Lactobacillus reuteri CCFM1403 with skin repairing effect and metagen of lactobacillus reuteri CCFM1403

Abstract

The invention discloses lactobacillus reuteri CCFM1403 with a skin repairing effect and a metagen of the lactobacillus reuteri CCFM1403, and belongs to the technical field of microorganisms and medicines. The lactobacillus reuteri CCFM1403 provided by the invention has the effect of improving the barrier function of the skin in two aspects of external use and oral administration, and is specifically shown in that when the thallus lysate of the lactobacillus reuteri CCFM1403 is externally used, the cell viability and the related barrier function of keratinocytes after injury and the expression of a structural protein gene level can be improved; and the DNFB-induced mouse skin barrier injury can be relieved in the aspects of TEWL in apparent aspect, water content of cuticle, skin barrier protein expression and the like by orally taking viable bacteria and postbiotics of the lactobacillus reuteri CCFM1403. Therefore, the lactobacillus reuteri 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 strain of Lactobacillus reuteri CCFM1403 with skin repairing function and postbiotics thereof, belonging to the technical field of microorganisms and the technical field of medicine. Background Art

[0002] The skin barrier is the first line of defense against potential pathogens, pollutants and contact allergens. In addition to the physical barrier provided by keratinocytes, the skin barrier contains innate immune elements such as antimicrobial peptides, pattern recognition receptor systems, lipids, ions designed to prevent pathogen colonization and protect the commensal microbiota. The physical barrier of keratinocytes depends on the formation of tight junctions (TJs). TJs separate the epidermal layer of the stratum granulosum from the external environment. It is now known that dysregulated TJs and stratum corneum (SC) proteins that disrupt the epithelial barrier are one of the potential causes of contact allergic skin diseases.

[0003] Currently, most skin barrier repairs come from Chinese herbal medicine ingredients to relieve oxidative damage to the skin. Among them, in terms of external use, CN118384321A discloses a sodium hyaluronate dressing for skin barrier repair, and CN118476992A discloses a moisturizing and repairing skin care composition, including phytosphingosine, ceramide and cholesterol. In oral applications, CN113616765A discloses a skin antibacterial liquid, including dandelion, blood lotus, officinal maple, red cypress, Bidens pilosa, red baiwu, white loquat, notoginseng, Uncaria rhynchophylla, safflower, vanilla, ginger, wormwood, Panax notoginseng, honeysuckle vine, hawthorn, heather, sea maple, ilex, and red rock bean; in terms of oral administration, CN104323267A discloses a specific Ganoderma lucidum polybacterium and its preparation method, which can help wound healing. Ganoderma lucidum water can phagocytize bacteria, inhibit bacterial growth, help wounds to reduce inflammation and relieve pain, and has good cosmetic effects. Long-term consumption can make the skin elastic.

[0004] In summary, although the existing research on skin barrier repair uses Chinese herbal ingredients, sodium hyaluronate, ceramide and other substances to alleviate oxidative damage and provide moisturizing and antibacterial effects, there are still some defects. Its mechanism of action is relatively simple, mainly focusing on surface repair, and cannot deeply regulate the skin microecology and immune function. In addition, there are individual differences in the absorption efficiency and bioavailability of oral Chinese herbal formulas, and the effect on skin barrier repair is not direct and long-lasting. Some Chinese herbal ingredients may have adverse reactions on sensitive skin, limiting the scope of application. The use of probiotic products that can repair the skin barrier by oral and external application to intervene in damaged skin can not only deeply repair the skin, regulate the microecology, and enhance immunity, but also have better naturalness and biocompatibility, suitable for a variety of skin types, and can achieve a more lasting and stable repair effect through internal and external synergy. Therefore, it is crucial to provide a strain that can repair the skin barrier by oral and external application. Summary of the invention

[0005] The invention provides application of Limosilactobacillus reuteri CCFM1403 and postbiotics thereof in preparing a product for repairing skin barrier.

[0006] The invention provides a strain of Limosilactobacillus reuteri CCFM1403, which is preserved in Guangdong Province Microbiological Culture Collection Center with a preservation number of GDMCC No: 65088.

[0007] The Lactobacillus reuteri CCFM1403 is derived from the 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 Lactobacillus reuteri of the genus Pediococcus, and is named Lactobacillus reuteri CCFM1403.

[0008] The colonies of Lactobacillus reuteri CCFM1403 on the MRS solid culture medium are raised, white, smooth and round.

[0009] The present invention also provides a postbiotic prepared by applying the Lactobacillus mucilaginosus CCFM1403.

[0010] In one embodiment, the postbiotics include bacterial lysate, killed or inactivated cells, fermentation supernatant, or any of the above powders prepared by drying.

[0011] In one embodiment, the inactivated or inactivated cells are prepared as follows: the Lactobacillus reuteri CCFM1403 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.

[0012] In one embodiment, the heat treatment conditions are: 65° C., 30 min.

[0013] In one embodiment, the method for preparing the bacterial lysate is: culturing the Lactobacillus reuteri CCFM1403 in a culture medium for a period of time, collecting bacterial cells, high-pressure homogenizing, and obtaining the bacterial lysate from the supernatant of the centrifugation.

[0014] In one embodiment, the fermentation supernatant is the supernatant obtained by culturing Lactobacillus soreuteri CCFM1403 in a culture medium for a period of time and then centrifuging the culture medium.

[0015] The present invention also provides a composition containing the Lactobacillus reuteri CCFM1403 and / or its postbiotics.

[0016] In one embodiment, the composition includes but is not limited to food, medicine, health care product or cosmetics.

[0017] In one embodiment, the food comprises the above composition and conventional auxiliary materials.

[0018] In one embodiment, the conventional auxiliary materials include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional enhancers.

[0019] In one embodiment, the health care product comprises the above composition and conventional auxiliary materials.

[0020] In one embodiment, the conventional auxiliary materials include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional enhancers.

[0021] In one embodiment, the composition is a probiotic powder.

[0022] In one embodiment, the bacterial powder is a solid powder postbiotic prepared by drying the prepared liquid postbiotic of Lactobacillus reuteri CCFM1403.

[0023] In one embodiment, drying includes but is not limited to spray drying, vacuum freeze drying, fluidized bed drying, vacuum drying preparation.

[0024] The invention also provides application of the composition in preparing a product for repairing skin barrier.

[0025] In one embodiment, the product comprises at least one of the following effects:

[0026] (1) Improve the cell viability of skin keratinocytes (HaCaT) damaged by sodium dodecyl sulfate (SDS) in vitro;

[0027] (2) Improve the barrier function and structural protein expression of skin keratinocytes (HaCaT) damaged by sodium dodecyl sulfate (SDS) in vitro;

[0028] (3) Reduce the manifestations of skin barrier damage in individuals (blood biochemical indicators, skin appearance);

[0029] (4) Increase the barrier function and structural proteins of individual skin tissue;

[0030] (5) Increase basement membrane protein in individual skin tissue;

[0031] (6) Reduce the individual's inflammatory response.

[0032] In one embodiment, the symptoms associated with skin barrier disruption include increased skin appearance, skin proteins, and inflammatory factors in the blood.

[0033] In one embodiment, the skin damage comprises skin dryness, decreased elasticity, sagging, wrinkles, oxidative damage, and protein loss.

[0034] In one embodiment, the application method includes topical application and oral administration.

[0035] In one embodiment, the content of Lactobacillus reuteri CCFM1403 in the product is not less than 1×10 7 CFU / mL.

[0036] In one embodiment, the dosage of the postbiotic fermentation supernatant prepared by Lactobacillus reuteri CCFM1403 in the product is not less than 720 μg / kg.

[0037] In one embodiment, the product is a medicine or a cosmetic.

[0038] In one embodiment, the medicine comprises the Lactobacillus reuteri CCFM1403, a drug carrier and / or a pharmaceutical excipient.

[0039] In one embodiment, the pharmaceutical excipient comprises an excipient and an additive.

[0040] 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.

[0041] In one embodiment, the cosmetic contains Lactobacillus reuteri CCFM1403, matrix raw materials and / or conventional excipients.

[0042] 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.

[0043] 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.

[0044] The present invention also provides application of the Lactobacillus reuteri CCFM1403 in food production.

[0045] Beneficial effects:

[0046] The Limosilactobacillus reuteri CCFM1403 and the postbiotics prepared therefrom of the present invention 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:

[0047] (1) Improve the cell activity of skin keratinocytes (HaCaT) damaged by sodium dodecyl sulfate (SDS);

[0048] (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;

[0049] (3) increase the levels of skin barrier function proteins (FLG, LOR, and IVL) in individuals with DNFB skin barrier damage;

[0050] (4) increase the levels of skin barrier structural proteins (ZO-1, OCCLUDIN, and CLDN) in individuals with DNFB skin barrier damage;

[0051] (5) Enhance the basement membrane barrier effect of individuals with skin barrier damage caused by DNFB;

[0052] (6) Repair the skin appearance of individuals with DNFB skin barrier damage;

[0053] (7) reduce skin thickness and TEWL in individuals with DNFB skin barrier loss;

[0054] (8) improve skin moisture content in individuals with DNFB skin barrier loss;

[0055] Therefore, postbiotics prepared by Limosilactobacillus reuteri CCFM1403 have great application prospects in products that alleviate skin barrier damage.

[0056] Biomaterial Deposit

[0057] Limosilactobacillus reuteri, taxonomically named Limosilactobacillus reuteri, was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 2, 2024, with the deposit number GDMCC No: 65088, 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 :Receptors for the onset pathway of the postbiotic repair effect of Lactobacillus reuteri CCFM1403;

[0063] Figure 6 :Animal experiment procedures;

[0064] Figure 7 :Effects of Lactobacillus reuteri CCFM1403 and its prepared postbiotics on mouse skin thickness;

[0065] Figure 8:Effects of Lactobacillus reuteri CCFM1403 and its prepared postbiotics on barrier function proteins in mouse skin tissue;

[0066] Fig. 9 :Effects of Lactobacillus reuteri CCFM1403 and its prepared postbiotics on the expression of barrier protein genes in the basal layer of mouse skin;

[0067] Fig.10 :Effects of Lactobacillus reuteri CCFM1403 and its prepared postbiotics on TEWL and stratum corneum water content of mouse skin tissue;

[0068] Fig.11 : Lactobacillus reuteri CCFM1403 and the postbiotics prepared therefrom act on the pathway receptors that repair the skin barrier;

[0069] “*” 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

[0070] The present invention will be further described below in conjunction with specific embodiments.

[0071] The human keratinocytes (HaCaT) involved in the following examples were purchased from Shanghai Cell Bank.

[0072] The BALB / c mice involved in the following examples were purchased from Vital River Company.

[0073] Lactobacillus reuteri 92, Lactobacillus reuteri AA38, Lactobacillus reuteri 82, Lactobacillus reuteri FSDLZ12M1, Lactobacillus reuteri FSDLZ13M1 and Lactobacillus reuteri CCFM1403 involved in the following examples are strains screened by the Food Biotechnology Center of Jiangnan University.

[0074] The culture medium involved in the following examples is as follows:

[0075] 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.

[0076] 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.

[0077] 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).

[0078] The cell recovery and culture methods involved in the following examples are as follows:

[0079] 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.

[0080] Example 1: Screening and identification of Lactobacillus reuteri CCFM1403

[0081] The strain sample screening was derived from the feces of healthy people. After pretreatment, it was stored in a -80°C refrigerator in 20% glycerol. After taking out and thawing, it was mixed and 0.5mL of the sample was added to 4.5mL of normal saline, and gradient dilution was performed with normal saline. The appropriate gradient dilution was selected and coated on MRS solid medium, and cultured at 37°C for 48h. The typical colonies of Lactobacillus reuteri were picked and streaked on MRS solid medium for purification, and single colonies were picked and transferred to MRS liquid medium for bacterial enrichment, and 30% glycerol was preserved 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 Lactobacillus reuteri by NCBI sequence alignment and named Lactobacillus reuteri CCFM1403. The Lactobacillus reuteri CCFM1403 has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 2, 2024, with a deposit number of GDMCC No: 65088.

[0082] Example 2: Preparation of postbiotics by Lactobacillus reuteri CCFM1403

[0083] (1) streaking Lactobacillus reuteri CCFM1403 from a bacterial preservation tube, culturing in a 37°C water-tight constant temperature incubator using a modified MRS solid medium for 48 hours to obtain a single colony; picking a single colony and inoculating it into a modified MRS liquid medium, culturing at 37°C for 12-18 hours, and obtaining a culture solution 1;

[0084] 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;

[0085] The seed solution was inoculated into the modified MRS liquid culture 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.

[0086] 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 freeze-dried powder of the fermentation supernatant of Lactobacillus reuteri CCFM1403 (referred to as CCFM1403-Q). The bacterial mud was resuspended in 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 CCFM1403-L).

[0087] The preparation method of live bacteria of Lactobacillus reuteri CCFM1403 is as follows: prepare the bacterial solution in the same manner as above, adjust the bacterial solution concentration to the same level as that of bacterial solution a, centrifuge the bacterial sludge obtained at 8000 r / min for 30 min, resuspend it in a ratio of 1 g:2 mL glycerol to obtain a live bacteria glycerol tube of Lactobacillus reuteri CCFM1403, recorded as CCFM1403-H.

[0088] The postbiotics of Lactobacillus reuteri CCFM1403 (bacterial lysate CCFM1403-L and fermentation supernatant CCFM1403-Q) were prepared by the above-mentioned means.

[0089] (2) According to the method of step (1), postbiotics of Lactobacillus reuteri 92, Lactobacillus reuteri AA38, Lactobacillus reuteri 82, Lactobacillus reuteri FSDLZ12M1, and Lactobacillus reuteri FSDLZ13M1 are simultaneously prepared.

[0090] Example 3: Establishment of in vitro sodium dodecyl sulfate (SDS)-damaged keratinocyte (HaCaT) model

[0091] (1) HaCaT cells in logarithmic growth phase were cultured at 1.2×10 4The 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;

[0092] Blank group: containing only cell culture medium without HaCaT cells;

[0093] Control group: containing cell culture medium and HaCaT cells but without SDS;

[0094] 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).

[0095] (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.

[0096] The cell viability was calculated according to the following formula: cell viability (%) = (OD value of treatment group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0097] (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.

[0098] Example 4: Effect of postbiotics prepared by Lactobacillus reuteri CCFM1403 on HaCaT cell activity

[0099] (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 was filled with PBS solution to prevent edge effect. After culturing for 36 h to allow the cells to adhere to the plate, a blank group, a control group, and a postbiotic treatment group were set up.

[0100] Blank group: containing only cell culture medium without HaCaT cells;

[0101] Control group: containing cell culture medium and HaCaT cells but without postbiotics;

[0102] Postbiotic treatment group: contains cell culture medium and HaCaT cells, as well as postbiotics.

[0103] Resuspend the postbiotics in cell culture medium (the amount of postbiotics after resuspending is the same as that of fermentation to a concentration of 5.0×10 7 CFU / ml of bacterial solution), and 100 μL of postbiotics prepared by Lactobacillus reuteri CCFM1403, Lactobacillus reuteri 92, Lactobacillus reuteri AA38, Lactobacillus reuteri 82, Lactobacillus reuteri FSDLZ12M1 and Lactobacillus reuteri FSDLZ13M1 were added respectively.

[0104] (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.

[0105] 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%

[0106] 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 Lactobacillus reuteri CCFM1403 (CCFM1403-Q and CCFM1403-L), Lactobacillus reuteri 92 (92-Q and 92-L), Lactobacillus reuteri 82 (82-Q and 82-L), Lactobacillus reuteri FSDLZ12M1 (FSDLZ12M1-Q and FSDLZ12M1-L) and Lactobacillus reuteri FSDLZ13M1 (FSDLZ13M1-Q and FSDLZ13M1-L) at a killed cell concentration of 5.0×10 7 The cell proliferation rates at CFU / ml were 100.26%, 98.81%, 99.89%, 100.34%, 100.01%, 100%, 100.33%, 99.99% and 100.33%, respectively.

[0107] 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.

[0108] Example 5: Effect of postbiotics prepared by Lactobacillus reuteri CCFM1403 on HaCaT cell damage caused by SDS

[0109] (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;

[0110] (2) After the cells adhered to the wall, different groups were treated with different medium changes and incubated for 6 h.

[0111] Control group: after changing the medium in step (1), containing cell culture medium and HaCaT cells, without postbiotic treatment, and without SDS modeling agent;

[0112] 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;

[0113] Postbiotic treatment group: The treatment method was the same as the model group.

[0114] (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.

[0115] 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 100 μL of postbiotics prepared by Lactobacillus reuteri CCFM1403, Lactobacillus reuteri 92, Lactobacillus reuteri AA38, Lactobacillus reuteri 82, Lactobacillus reuteri FSDLZ12M1 and Lactobacillus reuteri FSDLZ13M1 were added respectively.

[0116] (4) Add 10 μL of CCK8 solution to each well and incubate for 1.5 h, then measure the absorbance (OD) at 450 nm.

[0117] 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%.

[0118] Effects of postbiotics on HaCaT cell damage caused by SDS Figure 3Compared with the control group (cell viability 100%), the cell viability of the model group was 51.73%, and SDS modeling caused significant damage to HaCaT cells.

[0119] After adding CCFM1403-Q and CCFM1403-L to the treatment group, the cell viability was 59.09% and 87.3% respectively, among which CCFM1403-L significantly improved the viability of HaCaT cells compared with 51.73% in the model group, indicating that the postbiotics of Lactobacillus reuteri CCFM1403 can effectively alleviate the damage caused by SDS to HaCaT cells.

[0120] After treatment with postbiotics of other treatment groups including Lactobacillus reuteri 92 (92-Q and 92-L), Lactobacillus reuteri 82 (82-Q and 82-L), Lactobacillus reuteri FSDLZ12M1 (FSDLZ12M1-Q and FSDLZ12M1-L) and Lactobacillus reuteri FSDLZ13M1 (FSDLZ13M1-Q and FSDLZ13M1-L), the viability of HaCaT cells was 64.45%, 63.4%, 57.48%, 62.57%, 49.05%, 53.14%, 52.06% and 53.57%, respectively. That is, the postbiotics of other Lactobacillus reuteri do not have the excellent ability to alleviate the damage of HaCaT cells caused by SDS as shown by the postbiotics of Lactobacillus reuteri CCFM1403.

[0121] Example 6: Effect of postbiotics prepared by Lactobacillus reuteri CCFM1403 on the repair of barrier function and structural protein gene expression in SDS-damaged HaCaT cells

[0122] (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.

[0123] (2) After the cells adhered to the wall, different groups were treated with different medium changes and incubated for 6 h.

[0124] Control group: after changing the medium in step (1), containing cell culture medium and HaCaT cells, without postbiotic treatment, and without SDS modeling agent;

[0125] 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;

[0126] Postbiotic treatment group: The treatment method was the same as the model group.

[0127] (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.

[0128] 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 2 mL of postbiotics prepared by Lactobacillus reuteri CCFM1403-L and Lactobacillus reuteri GDLZ105-L (GDLZ105-L is a negative control of Lactobacillus reuteri CCFM1403-L) were added respectively.

[0129] (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-qPCR 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.

[0130] Table 1 Primer sequences

[0131]

[0132] 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 about 1, and the expression level in the model group decreased to 0.24 after SDS intervention, and the expression level in the negative control group was 0.17; the postbiotics (CCFM1403-L) prepared by Lactobacillus reuteri CCFM1403 upregulated the mRNA expression level of FLG to 3.16, which was 13.15 times that of the model group.

[0133] 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, the expression level in the model group decreased to 0.16 after SDS intervention, and the expression level in the negative control group was 0.34; the postbiotics (CCFM1403-L) prepared by Lactobacillus reuteri CCFM1403 significantly increased the expression level of IVL mRNA in HaCaT cells to 5.83.

[0134] 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 4 It 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 0.22 after SDS intervention, and the expression level in the negative control group was 0.55. The postbiotics prepared by Lactobacillus reuteri CCFM1403 (CCFM1403-L) significantly increased the expression level of LOR mRNA in HaCaT cells to 8.14.

[0135] 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 CCFM1403-L has a significant upregulation effect on ZO-1, CLDN and OCCLUDIN compared with the model group, increasing the mRNA to 2.04, 2.23 and 1.83 respectively.

[0136] From the above results, it can be seen that the postbiotics (bacterial lysate) prepared by Lactobacillus reuteri 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.

[0137] Example 7: Effects of postbiotics prepared by Lactobacillus reuteri CCFM1403 on cell pathway receptors

[0138] (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.

[0139] (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.

[0140] 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 from Lactobacillus reuteri CCFM1403 (CCFM1403-Q and CCFM1403-L) were added respectively.

[0141] (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 levels of AHR, OVOL1 and STAT3 were 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.

[0142] Table 2: Primer sequences

[0143]

[0144] The AHR signaling pathway plays a role in maintaining the integrity of the skin barrier. Low AHR expression is associated with increased skin barrier permeability, enhanced susceptibility to infection, and increased pathological features in atopic dermatitis models. As a transcription factor, OVOL1 is involved in regulating gene expression related to skin barrier function. At the same time, OVOL1 was identified as a direct transcriptional target of AHR, and the expression of OVOL1 affects AHR regulation of related gene expression in keratinocytes. The absence of OVOL1 in keratinocytes impedes the barrier-promoting function of AHR. MAPK is a classic pathway for upregulating skin barrier structural proteins, and downregulation of ERK content can serve as a characteristic target in this pathway. According to Figure 5 The results show that the bacterial lysate of CCFM1403 can significantly upregulate the gene expression of AHR and OVOL1 to 3.32 and 1.88, respectively, activating the expression of this pathway. At the same time, the bacterial lysate of CCFM1403 can upregulate ERK to 2.82.

[0145] In summary, CCFM1403 bacterial lysate repairs the HaCaT cell barrier through the AHR-OVOL1 receptor and MAPK pathway.

[0146] Example 8: Effects of Lactobacillus reuteri CCFM1403 and postbiotics prepared therefrom on the skin barrier appearance of mice with skin barrier damage

[0147] The preparation method of the postbiotics (CCFM1403Q and CCFM1403S) of Lactobacillus reuteri CCFM1403 involved in the following embodiments is the same as that in Example 2, except that the cultured bacterial solution is centrifuged at 8000r / min for 30min to obtain bacterial sludge, the bacterial sludge is resuspended with double distilled water with 75% volume of the original bacterial solution, and the resuspended solution is heat treated at 65°C for 30min without high-pressure homogenization to obtain inactivated bacteria.

[0148] (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:

[0149] Blank group (Control): Use normal saline as control;

[0150] Model group: normal saline was used as the control;

[0151] CCFM1403H group: live Lactobacillus reuteri CCFM1403 was used, with a dose of 5×10 9 CFU;

[0152] CCFM1403S group: Lactobacillus reuteri CCFM1403 postbiotics (inactivated bacteria) were used, with a dose of: viable bacteria count 5×10 9 CFU;

[0153] CCFM1403Q group: using Lactobacillus reuteri CCFM1403 metabolites (fermentation supernatant), the dose is: 72 mg / kg mouse body weight;

[0154] Among them, the inactivated bacteria or metabolites in the above groups are prepared from bacterial liquid with the corresponding amount of live bacteria.

[0155] The experiment lasted for four weeks, and gavage was performed every day from the 7th day to the 28th day. Before modeling, the skin on the back of the mice was shaved with a razor and depilatory cream, with the size of 2 cm long x 1.5 cm wide. A basic solution was prepared with acetone and olive oil (4:1 v / 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, 25 μL of 0.5% DNFB drug was applied to the back skin of the mice. Afterwards, 25 μL 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.

[0156] After the experiment, the mice were killed, and the skin of the shaved back was removed for H&E staining and the thickness of the epidermis was calculated using imageJ. Figure 7 The epidermal thickness of the model group (129.51) was significantly higher than that of the control group, and oral administration of CCFM1403-S (32.85), CCFM1403-H (47.07) and CCFM1403-Q (99.58) could significantly reduce the thickness of the epidermis.

[0157] Example 9: Effects of fermented Lactobacillus CCFM1403 and postbiotics prepared therefrom on the levels of barrier function proteins in the skin of mice with skin barrier damage

[0158] 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 and the blood supernatant was taken for ELISA detection. The back skin tissue was cut and ground into a homogenate at a weight-volume ratio of 1:10 with PBS, centrifuged at 3000r / min for 20 minutes, and the skin supernatant was taken for ELISA kit detection. The content of filaggrin (FLG), loricrin (LOR) and involucrin (IVL) in mouse skin was detected by Elisa kit. Figure 8 shown.

[0159] (1) Skin FLG content: The FLG content in the control group was 3969.97pg / 100mg, and that in the model group was 2725pg / 100mg. Oral administration of CCFM1403-S could significantly increase the FLG content to 3938pg / 100mg, and CCFM1403-Q could increase the FLG content to 3751.72pg / 100mg. Although CCFM1403-H could increase the FLG content to 2829.72pg / 100mg, there was no significant difference compared with the model group.

[0160] (2) Skin IVL content: The IVL content in the control group was 717.99 pg / 100 mg, and that in the model group was 613.12 pg / 100 mg. Only oral administration of CCFM1403-Q increased IVL to 723.33 pg / 100 mg, which was significantly different from the model group. However, although CCFM1403-S and CCFM1403-Q could increase IVL content to 659.75 pg / 100 mg and 626.05 pg / 100 mg, there was no significant difference from the model group.

[0161] (3) Skin LOR content: The LOR content in the control group was 510.89pg / 100mg, and that in the model group was 433.33pg / 100mg. Only the oral CCFM1403-S group showed significant differences from the model group, with a content of 505.92pg / 100mg. Although oral CCFM1403-H could increase the LOR content to 458.5pg / 100mg, it was not significantly different from the model group. The LOR content in the oral CCFM1403-Q group was 420.25pg / 100mg, which was unable to increase the index.

[0162] Example 10: Effects of Lactobacillus reuteri CCFM1403 and postbiotics prepared therefrom on the gene expression of basement membrane barrier proteins in mice with skin barrier damage

[0163] The animal experiment design and gavage groups involved in the following examples are the same as those in Example 9. Skin RNA was extracted using the Novozyme kit and reversely transcribed into cDNA to detect the gene expression of fibronectin and laminin in the skin, wherein the primer sequences are described in Table 3 below. Fibronectin and laminin are proteins in the basal layer of the skin that are essential for the health of the skin barrier.

[0164] Table 3: Primer sequences

[0165]

[0166] By detecting the mRNA content of fibronectin and adhesion protein, it can be seen that the relative expression of fibronectin mRNA of oral CCFM1403-S and CCFM1403-H is 3.96 and 1.98, which are significantly upregulated compared with the model group (0.54), while CCFM1403-Q has no significant difference with the model group. As for adhesion protein, the content of oral CCFM1403-S, CCFM1403-H and CCFM1403-Q is 1.34, 2.63 and 1.40, respectively, which are all well upregulated compared with the model group. Fig. 9 shown.

[0167] Example 11: Effects of Lactobacillus reuteri CCFM1403 and postbiotics prepared therefrom on the skin condition of mice with skin barrier damage

[0168] The animal experiment design and oral gavage groups involved in the following examples are the same as those in Example 9. 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 CCFM1403-S, CCFM1403-H and CCFM1403-Q are shown in the figure. Fig.10 The TEWL of the oral CCFM1403-S group decreased to 14.55 g / m 2 / h, compared with the model group (47.77g / m 2 / h) decreased by 3.31 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 CCFM1403-S was 26.27, which was 1.56 times that of the model group (15.6). However, there was no significant difference in TEWL and stratum corneum water content between oral CCFM1403-H and CCFM-S and the model group.

[0169] Therefore, oral administration of CCFM1403-S has a good repairing effect on the skin surface condition.

[0170] Example 12: Lactobacillus reuteri CCFM1403 fermentation supernatant repairs skin barrier damage in mice via TSR2A and OX40 receptors and JAK / STAT3

[0171] The animal experiment design and gavage groups involved in the following examples are the same as those in Example 8. Skin RNA was extracted using the Novozyme kit and reversely transcribed into cDNA to detect the gene expression of STAT3, TSR2A and OX40 in the skin, wherein the primer sequences are described in Table 4 below.

[0172] According to the results obtained in the above examples, all indicators were analyzed to show that oral CCFM1403-S has a good skin barrier repair effect, so the oral CCFM1403-S group was selected to analyze the repair mechanism. Fig.11 It can be seen that oral CCFM1403-Q can significantly downregulate STAT3 in the JAK / STAT pathway to 0.77, and the expression of STAT3 in the model group is 4.39. For the two receptors OX40 and TSR2A, oral CCFM1403-S can also significantly downregulate them to 0.81 and 0.74 compared with the model group (1.84 and 1.69).

[0173] Based on this, it can be inferred that CCFM1403-Q can repair the skin barrier in the DNFB-damaged mouse skin model by downregulating the expression of STAT3 in the JAK / STAT pathway and downregulating OX40 and TSR2A receptors.

[0174] Table 4: Primer sequences

[0175]

[0176] 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 Lactobacillus reuteri CCFM1403, which is deposited in Guangdong Microbiological Culture Collection Center with the deposit number: GDMCC No: 65088.

2. A microbial preparation containing the Lactobacillus reuteri CCFM1403 according to claim 1.

3. A postbiotic prepared by the Lactobacillus reuteri CCFM1403 according to claim 1.

4. The postbiotic according to claim 3, characterized in that The postbiotics include bacterial lysate, inactivated or inactivated cells, fermentation supernatant, or any of the above powders prepared by drying.

5. A product containing Lactobacillus reuteri CCFM1403 and / or its postbiotics according to claim 1, characterized in that: The products include food, medicine, health care products or daily chemical products.

6. The product according to claim 5, characterized in that The daily chemical products include skin care products, cosmetics or toiletries.

7. The product according to claim 5, characterized in that The product further comprises conventional auxiliary materials; the conventional auxiliary materials include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional enhancers.

8. Use of Lactobacillus reuteri CCFM1403 or its postbiotics according to claim 1 in preparing products for repairing skin barrier.

9. The use according to claim 8, characterized in that The products include oral products or external application products.

10. The use according to claim 9, characterized in that Repairing the skin barrier includes at least one of the following functions: (1) Improve the cell activity of skin keratinocytes damaged by sodium dodecyl sulfate; (2) Improve the barrier function and structural protein expression of skin keratinocytes after sodium dodecyl sulfate injury; (3) reduce the manifestation of skin barrier damage in individuals; (4) Increase the barrier function and structural proteins of individual skin tissue; (5) Increase basement membrane protein in individual skin tissue; (6) Reduce the individual's inflammatory response.

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