regulating the balance of skin microecology, play a role through anti-oxidation, reducing extracellular matrix degradation and inhibiting the expression of inflammatory factors.

[0003] Among the inventions for skin barrier repair, most of them produce excellent effects by external use. For example, CN118304225A discloses an essential oil composition including levorotatory linalool, olive oil, jojoba seed oil, composite plant essential oil, phospholipids, glycerol, polyglycerol monolaurate and deionized water, and the composite plant essential oil is composed of perilla, Acanthopanax senticosus seeds, Guang Fo Shou, Liangfen grass, chamomile, Blumea balsamina and grapefruit peel, which has significant effects of removing acne, soothing sensitive muscles and promoting skin repair. CN118477030A discloses a high moisturizing repair mask containing Huoshan Dendrobium and a preparation method thereof, which has excellent antioxidant capacity, can provide more comprehensive protection for the skin and accelerate skin repair. Although these external products have certain effects in repairing damaged skin barriers, there is still room for improvement. First, their mechanism of action is relatively simple, and they mainly work by applying them on the skin surface. Secondly, external products usually have significant effects in the short term, but their ability to repair chronic or long-term damaged skin barriers is insufficient. In addition, topical products can only act on the outer layer of the skin and cannot regulate skin health from the inside, and the integrity of the skin barrier is closely related to the health of the body's internal organs. For this reason, it is particularly necessary to provide a probiotic repair product that can be taken orally and applied externally. Probiotic products that combine the synergistic effects of internal and external factors can repair the skin barrier more comprehensively and long-term, providing a more comprehensive repair effect. Summary of the invention

[0004] The invention provides application of fermented mucus lactobacillus (Limosilactobacillus fermentum) CCFM1401 and postbiotics thereof in preparing a product for repairing skin barrier.

[0005] The present invention provides a strain of fermented mucus lactobacillus (Limosilactobacillus fermentum) CCFM1401, which is deposited in Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC No: 64895 and a deposit date of July 22, 2024.

[0006] The fermented mucus Lactobacillus CCFM1401 is derived from feces of healthy people. The strain is sequenced and analyzed, and the obtained sequence is compared with the nucleic acid sequence in NCBI. The result shows that it is fermented mucus Lactobacillus of the genus Lactobacillus, and is named fermented mucus Lactobacillus CCFM1401.

[0007] The colonies of the fermented mucus lactobacillus CCFM1401 on the MRS solid culture medium are raised, white, smooth and round.

[0008] The present invention also provides a postbiotic prepared by applying the fermented mucus lactobacillus CCFM1401.

[0009] In one embodiment, the postbiotics include bacterial lysate, killed or inactivated cells, fermentation supernatant, or any of the above powders prepared by drying.

[0010] In one embodiment, the inactivated or inactivated cells are prepared as follows: the fermented mucus Lactobacillus CCFM1401 is cultured in a culture medium for a period of time, bacterial cells in the cell culture medium are collected, and inactivated bacterial cells are obtained after heat treatment.

[0011] In one embodiment, the heat treatment conditions are: 65° C., 30 min.

[0012] In one embodiment, the method for preparing the bacterial lysate is: culturing the fermented mucus Lactobacillus CCFM1401 in a culture medium for a period of time, collecting bacterial cells, high-pressure homogenizing, and centrifuging the supernatant to obtain the bacterial lysate.

[0013] In one embodiment, the fermentation supernatant is the supernatant obtained by culturing Lactobacillus mucilaginosus CCFM1401 in a culture medium for a period of time and then centrifuging the culture medium.

[0014] The present invention also provides a composition containing the fermented mucus lactobacillus CCFM1401 and / or its postbiotics.

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

[0016] In one embodiment, the food comprises the above composition and conventional auxiliary materials.

[0017] In one embodiment, the conventional auxiliary materials include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional enhancers.

[0018] In one embodiment, the health care product comprises the above composition and conventional auxiliary materials.

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

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

[0021] In one embodiment, the bacterial powder is a solid powder postbiotic prepared by drying the prepared liquid postbiotic of fermented Lactobacillus mucilaginosus CCFM1401.

[0022] In one embodiment, drying includes but is not limited to spray drying, vacuum freeze drying, fluidized bed drying, vacuum drying preparation.

[0023] The invention also provides application of the composition in preparing a product for repairing skin barrier.

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

[0025] (1) Improve the cell viability of skin keratinocytes (HaCaT) damaged by sodium dodecyl sulfate (SDS) in vitro;

[0026] (2) Improve the barrier function and structural protein expression of skin keratinocytes (HaCaT) damaged by sodium dodecyl sulfate (SDS) in vitro;

[0027] (3) Reduce the manifestations of skin barrier damage in individuals (blood biochemical indicators, skin appearance);

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

[0029] (5) Increase basement membrane protein in individual skin tissue;

[0030] (6) Reduce the individual's inflammatory response.

[0031] In one embodiment, the symptoms associated with skin barrier disruption include increased skin appearance, skin proteins, and inflammatory factors in the blood.

[0032] In one embodiment, the skin damage comprises skin dryness, decreased elasticity, sagging, wrinkles, oxidative damage, and protein loss.

[0033] In one embodiment, the application method includes topical application and oral administration.

[0034] In one embodiment, the content of fermented mucus lactobacillus CCFM1401 in the product is not less than 1×10 7 CFU / mL.

[0035] In one embodiment, the dosage of postbiotics prepared by fermenting Lactobacillus mucilaginosus CCFM1401 in the product is not less than 430 μg / kg body weight.

[0036] In one embodiment, the product is a medicine or a cosmetic.

[0037] In one embodiment, the medicine comprises the fermented Lactobacillus mucilaginosus CCFM1401, a drug carrier and / or a pharmaceutical excipient.

[0038] In one embodiment, the pharmaceutical excipient comprises an excipient and an additive.

[0039] In one embodiment, the pharmaceutical excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids and release retardants.

[0040] In one embodiment, the cosmetic contains fermented Lactobacillus mucilaginosus CCFM1401, matrix raw materials and / or conventional excipients.

[0041] In one embodiment, the matrix raw materials include oil raw materials, wax raw materials, synthetic oil raw materials, powdery raw materials, colloid raw materials, coagulants, and surfactants.

[0042] In one embodiment, the conventional auxiliary materials include one or more of moisturizers, whitening agents, flavoring agents, adhesives, lubricants, preservatives, film agents, antioxidants, emulsifiers, and cosmetic nutritional additives.

[0043] The present invention also provides application of the fermented mucus lactobacillus CCFM1401 in food production.

[0044] The present invention also provides the use of the fermented mucus lactobacillus CCFM1401 and / or its postbiotics in preparing products for repairing skin barrier or repairing skin damage.

[0045] The present invention also provides application of the fermented mucus lactobacillus CCFM1401 and / or its postbiotics in preparing a product for improving skin hydration.

[0046] Beneficial effects:

[0047] The fermented mucus lactobacillus (Limosilactobacillus fermentum) CCFM1401 of the present invention and the postbiotics prepared therefrom have the ability to alleviate skin barrier damage and increase the expression of related proteins for external and oral use, which is specifically embodied in:

[0048] (1) Improve the cell activity of skin keratinocytes (HaCaT) damaged by sodium dodecyl sulfate (SDS);

[0049] (2) In vitro, the mRNA expressions of FLG, IVL, LOR, ZO-1, Occludin, and CLDN in skin keratinocytes (HaCaT) damaged by sodium dodecyl sulfate (SDS) were increased;

[0050] (3) increase the levels of skin barrier function proteins (FLG and LOR) in individuals with skin barrier damage;

[0051] (4) increase the levels of skin barrier structural proteins (ZO-1, OCCLUDIN, and CLDN) in individuals with skin barrier damage;

[0052] (5) Enhance the basement membrane barrier effect in individuals with skin barrier damage;

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

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

[0055] Therefore, postbiotics prepared by fermentation of Limosilactobacillus fermentum CCFM1401 have great application prospects in products that alleviate host skin barrier damage.

[0056] Biomaterial Deposit

[0057] Limosilactobacillus fermentum CCFM1401, taxonomically named Limosilactobacillus fermentum, was deposited in Guangdong Provincial Microbiological Culture Collection Center on July 22, 2024, with the deposit number GDMCC No: 64895, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 :Evaluation of the effects of different concentrations of SDS on HaCaT cell damage;

[0059] Figure 2 :Effects of different postbiotics on the proliferation of HaCaT cells;

[0060] Figure 3 :The effects of different postbiotics on the viability of HaCaT cells after SDS injury;

[0061] Figure 4 :Different postbiotics repair the barrier function and structural protein gene expression of HaCaT cells after SDS injury;

[0062] Figure 5 :The receptors for the onset pathway of the postbiotic repair effect of fermented Lactobacillus mucilaginosus CCFM1401;

[0063] Figure 6 :Animal experiment procedures;

[0064] Figure 7 :Effects of fermented Lactobacillus mucilaginosus CCFM1401 and its prepared postbiotics on barrier function proteins in mouse skin tissue;

[0065] Figure 8 :Effects of fermented Lactobacillus mucilaginosus CCFM1401 and its prepared postbiotics on barrier structural proteins in mouse skin tissue;

[0066] Fig. 9 :Effects of fermented Lactobacillus mucilaginosus CCFM1401 and its postbiotics on the expression of barrier protein genes in the basal layer of mouse skin;

[0067] Fig.10 :Effects of fermented Lactobacillus mucilaginosus CCFM1401 and its prepared postbiotics on TEWL and stratum corneum water content of mouse skin tissue;

[0068] Fig.11 :Effects of fermented Lactobacillus mucilaginosus CCFM1401 and its prepared postbiotics on inflammation in mouse skin tissue and serum;

[0069] Fig.12 : The pathway receptors of fermented Lactobacillus mucilaginosus CCFM1401 and the postbiotics prepared therefrom for repairing the skin barrier;

[0070] “*” indicates statistical difference compared with the Model group (P<0.05), “**” indicates significant statistical difference compared with the Model group (P<0.01), “***” indicates extremely significant statistical difference compared with the Model group (P<0.001); “****” indicates extremely significant statistical difference compared with the Model group (P<0.0001). DETAILED DESCRIPTION

[0071] The present invention will be further described below in conjunction with specific embodiments.

[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 Vital River Company.

[0074] The fermented mucus Lactobacillus FJSNTL31, fermented mucus Lactobacillus FJSNTL41 and fermented mucus Lactobacillus CCFM1401 involved in the following examples are strains screened by the Food Biotechnology Center of Jiangnan University.

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

[0076] Improved MRS liquid medium: yeast powder 5.0 g / L, peptone 10.0 g / L, glucose 20.0 g / L, anhydrous sodium acetate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, dipotassium hydrogen phosphate 2.6 g / L, manganese sulfate monohydrate 0.25 g / L, magnesium sulfate heptahydrate 0.5 g / L and Tween-80 1 mL / L, pH 6.2-6.4.

[0077] Improved MRS solid medium: yeast powder 5.0 g / L, peptone 10.0 g / L, glucose 20.0 g / L, anhydrous sodium acetate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, dipotassium hydrogen phosphate 2.6 g / L, manganese sulfate monohydrate 0.25 g / L, magnesium sulfate heptahydrate 0.5 g / L, Tween-80 1 mL / L and agar 20.0 g / L, pH 6.2-6.4.

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

[0079] The cell recovery and culture methods involved in the following examples are as follows:

[0080] First, take out the frozen human keratinocyte cell line (HaCaT) from the -80℃ refrigerator, thaw quickly in a 37℃ water bath, then add 5mL of cell culture medium and centrifuge at 1000r / min for 3min, discard the supernatant. Add 1mL of cell culture medium to resuspend the cells and place them in a culture dish, and place them in a 37℃ incubator containing 5% CO2 for culture. When the cells regain vitality and grow for 1 to 2 days to reach 70% to 80% fusion, cell passage is performed.

[0081] Example 1: Fermentation of Lactobacillus mucilaginosus CCFM1401 to prepare postbiotics

[0082] 1. Screening and identification of fermentative mucus Lactobacillus CCFM1401

[0083] The strain samples were screened from the feces of healthy people. After pretreatment, they were stored in a -80°C refrigerator in 20% glycerol. After thawing, they were mixed and 0.5 mL of the sample was added to 4.5 mL of normal saline. The sample was gradiently diluted with normal saline, and the appropriate gradient dilution was selected and spread on MRS solid culture medium. The culture was cultured at 37°C for 48 hours, and the typical colonies of fermented mucus lactobacillus were picked and streaked on MRS solid culture medium for purification. Single colonies were picked and transferred to MRS liquid culture medium for bacterial enrichment and preserved in 30% glycerol to obtain the strain. The strain genome was extracted for 16S rDNA amplification and sequencing (performed by Suzhou Jinweizhi Biotechnology Co., Ltd.). The results were determined to be fermented mucus lactobacillus through NCBI sequence alignment and named fermented mucus lactobacillus (Limosilactobacillus fermentum) CCFM1401, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on July 22, 2024, with the deposit number GDMCC No.: 64895. The storage address is 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou.

[0084] 2. Preparation of postbiotics from fermented Lactobacillus mucilaginosus CCFM1401

[0085] (1) streaking the fermented mucus Lactobacillus CCFM1401 from the bacterium preservation tube and culturing it in a water-tight constant temperature incubator at 37° C. for 48 h using a modified MRS solid medium to obtain a single colony; picking a single colony and inoculating it into a modified MRS liquid medium, culturing it at 37° C. for 12-18 h, and obtaining a culture solution 1;

[0086] The culture solution 1 was inoculated into the modified MRS liquid medium at an inoculum volume of 2% (v / v), and cultured at 37° C. for 12 h to obtain a seed solution;

[0087] The seed solution was inoculated into the improved MRS liquid medium at 2% (v / v) for expansion and cultured at 37°C for 18 hours. The number of viable bacteria was recorded and the bacterial solution a was obtained.

[0088] The bacterial solution a was centrifuged at 8000r / min for 30min, the supernatant and the bacterial mud were collected, the supernatant was heat treated (65°C for 30min), and the powder was freeze-dried for standby use to prepare the fermentation supernatant freeze-dried powder of fermented mucus lactobacillus CCFM1401 (referred to as CCFM1401-Q). The bacterial mud was resuspended with double distilled water with 75% volume of the original bacterial solution, the resuspended liquid was heat treated (65°C for 30min), and then homogenized at high pressure in a high pressure homogenizer (1000MPa, 10P times), and after homogenization, the supernatant was centrifuged at 8000r / min for 30min to obtain the bacterial lysate (referred to as CCFM1401-L).

[0089] The preparation method of live fermented Lactobacillus mucus CCFM1401 bacteria is as follows: prepare the bacterial solution according to the same method as above, adjust the bacterial solution concentration to the same level as bacterial solution a, centrifuge the bacterial sludge at 8000r / min for 30min, and then reselect the bacterial sludge at a ratio of 1g:2mL glycerol to obtain a live fermented Lactobacillus mucus CCFM1401 bacteria glycerol tube, recorded as CCFM1401-H.

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

[0091] (2) According to the method of step (1), postbiotics of fermented Lactobacillus mucilaginosus FJSNTL31 and fermented Lactobacillus mucilaginosus FJSNTL41 are simultaneously prepared.

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

[0093] (1) HaCaT cells in logarithmic growth phase were cultured at 1.2×10 4 The cells were seeded in a 96-well plate at a concentration of 10 cells / 100 μL / well, and the outermost circle of the well plate was filled with PBS solution to prevent edge effect. After culturing for 36 h until the cells adhered to the wall, a blank group, a control group, and an SDS-treated group were set up;

[0094] Blank group: containing only cell culture medium without HaCaT cells;

[0095] Control group: containing cell culture medium and HaCaT cells but without SDS;

[0096] Treatment groups: contained cell culture medium, HaCaT cells and SDS (at concentrations of 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL and 50 μg / mL).

[0097] (2) The above-mentioned well plates were incubated in an incubator at 37°C for 6 h, 12 h and 24 h respectively. After the incubation, 10 μL of CCK8 solution was added to each well and incubated for 1.5 h, and the absorbance (OD) at 450 nm was measured.

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

[0099] (3) After nonlinear regression fitting, the effect on cell activity is as follows Figure 1 As shown, compared with the control group (cell proliferation rate 100%), the cell viability of the model group treated with 15 μg / mL SDS for 6 hours was 60.6%, and SDS modeling caused significant damage to HaCaT cells.

[0100] Example 3: Effect of postbiotics prepared by fermentation of Lactobacillus mucilaginosus CCFM1401 on HaCaT cell activity

[0101] (1) HaCaT cells in logarithmic growth phase were cultured at 1.2×10 4 The cells were inoculated into a 96-well plate at a concentration of 10 cells / 100 μL / well, and the outermost circle of the well plate was filled with PBS solution to prevent edge effect. After culturing for 36 h until the cells adhered to the wall, a blank group, a control group, and a postbiotic treatment group were set up;

[0102] Blank group: containing only cell culture medium without HaCaT cells;

[0103] Control group: containing cell culture medium and HaCaT cells but without postbiotics;

[0104] Postbiotic treatment group: contains cell culture medium and HaCaT cells, as well as postbiotics.

[0105] The postbiotics were resuspended in cell culture medium (the amount of postbiotics after resuspension was the same as that of the fermentation to a concentration of 5.0×10 7 CFU / ml of bacterial liquid), and 100 μL of postbiotics prepared by fermented Lactobacillus mucilaginosus FJSNTL31, fermented Lactobacillus mucilaginosus FJSNTL41 and fermented Lactobacillus mucilaginosus CCFM1401 were added respectively.

[0106] (2) The above-mentioned well plates were incubated in an incubator at 37°C for 24 h. After the incubation, 10 μL of CCK8 solution was added to each well and incubated for 1.5 h, and the absorbance (OD) at 450 nm was measured.

[0107] The cell viability was calculated according to the following formula: Cell viability (%) = (OD value of the treatment group - OD value of the blank group) / (OD value of the control group - OD value of the blank group) × 100%

[0108] Effects on cell viability Figure 2 As shown in the figure, compared with the control group (cell proliferation rate 100%), the addition of postbiotics consisting of fermented mucus lactobacillus CCFM1401 (CCFM1401-Q and CCFM1401-L), fermented mucus lactobacillus FJSNTL31 (FJSNTL31-Q and FJSNTL31-L) and fermented mucus lactobacillus FJSNTL41 (FJSNTL41-Q and FJSNTL41-L) at a killed cell concentration of 5.0×10 7 The cell proliferation rates at CFU / ml were 99.92%, 100%, 99.91%, 100.13% and 99.72% respectively.

[0109] According to the ISO 10993-5:2009 toxicity grading evaluation method, if the cell viability is greater than 70%, it can be considered non-toxic. The above results show that the inactivated bacterial concentration is 5.0×10 7 The viability of HaCaT cells at the postbiotic concentration of CFU / ml was higher than 95%. Considering its non-cytotoxicity, the inactivated bacterial concentration can be selected as 5.0×10 7 CFU / ml is the appropriate postbiotic concentration for subsequent cell experiments.

[0110] Example 4: Effect of postbiotics prepared by fermentation of Lactobacillus mucilaginosus CCFM1401 on HaCaT cell damage caused by SDS

[0111] 1. Postbiotics prepared by fermentation of Lactobacillus mucilaginosus CCFM1401 maintain the cell viability of SDS-damaged HaCaT cells

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

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

[0114] Control group: after changing the medium in step (1), containing cell culture medium and HaCaT cells, without postbiotic treatment, and without SDS modeling agent;

[0115] Model group: the medium in step (1) was replaced with a cell culture medium containing 15 μg / mL SDS, containing the original HaCaT cells without postbiotic treatment; the preparation method of the cell culture medium containing the SDS modeling agent: SDS was mixed evenly in the culture medium and sterilized by a 0.22 μm water filter membrane, so that the final concentration of SDS in the cell culture medium was 15 μg / mL;

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

[0117] (3) After the incubation, the original culture medium was discarded and the cells were washed once with PBS. The corresponding postbiotic samples were added to the postbiotic treatment group, and the cell culture medium was added to the control group and the model group, and the cells were incubated again for 24 h.

[0118] Among them, the postbiotic treatment group used cell culture medium to resuspend the postbiotics (the amount of postbiotics after resuspending was the same as that of fermentation to a concentration of 5.0×10 7 CFU / ml of bacterial liquid), and 100 μL of postbiotics prepared by fermented Lactobacillus mucilaginosus FJSNTL31, fermented Lactobacillus mucilaginosus FJSNTL41 and fermented Lactobacillus mucilaginosus CCFM1401 were added respectively.

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

[0120] The cell viability was calculated according to the following formula: cell viability of model group (%) = (OD value of model group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%; cell viability of treatment group (%) = (OD value of treatment group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0121] Effects of postbiotics on HaCaT cell damage caused by SDS Figure 3 Compared with the control group (cell viability 100%), the cell viability of the model group was 68.91%, and SDS modeling caused significant damage to HaCaT cells.

[0122] After adding CCFM1401-Q and CCFM1401-L, the cell viability of the treatment group was 56.10% and 81.15%, respectively. Compared with 68.91% in the model group, CCFM1401-L significantly improved the viability of HaCaT cells, indicating that the postbiotics of fermented Lactobacillus mucilaginosus CCFM1401 can effectively alleviate the damage caused by SDS to HaCaT cells.

[0123] After treatment with postbiotics from fermented Lactobacillus mucilaginosus FJSNTL31 (FJSNTL31-Q and FJSNTL31-L) and fermented Lactobacillus mucilaginosus FJSNTL41 (FJSNTL41-Q and FJSNTL41-L) in other treatment groups, the viability of HaCaT cells was 65.77%, 67.82%, 74.75% and 63.53%, respectively. That is, the postbiotics from other fermented Lactobacillus mucilaginosus did not have the excellent ability to alleviate the damage of SDS to HaCaT cells as shown by the postbiotics from fermented Lactobacillus mucilaginosus CCFM1401.

[0124] 2. Effect of postbiotics prepared by fermentation of Lactobacillus mucilaginosus CCFM1401 on the repair of barrier function and structural protein gene expression in SDS-damaged HaCaT cells

[0125] (1) HaCaT cells were cultured at 1.8×10 4 Cells / well were seeded on 6-well plates and cultured for 36 h until the cells adhered to the plate.

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

[0127] Control group: after changing the medium in step (1), containing cell culture medium and HaCaT cells, without postbiotic treatment, and without SDS modeling agent;

[0128] Model group: the medium in step (1) was replaced with a cell culture medium containing 15 μg / mL SDS, containing the original HaCaT cells without postbiotic treatment; the preparation method of the cell culture medium containing the SDS modeling agent: SDS was mixed evenly in the culture medium and sterilized by 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 the incubation, the original culture medium was discarded and the cells were washed once with PBS. The corresponding postbiotic samples were added to the postbiotic treatment group, and the cell culture medium was added to the control group and the model group, and the cells were incubated again for 24 h.

[0131] Among them, the postbiotic treatment group used cell culture medium to resuspend the postbiotics (the amount of postbiotics after resuspending was the same as that of fermentation to a concentration of 5.0×10 7 CFU / ml of bacterial solution), and 2 mL of postbiotics prepared by fermentation of Lactobacillus mucilaginosus CCFM1401-L were added respectively.

[0132] (4) After the incubation, the culture supernatant was discarded, each well was quickly washed three times with PBS, 1 mL of cell lysis solution was added to each well, and the well was repeatedly blown and vortexed. The cell lysis solution was aspirated to extract RNA, 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. -△△Ct The mRNA expression levels of FLG, IVL, LOR, ZO-1, OCCLUDIN and CLDN were calculated using the formula, wherein the internal reference was GAPDH, and the primers were described in Table 1 below. The results are shown in Table 1. Figure 4 shown.

[0133] Table 1 Primer sequences

[0134]

[0135] According to the data results of step 1, only Lactobacillus fermentum CCFM1401-L has the effect of upregulating cell activity, so this group was selected for the subsequent qPCR tests of cell barrier and functional proteins.

[0136] Filaggrin (FLG) is essential for the skin barrier function. It is an important molecule that connects keratin fibers in the stratum corneum of human skin. With the help of FLG monomers, keratin fibers are regularly aggregated to form a solid physical barrier in the outermost layer of the epidermis, which can prevent the loss of epidermal moisture and the invasion of external allergens. The loss of FLG is associated with many skin diseases, such as eczema (atopic dermatitis) and ichthyosis. The defects or loss of FLG protein in these diseases can lead to weakened skin barrier function, thereby increasing the skin's sensitivity to external stimuli. Therefore, the goal is to alleviate the damage of SDS to keratinocytes by increasing the gene expression of FLG. The results of FLG expression are shown in Figure 2. Figure 4 It can be seen that the expression level of FLG mRNA in the control group was 1, and the expression level in the model group decreased to 0.29 after SDS intervention; the postbiotic CCFM1401-L prepared by fermenting Lactobacillus mucilaginosus CCFM1401 significantly increased the expression level of FLG mRNA in HaCaT cells to 3.09, which was 10.6 times that of the model group.

[0137] Involucrin (IVL) is cross-linked with loricrin under the catalysis of keratinocyte transglutaminase to form an insoluble cornified envelope, which constitutes the unique keratin 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. Figure 4 It can be seen that the expression level of IVL mRNA in the control group was about 1, and the expression level in the model group decreased to 0.18 after SDS intervention; the postbiotic CCFM1401-L prepared by fermenting Lactobacillus mucilaginosus CCFM1401 significantly increased the expression level of IVL mRNA in HaCaT cells to 5.3, which was 29 times higher than that in the model group.

[0138] Loricrin (LOR) is involved in the formation of the cornified envelope during keratinization, a tough, water-insoluble outer membrane that is essential for maintaining the integrity of the skin barrier. In addition, it helps maintain the hydration state of the skin and maintains the softness and elasticity of the skin by working with other keratinocyte differentiation proteins. Defects or absence of LOR proteins in these diseases can lead to a weakening of the skin barrier function, thereby increasing the sensitivity of the skin to external stimuli. Its abnormal expression or loss of function is associated with certain skin diseases, such as ichthyosis and certain types of keratosis. LOR expression results are determined by Figure 4It can be seen that the expression level of LOR mRNA in the control group was about 1, and the expression level in the model group decreased to 0.24 after SDS intervention. The postbiotic CCFM1401-L prepared by fermenting Lactobacillus mucilaginosus CCFM1401 significantly increased the expression level of LOR mRNA in HaCaT cells to 10.47.

[0139] Zonula occludens 1 (ZO-1), occludens connexin (OCCLUDIN) and occludens DN (CLDN) are key components of tight junctions (TJs) between cells 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 are not only involved in the physical connection between cells, but also in the signal transduction process within cells, affecting cell proliferation, differentiation and migration. When the skin is damaged, these proteins are involved 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 Figure 2. Figure 4 It can be seen that CCFM1401-L has a significant upregulation effect on ZO-1, CLDN and OCCLUDIN compared with the model group, increasing the mRNA to 1.99, 1.52 and 2.25 respectively.

[0140] From the above results, it can be seen that the postbiotics (bacterial lysate) prepared by fermented lactobacillus mucilaginosus can upregulate 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 repair the damage of SDS to HaCaT cells by alleviating cellular protein dysfunction and alleviating cell apoptosis.

[0141] Example 5: Effects of postbiotics prepared by fermentation of Lactobacillus mucilaginosus CCFM1401 on cell pathway receptors

[0142] (1) HaCaT cells were cultured at 1.8×10 4 Cells / well were seeded on 6-well plates and cultured for 36 h until the cells adhered to the plate.

[0143] (2) After the cells adhered to the wall, the original culture medium was discarded, and the corresponding postbiotic samples were added to the postbiotic treatment group and the cell culture medium was added to the control group, and the cells were incubated again for 24 h.

[0144] Among them, the postbiotic treatment group used cell culture medium to resuspend the postbiotics (the amount of postbiotics after resuspending was the same as that of fermentation to a concentration of 5.0×10 7 CFU / ml of bacterial solution), and 2 mL of postbiotics prepared by fermentation of Lactobacillus mucilaginosus CCFM1401-L were added respectively.

[0145] (3) After the incubation, the culture supernatant was discarded, each well was quickly washed three times with PBS, 1 mL of cell lysis solution was added to each well, and the cells were repeatedly pipetted and the cell lysis solution was aspirated to extract RNA, which was 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. -△△Ct The mRNA expression of STAT3 was calculated using the formula, wherein the internal reference was GAPDH, and the primers were described in Table 2 below. The results are shown in Table 2. Figure 5 shown.

[0146] Table 2: Primer sequences

[0147]

[0148] JAK / STAT is also a classic pathway for maintaining the skin barrier and inhibiting inflammation. Downregulation of STAT3 content can serve as a characteristic target in this pathway. Figure 5 The results show that CCFM1401 bacterial lysate can downregulate the expression of STAT3 to 0.43.

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

[0150] Example 6: Fermentation of Lactobacillus mucilaginosus CCFM1401 and postbiotics prepared therefrom alleviate skin barrier damage

[0151] 1. Effects of fermented Lactobacillus mucilaginosus CCFM1401 and its prepared postbiotics on the barrier function level in the skin of mice with skin barrier damage

[0152] The preparation method of the postbiotics (CCFM1401-Q and CCFM1401-S) of the fermented mucus Lactobacillus CCFM1401 involved in the following embodiments is the same as that in Example 1, the difference is that the cultured bacterial solution is centrifuged at 8000r / min for 30min to obtain the bacterial mud, the bacterial mud is resuspended with double distilled water with 75% volume of the original bacterial solution, and the resuspension is heat treated at 65°C for 30min without high-pressure homogenization.

[0153] (1) 45 healthy female BALB / c mice aged 7 weeks were randomly divided into 9 cages, with 5 mice in each cage. The 9 cages were as follows: except for the model group (Model) which had 2 cages, the blank group and the other groups each had 1 cage, which were:

[0154] Blank group (Control): Use normal saline as control;

[0155] Model group: normal saline was used as the control;

[0156] CCFM1401-H group: fermented mucus Lactobacillus CCFM1401 live bacteria, dose: 1×10 9 CFU;

[0157] CCFM1401-S group: fermented Lactobacillus mucilaginosus CCFM1401 postbiotics (inactivated bacteria), the dosage is: bacteria count 1×10 9 CFU;

[0158] CCFM1401-Q group: fermented Lactobacillus mucilaginosus CCFM1401 metabolites (fermentation supernatant) were used at a dose of 43 mg / kg mouse body weight;

[0159] Among them, the postbiotics (inactivated bacteria) or metabolites in the above groups are inactivated bacteria or metabolites prepared from bacterial liquid with the corresponding amount of live bacteria.

[0160] The experiment lasted for four weeks, and gavage was performed on the 7th to 28th day. Before modeling, the back skin of the mice was shaved with a razor and depilatory cream, with the size of 2cm long x 1.5cm wide. A basic solution was prepared with acetone and olive oil (4:1v / v), and then DNFB and the basic solution were mixed to prepare 0.5% and 0.2% DNFB sensitizing drugs. Modeling started in the 3rd week, and on the first day of modeling, 25μL of 0.5% DNFB drug was applied to the back skin of the mice. Afterwards, 25L of 0.2% DNFB drug was used on days 18, 21, 24 and 27. The blank group mice used an equal amount of basic solution. The specific experimental methods are as follows. Figure 6 shown.

[0161] After the experiment, the mice were killed and blood was collected from the eyeballs. After standing for 40 minutes, the blood was centrifuged at 3000r / min for 20 minutes to obtain the blood supernatant for ELISA detection; the back skin tissue was cut and ground into a homogenate at a weight-to-volume ratio of 1:10 with PBS, and the homogenate was centrifuged at 3000r / min for 20 minutes and the skin supernatant was obtained for detection by an ELISA kit.

[0162] The contents of filaggrin (FLG) and loricrin (LOR) in mouse skin were detected by Elisa kit. Figure 7 shown.

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

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

[0165] 2. Effects of Lactobacillus fermentum CCFM1401 and its postbiotics on the levels of barrier structural proteins in the skin of mice with skin barrier damage

[0166] The animal experiment design, gavage groups and skin homogenate sample preparation used in the following examples are the same as those in step 1. The levels of barrier structural proteins ZO-1, CLDN and Occludin in the skin of mice with skin barrier damage are as follows: Figure 8 shown.

[0167] (1) Skin ZO-1 content: The ZO-1 content in the control group was 58.13 pg / 100 mg, and that in the model group was 47.78 pg / 100 mg. The oral CCFM1401-S, CCFM1401-H, and CCFM1401-Q groups had significantly higher ZO-1 content than that in the model group, which were 58.51 pg / 100 mg, 61.7 pg / 100 mg, and 52.9 pg / 100 mg, respectively. The highest ZO-1 content was found in the oral CCFM1401-H group.

[0168] (2) Skin CLDN content: The CLDN content in the control group was 97.05 pg / 100 mg, and that in the model group was 67.59 pg / 100 mg. The CLDN content in the oral CCFM1401-S, CCFM1401-H, and CCFM1401-Q groups was significantly higher than that in the model group, which were 98.90 pg / 100 mg, 92.26 pg / 100 mg, and 78.22 pg / 100 mg, respectively.

[0169] (3) Occludin content in the skin: The occludin content in the control group was 87.78 pg / 100 mg, and that in the model group was 69.15 pg / 100 mg. Compared with the model group, the occludin content in the oral CCFM1401-S, CCFM1401-H, and CCFM1401-Q groups was significantly higher than that in the model group, which were 83.34 pg / 100 mg, 88.23 pg / 100 mg, and 78.47 pg / 100 mg, respectively.

[0170] In summary, based on the comprehensive indicators of skin structural proteins, it can be seen that fermented mucus Lactobacillus CCFM1401 and the postbiotics prepared therefrom can increase the content of skin structural proteins, and the effects of the dead bacteria and live bacteria groups are better.

[0171] 3. Effects of fermented Lactobacillus mucilaginosus CCFM1401 and its prepared postbiotics on the gene expression of basement membrane barrier proteins in mice with skin barrier damage

[0172] The animal experiment design and gavage groups involved in the following examples are the same as step 1. Skin RNA was extracted using the Novozyme kit and reversely transcribed into cDNA to detect the gene expression of fibronectin, globin and laminin in the skin, wherein the primer sequences are described in Table 3 below. The relative mRNA content of fibronectin and laminin is shown in Fig. 9 shown.

[0173] Table 3: Primer sequences

[0174]

[0175] Fibronectin and adhesion protein are proteins in the basal layer of the skin that are essential for the health of the skin barrier. By detecting the mRNA content of fibronectin and adhesion protein, it can be seen that the relative expression of fibronectin mRNA of oral CCFM1401-S, CCFM1401-H and CCFM1401-Q was 3.96, 1.98 and 1.38, which were significantly upregulated compared with the model group (0.46), among which the expression of oral CCFM1401-S was 8.62 times that of the model group. For adhesion protein, the relative expression of oral CCFM1401-S, CCFM1401-H and CCFM1401-Q mRNA was upregulated to 1.7, 2.63 and 1.40, which were significantly different from the model group (0.28).

[0176] According to the above results, the oral CCFM1401-S and CCFM1401-Q groups had better effects in the targets of basement membrane barrier repair.

[0177] 4. Effects of fermented Lactobacillus mucilaginosus CCFM1401 and its prepared postbiotics on TEWL and stratum corneum water content in mice with skin barrier damage

[0178] The animal experiment design and oral gavage groups involved in the following examples are the same as step 1. The skin surface state test was performed using a skin tester Cutometer DUAL MPA580 equipped with a TEWL probe and a stratum corneum moisture content CM825 probe. The skin TEWL and moisture content of oral CCFM1401-S, CCFM1401-H and CCFM1401-Q are shown in the figure. Fig.10 The TEWL of the oral CCFM1401-S group decreased to 14.55 g / m 2 / h (equivalent to the control group), and lower than the model group (46.48 g / m 2 / h) decreased by 3.19 times. At the same time, this group also had a significant increase in the water content of the stratum corneum compared with the model group. The water content of oral CCFM1401-S was 26.26, which was 1.49 times that of the model group (17.53). However, there was no significant difference in TEWL and stratum corneum water content between oral CCFM1401-H and CCFM-S and the model group.

[0179] In summary, oral CCFM1401-S has a good repairing effect on the skin surface condition.

[0180] 5. Effects of fermented Lactobacillus mucilaginosus CCFM1401 and its prepared postbiotics on blood and skin inflammation in mice with skin barrier damage

[0181] The animal experiment design, gavage groups and skin homogenate sample preparation used in the following examples are the same as step 1. The three indicators of serum IgE, skin histamine and spleen index reflect the inflammation of the skin and the whole body to a certain extent. Fig.11 shown.

[0182] Oral administration of CCFM1401-S can significantly reduce the IgE content compared with the model group. The IgE content of the model group was 1.46μg / mL, and the IgE content of the CCFM1401-S group was 0.98μg / mL. However, the oral contents of CCFM1401-H and CCFM1401-Q were 1.31μg / mL and 1.32μg / mL, respectively, which were not significantly different from the model group.

[0183] In terms of skin histamine, compared with the control group (11.00ng / 100mg), the histamine content in the model group's skin increased significantly to 14.74ng / 100mg. Oral administration of CCFM1401-S and CCFM1401-H significantly reduced the histamine content in the skin to 11.76ng / 100mg and 10.98ng / 100mg compared with the model group. However, oral administration of CCFM1401-Q could not significantly reduce the histamine content in the skin, and its content was 13.25ng / 100mg.

[0184] The spleen index is a systemic inflammatory response, with the blank group spleen index being 0.38 and the model group spleen index being 0.59, indicating that DNFB damage to the skin can cause systemic inflammatory responses. Oral administration of CCFM1401-S significantly reduced the spleen index by 0.49, while oral administration of CCFM1401-H and CCFM1401-Q could not reduce the spleen index, with values ​​of both being 0.6.

[0185] In summary, oral administration of CCFM1401-S can alleviate the inflammation in the DNFB-damaged mouse skin barrier model to varying degrees and has a better anti-inflammatory effect.

[0186] 6. Fermentation supernatant of Lactobacillus mucilaginosus CCFM1401 repairs skin barrier damage in mice via JAK / STAT pathway and OX40 receptor

[0187] The animal experiment design and gavage groups involved in the following examples are the same as step 1. Skin RNA was extracted using the Novozyme kit and reversely transcribed into cDNA to detect the gene expression of STAT3 and OX40 in the skin, wherein the primer sequences are described in Table 4 below.

[0188] According to the results obtained in the above examples, all indicators were analyzed to show that oral CCFM1401-S has a good skin barrier repair effect, so the oral CCFM1401-S group was selected to analyze the repair mechanism. Fig.12 It can be seen that oral administration of CCFM1401-S can significantly downregulate the mRNA expression of STAT3 and OX40 to 0.77 and 0.8, which are 5.69 and 2.39 times lower than those of 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 downregulating the expression of two different pathway receptors, STAT3 and OX40.

[0190] Table 4: Primer sequences

[0191]

[0192] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Limosilactobacillus fermentum CCFM1401, characterized in that: The fermented mucus Lactobacillus CCFM1401 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on July 22, 2024, with the collection number GDMCC No: 64895.

2. A microbial preparation containing the fermented mucus lactobacillus CCFM1401 according to claim 1.

3. The microbial preparation according to claim 2, characterized in that The content of fermented mucus lactobacillus CCFM1401 in the microbial preparation is ≥1×10 6 CFU / mL or 1×10 6 CFU / g.

4. The postbiotic prepared by fermenting Lactobacillus mucilaginosus CCFM1401 according to claim 1.

5. The postbiotic according to claim 4, characterized in that The postbiotics include bacterial lysate, inactivated or inactivated cells, fermentation supernatant, or any powder prepared by drying of fermented Lactobacillus mucilaginosus CCFM1401.

6. A composition comprising the fermented mucus Lactobacillus CCFM1401 according to claim 1, or the microbial preparation according to claim 2 or 3, or the postbiotics according to claim 4 or 5.

7. The composition according to claim 6, characterized in that The composition includes food, medicine, health care product or daily chemical product.

8. The composition according to claim 6, characterized in that The daily chemical products include skin care products, cosmetics or toiletries; the medicines include smear-type medicines or oral medicines.

9. Use of the fermented mucus lactobacillus CCFM1401 and / or its postbiotics according to claim 1 in preparing products for repairing skin barrier or repairing skin damage.

10. Use of the fermented Lactobacillus mucilaginosus CCFM1401 and / or its postbiotics according to claim 1 in the preparation of a product for improving skin hydration.

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