Postbiotics prepared from bifidobacterium adolescentis CCFM1404 and having skin health improving effect

Abstract

The invention discloses a metagen which is prepared from bifidobacterium adolescentis CCFM1404 and has the effect of improving skin health, and belongs to the technical field of microorganisms and medicines. The metagen (especially the thallus lysate) of the bifidobacterium adolescentis CCFM1404, provided by the invention, has a good skin barrier repairing effect, which is specifically shown in that the cell viability of keratinocytes after injury and the expression of FLG, IVL and LOR gene levels can be improved by externally applying the thallus lysate of the bifidobacterium adolescentis CCFM1404; the DNFB-induced mouse skin barrier injury can be relieved in the aspects of apparent TEWL and epidermis thickness, up-regulation of epidermis and basal layer protein and the like by oral administration of metabiotics (including thallus lysate and fermentation supernatant) of bifidobacterium adolescentis. Therefore, the bifidobacterium adolescentis 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 postbiotic prepared by a strain of Bifidobacterium adolescentis CCFM1404 and having the function of improving skin health, belonging to the technical field of microorganisms and the technical field of medicine. Background Art

[0002] An important function of the skin is to form an effective barrier between the organism and the environment. The outer layer of the skin, the epidermis, is composed of stratified squamous epithelial cells. Keratinocytes constitute the main part of the human skin epidermis (accounting for about 90% of all epidermal cells) and form a barrier to protect against damage from foreign organisms, heat, ultraviolet radiation, pathogenic bacteria, fungi, parasites and viruses. Keratinocytes can produce a large number of antimicrobial molecules, which further help to form a strong protective blockade, promote the proliferation of fibroblasts and keratinocytes by regulating cell signaling pathways, improve the skin's repair ability, and accelerate wound healing and tissue reconstruction.

[0003] As far as skin barrier repair is concerned, there are inventions in both external and oral applications that have an improvement effect on the skin barrier. Patent CN117618306A discloses a soothing repair composition and a preparation method thereof, the composition ingredients include ginseng, coptis root, salvia miltiorrhiza, Dunaliella salina extract, hydrolyzed lupine protein, argan oil and modified filaggrin peptide, etc., which are used for comprehensive external repair of the skin barrier. CN118516436A discloses an enzymatic hydrolyzate of sea cucumber collagen peptide with uniform molecular weight (250-1000Da) and high purity (accounting for 96%), which has the effect of strengthening the repair of the skin barrier and can be used to prepare oral drugs for repairing the skin barrier. Although these technologies have demonstrated the potential for skin barrier repair, there are also some defects that need to be improved. First, the macromolecular components in the external composition, such as filaggrin peptide and plant extracts, have poor permeability and are difficult to penetrate into the skin, limiting the repair effect. Secondly, plant extracts such as ginseng and coptis root have poor stability and are easily affected by the environment and fail. In addition, different individuals react differently to these ingredients, which may cause allergies, especially in people with sensitive skin. As for the oral form of sea cucumber collagen peptides, although the molecular weight is uniform and the purity is high, the absorption efficiency may be affected during the digestion process, reducing the bioavailability and affecting the repair effect. Finally, the repair effect of these technologies is more significant in the short term, but the ability to continuously repair long-term skin barrier damage still needs further verification. Therefore, there is still a lot of room for improvement in skin permeability, ingredient stability, individual compatibility and absorption efficiency of oral products. The use of microbial products with high biocompatibility to achieve the function of repairing the skin barrier has great advantages in preventing allergies, improving repair efficiency, and maintaining long-term repair effects. No microbial products have been found in the prior art that can achieve efficient repair of the skin barrier both by oral administration and by external application. Summary of the invention

[0004] The present invention provides an application of Bifidobacterium adolescentis CCFM1404 and postbiotics thereof in the preparation of a product for repairing the skin barrier.

[0005] The present invention provides a strain of Bifidobacterium adolescentis CCFM1404, which is deposited in Guangdong Provincial Microbiological Culture Collection Center with the collection number: GDMCC No: 65089.

[0006] The Bifidobacterium adolescentis CCFM1404 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 Bifidobacterium adolescentis of the genus Pediococcus, and is named Bifidobacterium adolescentis CCFM1404.

[0007] The colonies of Bifidobacterium adolescentis CCFM1404 on the MRS solid culture medium are raised, white, smooth and round.

[0008] The present invention also provides a postbiotic prepared by applying the Bifidobacterium adolescentis CCFM1404.

[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 Bifidobacterium adolescentis CCFM1404 is cultured in a culture medium for a period of time, bacterial cells in the cell culture fluid 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 Bifidobacterium adolescentis CCFM1404 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.

[0013] In one embodiment, the fermentation supernatant is the supernatant obtained by culturing the Bifidobacterium adolescentis CCFM1404 in a culture medium for a period of time and then centrifuging the culture medium.

[0014] The present invention also provides a product containing the Bifidobacterium adolescentis CCFM1404 and / or its postbiotics.

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

[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 dosage form of the product includes at least one of creams, lotions, oils, aqueous solutions, gels, powders, and freeze-dried solutions.

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

[0022] In one embodiment, the bacterial powder is a solid powder postbiotic prepared by drying the prepared Bifidobacterium adolescentis CCFM1404 liquid postbiotic.

[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) In vitro, the expression of barrier function protein mRNA in skin keratinocytes (HaCaT) damaged by sodium dodecyl sulfate (SDS) was increased;

[0028] (3) Increase the barrier function structural proteins of individual skin tissue;

[0029] (4) increase the basement membrane protein of individual skin tissue;

[0030] (5) reduce the individual's inflammatory response;

[0031] (6) Reduce epidermal thickness and TEWL of individual skin tissue.

[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 Bifidobacterium adolescentis CCFM1404 in the product is not less than 1×10 7 CFU / mL.

[0036] In one embodiment, the dosage of postbiotics prepared by Bifidobacterium adolescentis CCFM1404 in the product is not less than 250 μg / kg.

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

[0038] In one embodiment, the medicine comprises the Bifidobacterium adolescentis CCFM1404, 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 Bifidobacterium adolescentis CCFM1404, 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 Bifidobacterium adolescentis CCFM1404 in food production.

[0045] Beneficial effects:

[0046] The Bifidobacterium adolescentis CCFM1404 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) increase the mRNA expression of FLG, IVL, and LOR in skin keratinocytes (HaCaT) damaged by sodium dodecyl sulfate (SDS) in vitro;

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

[0050] (4) Enhance the basement membrane barrier effect in individuals with skin barrier damage;

[0051] (5) Repair the skin appearance of individuals with skin barrier damage;

[0052] (6) Reduce the inflammatory response caused by individuals with skin barrier damage.

[0053] Therefore, postbiotics prepared from Bifidobacterium adolescentis CCFM1404 have great application prospects in products that alleviate host skin barrier damage.

[0054] Biomaterial Deposit

[0055] Bifidobacterium adolescentis CCFM1404, taxonomically named Bifidobacterium adolescentis, was deposited in the Guangdong Provincial Microbiological Culture Collection on September 2, 2024, with the collection number GDMCC No: 65089, and the collection address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 :Evaluation of the effects of different concentrations of SDS on HaCaT cell damage;

[0057] Figure 2:Effects of different postbiotics on the proliferation of HaCaT cells;

[0058] Figure 3 :The effects of different postbiotics on the viability of HaCaT cells after SDS injury;

[0059] Figure 4 :The expression of barrier structure protein genes in HaCaT cells after SDS injury was repaired by different postbiotics;

[0060] Figure 5 :Animal experiment procedures;

[0061] Figure 6 :Effects of Bifidobacterium adolescentis CCFM1404 and its prepared postbiotics on barrier function proteins in mouse skin tissue;

[0062] Figure 7 :Effects of Bifidobacterium adolescentis CCFM1404 and its prepared postbiotics on barrier protein gene expression in mouse skin basal tissue;

[0063] Figure 8 :Effects of Bifidobacterium adolescentis CCFM1404 and its prepared postbiotics on inflammation in mouse skin tissue and serum;

[0064] Fig. 9 : Effects of Bifidobacterium adolescentis CCFM1404 and its prepared postbiotics on TEWL and H&E staining sections of mouse skin tissue;

[0065] “*” 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

[0066] The present invention will be further described below in conjunction with specific embodiments.

[0067] The human keratinocytes (HaCaT) involved in the following examples were purchased from Shanghai Cell Bank.

[0068] The BALB / c mice involved in the following examples were purchased from Vital River Company.

[0069] The Bifidobacterium adolescentis CCFM1404 involved in the following examples is a strain screened by the Food Biotechnology Center of Jiangnan University, and the Bifidobacterium adolescentis CCFM1302 has been disclosed in patent CN116426419A.

[0070] The culture medium involved in the following examples is as follows:

[0071] 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, cysteine ​​1 g / L and Tween-80 1 mL / L, pH 6.2-6.4.

[0072] 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, cysteine ​​1 g / L and agar 20.0 g / L, pH 6.2-6.4.

[0073] 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).

[0074] Example 1: Preparation of postbiotics by Bifidobacterium adolescentis CCFM1404

[0075] 1. Screening and identification of Bifidobacterium adolescentis CCFM1404

[0076] 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, 0.5 mL of the sample was mixed and added to 4.5 mL of normal saline. The normal saline was used for gradient dilution, and the appropriate gradient dilution solution was selected and spread on MRS solid culture medium. It was cultured at 37°C for 48 hours, and the typical colonies of Bifidobacterium adolescentis 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 Bifidobacterium adolescentis through NCBI sequence alignment and named Bifidobacterium adolescentis CCFM1404. It was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 2, 2024, with the deposit number GDMCC No: 65089.

[0077] 2. Preparation of postbiotics with Bifidobacterium adolescentis CCFM1404

[0078] (1) streaking Bifidobacterium adolescentis CCFM1404 from a bacterial preservation tube and culturing it in a water-tight constant temperature incubator at 37°C for 48 hours 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 hours, and obtaining a culture solution 1;

[0079] 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;

[0080] 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.

[0081] 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 lyophilized powder of the fermentation supernatant of Bifidobacterium adolescentis CCFM1404 (referred to as CCFM1404-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 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 CCFM1404-L).

[0082] The preparation method of live bacteria of Bifidobacterium adolescentis CCFM1404 is as follows: prepare the bacterial solution according to the same method as above, adjust the concentration of the bacterial solution to the same level as that of 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 glycerol tube of live bacteria of Bifidobacterium adolescentis CCFM1404, which is recorded as CCFM1404-H.

[0083] The Bifidobacterium adolescentis CCFM1404 postbiotics (bacterial lysate CCFM1404-L and fermentation supernatant CCFM1404-Q) were prepared by the above-mentioned means.

[0084] Example 2: Establishment of in vitro sodium dodecyl sulfate (SDS)-damaged keratinocyte (HaCaT) model

[0085] 1. Cell recovery and culture

[0086] 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.

[0087] 2. Establishment of SDS-injured HaCaT cell model

[0088] (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;

[0089] Blank group: containing only cell culture medium without HaCaT cells;

[0090] Control group: containing cell culture medium and HaCaT cells but without SDS;

[0091] 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).

[0092] (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.

[0093] 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%.

[0094] (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.

[0095] Example 3: Effect of postbiotics prepared by Bifidobacterium adolescentis CCFM1404 on HaCaT cell activity

[0096] (1) HaCaT cells in logarithmic growth phase were cultured at 1.2×10 4The 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;

[0097] Blank group: containing only cell culture medium without HaCaT cells;

[0098] Control group: containing cell culture medium and HaCaT cells but without postbiotics;

[0099] Postbiotic treatment group: contains cell culture medium and HaCaT cells, as well as postbiotics.

[0100] 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 liquid), and 100 μL of postbiotics prepared by Bifidobacterium adolescentis CCFM1404 was added respectively.

[0101] (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.

[0102] 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%

[0103] Effects on cell viability Figure 2 As shown in Figure 2, compared with the control group (cell proliferation rate 100.94%), the addition of postbiotics from Bifidobacterium adolescentis CCFM1404 (CCFM1404-Q and CCFM1404-L) at a killed bacterial concentration of 5.0×10 7 The cell proliferation rates at CFU / ml were 101.11% and 100.99% respectively.

[0104] 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.

[0105] Example 4: Effect of postbiotics prepared from Bifidobacterium adolescentis CCFM1404 on HaCaT cell damage caused by SDS

[0106] (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, the outermost circle was filled with PBS solution to prevent edge effect, and cultured for 36 h until the cells adhered to the wall.

[0107] (2) After the cells adhered to the wall, different groups were treated with different medium changes and incubated for 6 h.

[0108] Control group: after changing the medium in step (1), containing cell culture medium and HaCaT cells, without postbiotic treatment, and without SDS modeling agent;

[0109] 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;

[0110] Postbiotic treatment group: The treatment method was the same as the model group.

[0111] (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.

[0112] 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 from Bifidobacterium adolescentis CCFM1404 was added respectively.

[0113] (4) Add 10 μL of CCK8 solution to each well and incubate for 1.5 h, then measure the absorbance (OD) at 450 nm.

[0114] 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%.

[0115] Effects of postbiotics on HaCaT cell damage caused by SDS Figure 3 Compared with the control group (cell viability 104.35%), the cell viability of the model group was 70.58%, and SDS modeling caused significant damage to HaCaT cells.

[0116] After adding CCFM1404-Q and CCFM1404-L to the treatment group, the cell viability was 96.81% and 96.75% respectively, compared with 70.58% in the model group. CCFM1404-L and CCFM1404-Q significantly improved the viability of HaCaT cells compared with the model group, indicating that the postbiotics prepared from the bacterial lysate and fermentation supernatant of Bifidobacterium adolescentis CCFM1404 can effectively alleviate the activity damage caused by SDS to HaCaT cells.

[0117] Example 5: Effect of postbiotics prepared by Bifidobacterium adolescentis CCFM1404 on the repair of barrier function and structural protein gene expression in SDS-damaged HaCaT cells

[0118] (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.

[0119] (2) After the cells adhered to the wall, different groups were treated with different medium changes and incubated for 6 h.

[0120] Control group: after changing the medium in step (1), containing cell culture medium and HaCaT cells, without postbiotic treatment, and without SDS modeling agent;

[0121] 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;

[0122] Postbiotic treatment group: The treatment method was the same as the model group.

[0123] (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.

[0124] 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 from Bifidobacterium adolescentis CCFM1404 (CCFM1404-Q and CCFM1404-L) were added respectively.

[0125] (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 and LOR were calculated using the formula, where the internal reference was GAPDH, and the primers were described in Table 1 below. The results are shown in Table 1. Figure 4 shown.

[0126] Table 1 Primer sequences

[0127]

[0128] 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 0.46 after SDS intervention, and the expression level in the negative control was 0.74; the postbiotics prepared by Bifidobacterium adolescentis CCFM1404 (CCFM1404-L and CCFM1404-Q) significantly increased the expression level of FLG mRNA in HaCaT cells to 7.17 and 3.14, and in this indicator, the fermentation supernatant and bacterial lysate of CCFM1404 both had a significant effect on upregulating the expression of FLG mRNA, but the upregulation effect of CCFM1404-L was better than that of CCFM1404-Q.

[0129] 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 4It 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 0.58 after SDS intervention, and the expression level in the negative control was 0.636; the postbiotics prepared by Bifidobacterium adolescentis CCFM1404 (CCFM1404-L and CCFM1404-Q) significantly increased the expression level of IVL mRNA in HaCaT cells to 11.26 and 9.73, which were 19 times and 16 times higher than those in the model group.

[0130] 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 0.2 after SDS intervention, and the expression level in the negative control was 0.52. Postbiotics prepared by Bifidobacterium adolescentis CCFM1404 (CCFM1404-L and CCFM1404-Q) significantly increased the expression level of LOR mRNA in HaCaT cells to 2.59 and 1.59.

[0131] From the above results, it can be seen that the postbiotics (bacterial lysate and fermentation supernatant) prepared by Bifidobacterium adolescentis can upregulate the expression of functional protein (FLG, IVL and LOR) mRNA when HaCaT cells are damaged by SDS, and CCFM1404-L has a better repair effect than CCFM1404-Q. It repairs the damage of SDS to HaCaT cells by alleviating cellular protein dysfunction and alleviating cell apoptosis.

[0132] Example 6: Effect of postbiotics prepared from Bifidobacterium adolescentis CCFM1404 on the level of barrier function proteins in the skin of mice with skin barrier damage

[0133] The preparation method of the postbiotics (CCFM1404-Q and CCFM1404-S) of Bifidobacterium adolescentis CCFM1404 involved in the following embodiments is the same as that in Example 1, except that the bacterial sludge obtained by centrifuging the cultured bacterial liquid at 8000 r / min for 30 min is resuspended in double distilled water with 75% volume of the original bacterial liquid, and the resuspended liquid is heat treated at 65°C for 30 min without high-pressure homogenization.

[0134] (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:

[0135] Blank group (Control): Use normal saline as control;

[0136] Model group: normal saline was used as the control;

[0137] CCFM1404-S group: Bifidobacterium adolescentis CCFM1404 postbiotics (inactivated bacteria), the dosage is: bacterial count 5×10 9 CFU / kg mouse body weight;

[0138] CCFM1404-Q group: using Bifidobacterium adolescentis CCFM1404 metabolite (fermentation supernatant), the dose is: 25 mg / kg mouse body weight;

[0139] Among them, the inactivated bacteria or metabolites in the above groups were 1×10 9 CFU viable bacteria equal to the amount of inactivated bacteria or metabolites prepared from the bacterial liquid.

[0140] 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 5 shown.

[0141] 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.

[0142] Elisa kit was used to detect the contents of filaggrin (FLG), loricrin (LOR) and involucrin (IVL) in mouse skin. Figure 6 shown.

[0143] Compared with the control group (3931.33pg / 100mg), the FLG content in the skin of the model group decreased significantly to 2761.84pg / 100mg. The oral CCFM1404-S and CCFM1404-Q groups significantly increased the FLG content compared with the model group, restoring the FLG content in the skin to 3518.6pg / 100mg and 4304.67pg / 100mg, respectively. Both groups can increase the FLG content.

[0144] The LOR content in the control group was 517.17pg / 100mg, which was 1.1 times that of the LOR content in the model group (436.67pg / 100mg). Oral administration of CCFM1404-S and CCFM1404-Q groups significantly increased its content to 474.17pg / 100mg and 517.75pg / 100mg compared with the model group. Both of them can increase the LOR content.

[0145] For IVL, the content in the control group was 722.39pg / 100mg, and the content in the model group was 610.9pg / 100mg. Only the CCFM1404-Q group could significantly increase the content of IVL compared with the model group, and this group restored the content to 823.33pg / 100mg.

[0146] The above results show that Bifidobacterium adolescentis CCFM1404 and postbiotics (lysate and supernatant) prepared therefrom can increase the content of functional proteins in the skin of mice with skin barrier damage.

[0147] Example 7: Effects of Bifidobacterium adolescentis CCFM1404 and postbiotics prepared therefrom on the gene expression of basement membrane barrier proteins in mice with skin barrier damage

[0148] The animal experiment design and gavage groups involved in the following examples are the same as those in Example 6. 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, laminin and beaded protein are proteins in the basal layer of the skin that are essential for the health of the skin barrier.

[0149] Table 3: Primer sequences

[0150]

[0151] By detecting the mRNA content of fibronectin and adhesion protein, it can be seen that the relative expression levels of fibronectin mRNA after oral administration of CCFM1404-S and CCFM1404-Q were 2.45 and 1.67, which were significantly upregulated compared with the model group (0.56), and the upregulation effect of CCFM1404-S was better. As for adhesion protein, the relative expression level of the model group was 0.31, and oral administration of CCFM1404-S and CCFM1404-Q increased its expression levels to 2.08 and 1.51, respectively. The model group was unable to downregulate the relative expression level of beaded protein, but oral administration of CCFM1404-S and CCFM1404-Q could increase its relative expression levels to 2.47 and 1.44. The mRNA expression levels of the three proteins are as follows Figure 7 shown.

[0152] In summary, CCFM1404-S has a better regulatory effect on basal layer proteins.

[0153] Example 8: Effects of Bifidobacterium adolescentis CCFM1404 and postbiotics prepared therefrom on blood and skin inflammation in mice with skin barrier damage

[0154] The animal experiment design, gavage groups and skin homogenate sample preparation used in the following examples are the same as those in Example 6. The three indicators of serum IgE, skin histamine and skin IL-4 reflect the inflammation of the skin to a certain extent. Figure 8 shown.

[0155] Oral administration of CCFM1404-Q can significantly reduce the IgE content compared with the model group. The IgE content of the model group was 1.32μg / mL, and the IgE content of the CCFM1404-S group was 1.13μg / mL, which was significantly different from the model group. However, oral administration of CCFM1404-Q was 1.43μg / mL, which could not reduce the IgE content.

[0156] In terms of skin histamine, the histamine content in the model group increased significantly to 13.73 ng / 100 mg compared with the control group (11.63 ng / 100 mg). Oral administration of CCFM1404-S significantly reduced the histamine content in the skin to 10.52, while oral administration of CCFM1404-Q group had a histamine content of 14018 ng / 100 mg, which was unable to reduce the histamine content in the skin.

[0157] As for the IL-4 content in the skin, the IL-4 content in the control group was 192.38pg / 100mg, and the IL-4 content in the model group was 222.52pg / 100mg. Oral administration of CCFM1404-S significantly downregulated the IL-4 content in the skin (170.69pg / 100mg), while oral administration of CCFM1404-Q did not downregulate the IL-4 content in the skin (223.19pg / 100mg) and had no significant difference from the model group.

[0158] In summary, oral administration of CCFM1404-S can alleviate the inflammation in the DNFB-damaged mouse skin barrier model to varying degrees, and has better anti-inflammatory effects compared with CCFM1404-Q.

[0159] Example 9: Effects of Bifidobacterium adolescentis CCFM1404 and postbiotics prepared therefrom on the skin barrier appearance of mice with skin barrier damage

[0160] The animal experimental design and gavage groups involved in the following examples are the same as those in Example 7. The skin surface state test was performed using a skin tester Cutometer DUAL MPA580 equipped with a TEWL probe. After the mice were killed, the skin of the shaved back was removed, H&E staining was performed, and the thickness of the epidermis was calculated using imageJ. The results are shown in the figure. Fig. 9 The TEWL value is inversely proportional to the health of the skin barrier. The TEWL value in the control group was 15.9 g / m 2 / h, the model group is 46.38g / m 2 / h, significantly improving the value. The TEWL value of oral CCFM1404-S is 29.69g / m 2 / h, which is significantly lower than that of the model group, and has a good skin barrier state. The TEWL of oral CCFM1404-Q is 49.67g / m 2 / h, which could not be downregulated compared with the model group. At the same time, according to the skin H&E staining, it can be clearly seen that oral CCFM1404-S can significantly reduce the thickness of the epidermis and has a good repair effect.

[0161] 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 Bifidobacterium adolescentis CCFM1404, characterized in that The Bifidobacterium adolescentis CCFM1404 is preserved in Guangdong Microbiological Culture Collection Center with the preservation number: GDMCC No: 65089.

2. A microbial preparation containing the Bifidobacterium adolescentis CCFM1404 according to claim 1.

3. A postbiotic comprising the Bifidobacterium adolescentis CCFM1404 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 Bifidobacterium adolescentis CCFM1404 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 6, characterized in that The product further comprises conventional auxiliary materials, which include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional enhancers.

8. The product according to claim 7, characterized in that The dosage form of the product includes at least one of creams, emulsions, oils, aqueous solutions, gels, powders, and freeze-dried solutions.

9. Use of Bifidobacterium adolescentis CCFM1404 or its postbiotics according to claim 1 in the preparation of products for repairing skin barrier.

10. The use according to claim 9, characterized in that The skin barrier repair comprises at least one of the following: (1) Improve the activity of skin keratinocytes; (2) regulating the expression of barrier-related proteins and genes in skin keratinocytes; (3) Reduce skin inflammation; (4) Reduce the epidermal thickness and TEWL of skin tissue.

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