Saliva combined lactobacillus for improving seborrheic dermatitis and atopic dermatitis as well as product and application thereof

Abstract

The invention discloses saliva combined lactobacillus for improving seborrheic and atopic dermatitis as well as a product and application thereof, and relates to the technical field of microorganisms. The invention discloses combined lactobacillus salivarius NHNK-612 which is preserved in the China Center for Type Culture Collection on April 25, 2024, and the preservation number of the combined lactobacillus salivarius NHNK-612 is CCTCC (China Center for Type Culture Collection) NO: M 2024799. The NHNK-612 has the functions of inhibiting malassezia growth and biofilm formation, flocculating malassezia, reducing related toxins of skin damaged by staphylococcus aureus, reducing skin cutin damaged by staphylococcus aureus induction, reducing skin cutin damaged by malassezia induction and relieving atopic dermatitis symptoms.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a saliva-associated lactobacillus for improving seborrheic dermatitis and atopic dermatitis, and a product and application thereof. Background Art

[0002] Seborrheic dermatitis is a chronic papular, scaly, superficial inflammatory skin disease that occurs in areas of sebum secretion. It is common in areas rich in sebaceous glands such as the head, face, and trunk. Studies have shown that seborrheic dermatitis is caused by an imbalance of normal flora on the skin surface on the basis of sebum secretion, and the dominant bacterial species cultured and identified from damaged skin is Malassezia furfur ( Malassezia furfur ) and Malassezia restricta ( Malassezia restricta ). Malassezia feeds on human sebum triglycerides, and the enzymes it secretes destroy the barrier function of the stratum corneum, causing an increase in free fatty acids and changes in chemical composition, which in turn causes skin inflammation and scaling. Compared with non-lesional skin and healthy controls, the density of Malassezia in seborrheic dermatitis lesions increases, which is positively correlated with the severity of seborrheic dermatitis.

[0003] Current studies have shown that lipophilic Malassezia plays a key role in the pathogenesis of seborrheic dermatitis, and other microorganisms such as Staphylococcus aureus are also involved in the pathogenesis of the disease. Staphylococcus aureus can secrete phenol-soluble modulins (PSMs). PSMα and PSMβ can stimulate keratinocytes to release pro-inflammatory cytokines such as IL-18 and IL-1β, leading to the amplification of the inflammatory response and further promoting the colonization of Staphylococcus aureus. Staphylococcus aureus protein A (SpA) is a surface protein of Staphylococcus aureus. It can affect the function of the GTPase Rho family to form the cytoskeleton by activating TNF receptor 1, thereby affecting the structure of epithelial cells and destroying the barrier function of the skin. In addition, SpA can also activate nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) by activating TNF receptor 1 to induce the release of a large number of cytokines and chemokines, thereby mediating inflammatory responses. Strains that produce α-hemolysin express high levels of hla Genes can cause damage to skin, epithelial cells, and immune cells, and the lack of hla The strain with the gene is less virulent.

[0004] Atopic dermatitis (AD) is an allergic skin disease. The prevalence of AD has been increasing in recent years. The prevalence of atopic dermatitis in children aged 1-7 years is 12.94%, and the prevalence in infants under 1 year old is as high as 30.48%. Many related studies have shown that the diversity of intestinal flora is negatively correlated with the onset and development of atopic dermatitis, and intestinal flora imbalance is particularly obvious in moderate and severe patients. Compared with healthy people, the proportion of Clostridium, Clostridium difficile and Escherichia coli in the intestinal environment of patients with atopic dermatitis is increased, and the proportion of Bacteroidetes is reduced. At the same time, the number of bacteria producing propionate and butyrate in the intestines of patients with severe atopic dermatitis is less.

[0005] The establishment of animal models is helpful for further studying the occurrence and development mechanism of AD lesions, as well as the preclinical development of potential preventive or therapeutic drugs. 2,4-dinitrochlorobenzene (DNCB) is a small molecule chemical substance and a strong hapten sensitizer that easily binds to tissue proteins in the skin, causing delayed allergic reactions. The skin of mice with successful modeling can be seen to have symptoms such as erythema, papules, edema, and scales, which can be used for the preclinical development of potential preventive or therapeutic drugs.

[0006] Marine microorganisms have developed complex molecular adaptations to cope with these harsh conditions, affecting their primary and secondary metabolic pathways. This has led to the evolution of unique physiological characteristics and metabolic processes, and marine microorganisms are more likely to synthesize structurally unique enzymes and secondary metabolites than terrestrial microorganisms. Marine microbial active substances have important application value in various fields such as medicine, food, and cosmetics. Therefore, it is of great practical significance to provide a marine industry-related product developed using microbial technology. Summary of the invention

[0007] In view of this, the present invention provides a saliva-associated lactobacillus, product and application for improving seborrheic dermatitis and atopic dermatitis.

[0008] The present invention provides a saliva-combined lactobacillus ( Ligilactobacillus salivarius ), which is Lactobacillus salivarius ( Ligilactobacillus salivarius ) NHNK-612, which was deposited in the China Center for Type Culture Collection (CCTCC for short, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Wuhan University, Postal Code 430072) on April 25, 2024, and its preservation number is CCTCC NO: M2024799.

[0009] The present invention also proposes the above-mentioned saliva combined with lactobacillus ( Ligilactobacillus salivarius ) in the preparation of products for improving seborrheic dermatitis and atopic dermatitis.

[0010] Furthermore, the improvement of seborrheic dermatitis and atopic dermatitis includes at least one of the functions of inhibiting the growth and biofilm formation of Malassezia, flocculating Malassezia, reducing toxins associated with skin damage caused by Staphylococcus aureus, reducing skin keratin damage induced by Staphylococcus aureus, reducing skin keratin damage induced by Malassezia, and alleviating symptoms of atopic dermatitis.

[0011] Further, the inhibition of Malassezia growth and biofilm formation includes inhibiting Malassezia restricta ( Malassezia restricta )、Malassezia furfur Malassezia furfur ) growth and biofilm formation.

[0012] Furthermore, the flocculant Malassezia includes at least one of Malassezia restricta and Malassezia furfur.

[0013] Further, the reduction of Staphylococcus aureus skin-damaging toxins includes down-regulating the Staphylococcus aureus α-hemolysin gene hla , Staphylococcus aureus protein A gene spa , phenol-soluble regulatory protein α gene psmα , phenol-soluble regulatory protein type β gene psmβ at least one of the expressions.

[0014] Furthermore, the method of reducing the damage to skin keratin induced by Staphylococcus aureus includes upregulating the cell matrix-related filaggrin gene that is damaged by Staphylococcus aureus-induced skin keratinocytes HaCaT. FLG , OVO-like protein family genes OVOL , tight junction protein gene OCLN , loricrin gene LOR , involucrin gene IVL , Moisture-related water channel protein gene AQP3 and glucocerebrosidase gene GBA down-regulation of interleukin-1β gene related to inflammatory factors IL-1β , interleukin-6 gene IL-6 , interleukin-8 gene IL-8 and tumor necrosis factor alpha gene TNF-α at least one of the expressions.

[0015] Furthermore, the reduction of Malassezia-induced skin keratin damage includes down-regulating the interleukin-1β gene associated with inflammatory factors that cause Malassezia-induced skin keratinocyte HaCaT damage. IL-1β , interleukin-6 gene IL-6 , tumor necrosis factor alpha gene TNF-α Matrix-associated filaggrin gene FLGand tight junction protein genes OCLN at least one of the expressions.

[0016] Furthermore, the function of alleviating the symptoms of atopic dermatitis includes improving the degree of swelling of the atopic dermatitis skin.

[0017] The present invention also provides a product for improving seborrheic dermatitis and atopic dermatitis, comprising the above-mentioned saliva combined with lactobacillus ( Ligilactobacillus salivarius ), and acceptable excipients and / or adjuvants.

[0018] The saliva-combined lactobacillus ( Ligilactobacillus salivarius ), and its deposit number is CCTCC NO: M 2024799. Experiments show that NHNK-612 has the functions of inhibiting the growth and biofilm formation of Malassezia, flocculating Malassezia, reducing the toxins associated with Staphylococcus aureus damaging the skin, reducing the keratin damage induced by Staphylococcus aureus, reducing the keratin damage induced by Malassezia, and alleviating the symptoms of atopic dermatitis.

[0019] Biological Deposit Description Lactobacillus salivae ( Ligilactobacillus salivarius ) NHNK-612 was deposited in the China Center for Type Culture Collection (CCTCC for short, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Wuhan University, Postal Code 430072) on April 25, 2024, and its deposit number is CCTCC NO: M 2024799. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The figures are experimental result diagrams of saliva combined with inactivated bacteria of Lactobacillus NHNK-612 combined with Malassezia restricta and Malassezia furfur in the present invention, wherein (a) is a Gram stain result diagram of saliva combined with inactivated bacteria of Lactobacillus NHNK-612, (b) is a Gram stain result diagram of Malassezia restricta, (c) is a Gram stain result diagram of saliva combined with inactivated bacteria of Lactobacillus NHNK-612 combined with Malassezia restricta, (d) is a Gram stain result diagram of Malassezia furfur, and (e) is a Gram stain result diagram of saliva combined with inactivated bacteria of Lactobacillus NHNK-612 combined with Malassezia furfur; Figure 2 The figures are the experimental results of the present invention that the saliva combined with Lactobacillus NHNK-612 improves the ear skin symptoms of mice with atopic dermatitis, wherein (a) is the ear morphology of mice in the normal group, (b) is the ear morphology of mice in the model group, (c) is the ear morphology of mice in the NHNK-612 coating group, and (d) is the ear morphology of mice in the NHNK-612 gavage group; Figure 3This is a measurement diagram of the thickness of the ear skin of mice with atopic dermatitis improved by saliva combined with Lactobacillus NHNK-612 in the present invention; Figure 4 The H&E staining images of the saliva combined with Lactobacillus NHNK-612 in the present invention to improve inflammation of the ear skin tissue of mice with atopic dermatitis, wherein (a) is the H&E staining image of the ear skin tissue of the normal group mice, (b) is the H&E staining image of the ear skin tissue inflammation of the model group mice, (c) is the H&E staining image of the ear skin tissue inflammation of the NHNK-612 coating group mice, and (d) is the H&E staining image of the ear skin tissue inflammation of the NHNK-612 gavage group mice; Figure 5 The figures are as follows: (a) is a toluidine blue staining figure of the ear skin tissue of mice in the normal group, (b) is a toluidine blue staining figure of the ear skin tissue of mice in the model group, (c) is a toluidine blue staining figure of the ear skin tissue of mice in the NHNK-612 application group, and (d) is a toluidine blue staining figure of the ear skin tissue of mice in the NHNK-612 gavage group.

[0021] The following specific implementation manner will further illustrate the present invention in conjunction with the above-mentioned drawings. DETAILED DESCRIPTION

[0022] The present invention provides saliva combined lactobacillus and application thereof. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to realize. It is particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described by preferred embodiments, and the relevant personnel can obviously change or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention, to realize and apply the technology of the present invention.

[0023] The salivary lactobacillus strain NHNK-612 of the present invention is derived from naturally fermented shrimp paste in Qingdao and is identified as salivary lactobacillus ( Ligilactobacillus salivarius ). This strain is Gram-positive and short rod-shaped under a microscope; it grows on MRS plates to form round colonies with smooth, translucent surfaces, white in color, and neat edges; it grows evenly in MRS liquid culture medium and forms white precipitates after long-term storage. The optimal growth temperature is 37°C.

[0024] Lactobacillus salivae ( Ligilactobacillus salivarius)NHNK-612, China Center for Type Culture Collection, address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, preservation date: April 25, 2024, preservation number: CCTCC NO: CCTCC NO: M 2024799.

[0025] Furthermore, the saliva-associated Lactobacillus NHNK-612 provided by the present invention exists in the form of sterilized inactivated bacteria, or in the form of fermentation / secretion products (i.e., supernatant), and is not sterilized live bacteria. The derivative form is preferably selected from: metabolites, metabolic biological products, prebiotics, cell walls and components thereof, extracellular polysaccharides, and compounds containing immunogenic components, preferably selected from: live bacteria, fermentation / secretion products, and inactivated bacteria.

[0026] Example 1 Isolation of NHNK-612 The naturally fermented shrimp paste from Qingdao was shaken and mixed, then diluted 10 times with physiological saline. After mixing again, 100 μL of the dilution was spread on MRS culture medium and placed in an anaerobic bag. After constant temperature culture at 37°C for 48 h, white colonies were picked and repeatedly streaked and screened until a uniform single colony was obtained, which was named NHNK-612.

[0027] Gram staining microscopy: strain NHNK-612 is a Gram-positive colony, which appears as a short rod under a microscope. When grown on an MRS plate, it forms white, smooth, round, translucent circular colonies with neat edges. It can grow evenly in a turbid MRS liquid culture medium, and the bacteria will form a white precipitate after long-term storage.

[0028] Example 2 Nucleic acid identification of NHNK-612 1. 16S rDNA gene sequence analysis: Pick a single colony in MRS liquid medium, culture at 37℃ overnight, collect the bacteria by centrifugation at 8000 rpm for 1min, and operate according to the instructions of the Gram-positive bacteria DNA extraction kit. The primers used were bacterial 16S sequencing universal primers 27F and 1492R, and the PCR amplification system was 20μL. The PCR amplification program was 95℃ pre-denaturation for 5min, 94℃ for 15s, 57℃ for 15s, 72℃ for 1min, 35 cycles; 72℃ extension for 10min.

[0029] 2. Results The PCR product was sequenced and compared with the standard sequences published in the GenBank database (BLASTN). It was concluded that the NHNK-612 strain was Lactobacillus salivarius ( Ligilactobacillus salivarius ).

[0030] Example 3 NHNK-612 inhibits the growth of Malassezia 1. Cultivation of Malassezia Preparation of mDixon medium: 36.0 g / L malt extract powder, 6.0 g / L peptone, 20.0 g / L ox bile powder, 2.0 g / L glycerol, 2.0 g / L oleic acid. Mix well and sterilize at 121℃ for 15 min. Cool and set aside.

[0031] Malassezia restricta Malassezia restricta ) The CICC 33081 frozen tube bacterial solution was inoculated into mDixon medium at 2% (v / v), and cultured at 30℃ for 3 days. After that, the OD was adjusted with mDixon medium. 600 =0.3.

[0032] Malassezia furfur Malassezia furfur ATCC 44344 frozen tube bacterial solution was inoculated into mDixon medium at 2% (v / v), and cultured at 30℃ for 3 days. After that, the OD was adjusted with mDixon medium. 600 =0.3. 2. Preparation of NHNK-612 inactivated bacteria The NHNK-612 glycerol bacterial suspension was inoculated into fresh MRS medium at 2% (v / v), cultured anaerobically at 37°C for 48 h, centrifuged and washed three times with sterile PBS, resuspended with sterile PBS and adjusted to OD 600 =1.0, sterilize at 121℃ for 15 min and set aside.

[0033] 3. Experiment on the inhibition of growth of Malassezia restricta and Malassezia furfur by NHNK-612 inactivated bacteria 3 mL of Malassezia restricta or Malassezia furfur suspension, 2 mL of mDixon medium and 1 mL of NHNK-612 inactivated bacteria were added to the centrifuge tube, and an equal volume of PBS was added to the control group. The experimental group and the control group were shaken and cultured at 30°C for 24 h and then the OD values ​​of the experimental group and the control group were measured. 600 The absorbance value at nm. Inhibition rate % = (1-absorbance value of experimental group / absorbance value of control group) * 100%. The results are shown in Table 1: Table 1: NHNK-612 inactivated bacteria inhibits the growth of Malassezia

[0034] The results showed that the inactivated bacteria NHNK-612 could inhibit the growth of Malassezia restricta and Malassezia furfur, with an inhibition rate of 11.89%~96.52%.

[0035] Example 4 NHNK-612 flocculation of Malassezia restricta and Malassezia furfur 1. Preparation of NHNK-612 inactivated bacteria The preparation method of NHNK-612 inactivated bacteria is shown in Example 3.

[0036] 2. Preparation of Malassezia restricta and Malassezia furfur suspensions Malassezia restricta Malassezia restricta ) The CICC 33081 frozen tube bacterial solution was inoculated into mDixon medium at 2% (v / v), cultured at 30°C for 3 days, and then centrifuged at 6000 rpm for 5 min to collect the cells. The cells were washed three times with PBS and the OD was adjusted. 600 =1.0.

[0037] Malassezia furfur Malassezia furfur ) ATCC 44344 frozen tube bacterial solution was inoculated into mDixon medium at 2% (v / v), cultured at 30℃ for 3 days, and then centrifuged at 6000 rpm for 5 min to collect the bacteria. The cells were washed three times with PBS and the OD was adjusted. 600 =1.0.

[0038] 3. Co-flocculation test The inactivated NHNK-612 bacteria were mixed with the bacterial suspensions of Malassezia restricta and Malassezia furfur at a volume ratio of 1:1. After 30 minutes of reaction, samples of the reaction solutions of NHNK-612 alone, Malassezia restricta or Malassezia furfur, and NHNK-612 and Malassezia restricta or Malassezia furfur were collected. The sampling range was the top 50 μL of the liquid surface. After aspiration, the samples were transferred to a 96-well plate and the absorbance at OD = 600 nm was measured. At the same time, the flocculation state of the bacteria was observed after the flocculation precipitate was stained with Gram stain. Figure 1 shown.

[0039] Calculation formula: Flocculation rate (%) = [(Ax+Ay)-2Amix] / (Ax+Ay)×100%; Note: Ax: OD measured by NHNK-612 alone at the reaction time 600 Value; Ay: OD measured by single Malassezia restricta or Malassezia furfur at the reaction time 600 Value; Amix: OD measured at the reaction time after NHNK-612 and Malassezia restricta or Malassezia furfur were mixed. 600 Numeric value.

[0040] The results are shown in Table 2 below: Table 2: Co-flocculation effect of NHNK-612 inactivated bacteria on Malassezia

[0041] The results showed that after 30 minutes of reaction, the inactivated bacteria of NHNK-612 were able to flocculate Malassezia restricta and Malassezia furfur, with flocculation rates ranging from 9.27% ​​to 13.38%.

[0042] Example 5 NHNK-612 inhibits Malassezia biofilm 1. Cultivation of Malassezia restricta and Malassezia furfur Malassezia restricta Malassezia restricta ) The CICC 33081 frozen tube bacterial solution was inoculated into mDixon medium at 2% (v / v), and cultured at 30℃ for 3 days. After that, the OD was adjusted with mDixon medium. 600 =0.3.

[0043] Malassezia furfur Malassezia furfur ATCC 44344 frozen tube bacterial solution was inoculated into mDixon medium at 2% (v / v), and cultured at 30℃ for 3 days. After that, the OD was adjusted with mDixon medium. 600 =0.3. 2. Preparation of NHNK-612 fermentation products and inactivated bacteria NHNK-612 glycerol tube bacterial solution was inoculated into fresh MRS medium at 2% (v / v) and cultured at 37°C for 48 h. After that, the OD was adjusted to 0. 600 =1.0, centrifuged at 5000rpm for 10min, and the supernatant was filtered through a 0.22μm filter membrane to obtain the fermentation product; the bacteria were washed three times with sterile PBS and adjusted to OD with MRS. 600 =1.0, and the inactivated bacteria are obtained after sterilization at 121℃ for 15min.

[0044] 3. NHNK-612 inhibits Malassezia biofilm Composite biofilm: 50 μL of Malassezia restricta and 50 μL of Malassezia furfur were mixed and added to a 96-well plate. 100 μL of NHNK-612 fermentation product or inactivated bacteria was added to each well of the experimental group, and an equal volume of PBS was added to the control group. Each group had 3 parallels and then cultured at 37°C for 24 hours.

[0045] After the incubation, the supernatant was discarded, 100 μL sterile PBS was added to each well for washing twice, and then 100 μL 4% paraformaldehyde fixative was added and fixed at room temperature for 30 minutes. The fixative was discarded, 100 μL crystal violet was added to each well, and staining was performed at room temperature for 30 minutes. After staining, the cells were washed twice with sterile PBS and dried, and 100 μL anhydrous ethanol was added to each well. After standing for 1 minute, the absorbance at 600 nm was measured. Inhibition rate % = (1-experimental group / control group) * 100%, and the results are shown in Table 3 below: Table 3: NHNK-612 inhibits Malassezia restricta-Malassezia furfur composite biofilm

[0046] The results showed that NHNK-612 could inhibit the formation of Malassezia restricta-Malassezia furfur composite biofilm, with an inhibition rate of 12.58%~21.04%.

[0047] Example 6 NHNK-612 down-regulates the expression of Staphylococcus aureus skin-damaging toxin genes 1. Preparation of NHNK-612 fermentation products and inactivated bacteria The preparation method of NHNK-612 fermentation product and inactivated bacteria refers to Example 5.

[0048] 2. Experiment on down-regulating the gene expression of Staphylococcus aureus-related toxins that damage the skin The glycerol culture of Staphylococcus aureus CGMCC 1.8721 was inoculated into fresh BHI medium at 1% (v / v), cultured at 37°C with shaking for 24 h, and the OD was adjusted to 0. 600 =0.3. Take 3mL of Staphylococcus aureus culture, add 1mL of fresh BHI medium, 1mL of NHNK-612 fermentation product or inactivated bacteria, add an equal volume of PBS to the control group, and culture at 37℃ for 24h. After the culture, centrifuge at 8000rpm for 1min to obtain the cells, extract total RNA according to the kit instructions, detect RNA concentration and purity, reverse transcribe into cDNA, use 16S rRNA as the internal reference gene, and use qPCR to detect psmα , psmβ , hla and spa The relative expression of the control group gene is F=1, using 2 -ΔΔCT The F value of each sample was calculated by this method.

[0049] Formula: F=2 -ΔΔCT ,in: △CT 实验 =CT 实验 -CT 内参（实验）; △CT 对照 =CT 对照 -CT 内参（对照）; △△CT=△CT 实验 -△CT 对照。

[0050] The results are shown in Tables 4 and 5 below: Table 4: Fermentation products down-regulate Staphylococcus aureus skin-damaging toxin genes

[0051] Table 5: Inactivated bacteria down-regulate Staphylococcus aureus skin-related toxin genes

[0052] The results showed that NHNK-612 could downregulate the genes of Staphylococcus aureus-related toxins that damage the skin hla , spa, psmα、psmβ The relative expression level was 0.13 to 0.86 times, thereby reducing the ability of Staphylococcus aureus to damage the skin.

[0053] Example 7 NHNK-612 regulates the expression of genes related to Staphylococcus aureus-induced damage to human skin keratinocytes 1. Preparation of Staphylococcus aureus liquid The glycerol culture of Staphylococcus aureus CGMCC 1.8721 was inoculated into fresh BHI medium at 1% (v / v), cultured at 37°C with shaking for 24 h, and then adjusted to OD 600 =0.1.

[0054] 2. Preparation of NHNK-612 fermentation products and inactivated bacteria The preparation method of NHNK-612 fermentation product and inactivated bacteria refers to Example 5.

[0055] 3. Cultivation of human skin keratinocytes HaCaT HaCaT cells were activated with DMEM medium containing 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin, and cultured at 37°C and 5% CO2. After the cells were fused to 80-90%, they were passaged or plated.

[0056] 4. NHNK-612 regulates the expression of genes related to Staphylococcus aureus-induced damage to human skin keratinocytes HaCaT cells were inoculated in a 6-well cell culture plate at 1×10^6 cells / well and cultured for 12 hours until the cells adhered to the wall. The cell culture medium was removed, washed twice with sterile PBS, and 1.9 mL of DMEM culture medium and 100 μL of NHNK-612 fermentation product or inactivated bacteria were added respectively. An equal volume of DMEM culture medium was added to the control group. After 3 hours of treatment, 100 μL of Staphylococcus aureus suspension was added and cultured for 24 hours at 37°C and 5% CO2. After the culture was completed, the supernatant was discarded, washed twice with sterile PBS, and then 1 mL of cell RNA extraction reagent was added to each well. Total RNA was extracted according to the reagent instructions and the concentration and purity were determined. After the extraction was completed, it was reverse transcribed into cDNA, and GADPH was used as the internal reference gene to determine the inflammation-related genes by qPCR. TNF-α , IL-6 , IL-8 , IL-1β and moisturizing repair-related genes FLG , OVOL , OCLN , LOR , AQP3 , GBA , IVL The relative expression of the control group gene is F=1, using 2 -ΔΔCT The F value of each sample was calculated by the method described in Example 6.

[0057] The results are shown in Tables 6 and 7 below: Table 6: NHNK-612 fermentation products regulate Staphylococcus aureus-induced HaCaT cell injury-related genes

[0058] Table 7: NHNK-612 inactivated bacteria regulates Staphylococcus aureus-induced HaCaT cell injury-related genes

[0059] The results showed that NHNK-612 down-regulated the genes related to inflammatory factors in skin keratinocytes HaCaT induced by Staphylococcus aureus IL-1β, IL-6, IL-8, TNF-α , upregulating cell matrix-related genes FLG, OVOL, OCLN, LOR, IVL , and moisturizing-related genes AQP3 and GBA expression.

[0060] Example 8 NHNK-612 regulates the expression of genes related to damage to human skin keratinocytes induced by Malassezia restricta 1. Preparation of Malassezia restricta liquid Malassezia restricta Malassezia restricta ) The CICC 33081 frozen tube bacterial solution was inoculated into mDixon medium at 2% (v / v), cultured at 30℃ for 3 days, centrifuged at 5000 rpm for 5 min, and washed twice with PBS and adjusted to OD 600 =0.1.

[0061] 2. Preparation of NHNK-612 fermentation products and inactivated bacteria The preparation method of NHNK-612 fermentation product and inactivated bacteria refers to Example 5.

[0062] 3. Cultivation of human skin keratinocytes HaCaT HaCaT cells were activated with DMEM medium containing 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin, and cultured at 37°C and 5% CO2. After the cells were fused to 80-90%, they were passaged or plated.

[0063] 4. NHNK-612 regulates the damage of human skin keratinocytes induced by Malassezia restricta HaCaT cells were inoculated in a 6-well cell culture plate at 1×10^6 cells / well and cultured for 12 hours until the cells adhered to the wall. The cell culture medium was removed, washed twice with sterile PBS, and 1.9 mL of DMEM culture medium and 100 μL of NHNK-612 fermentation product or inactivated bacteria were added respectively. An equal volume of DMEM culture medium was added to the control group. After 3 hours of treatment, 100 μL of restricted Malassezia suspension was added and cultured for 24 hours at 37°C and 5% CO2. After the culture was completed, the supernatant was discarded, washed twice with sterile PBS, and then 1 mL of cell RNA extraction reagent was added to each well. Total RNA was extracted according to the reagent instructions and the concentration and purity were determined. After the extraction was completed, it was reverse transcribed into cDNA, and GADPH was used as the internal reference gene to determine the inflammation-related genes by qPCR. TNF-α , IL-6 , IL-1β Matrix-related genes FLG and OCLN The relative expression of the control group gene is F=1, using 2 -ΔΔCT The F value of each sample was calculated by the method described in Example 6.

[0064] The results are shown in Tables 8 and 9 below: Table 8: NHNK-612 fermentation products regulate HaCaT cell damage-related genes induced by Malassezia restricta

[0065] Table 9: NHNK-612 inactivated bacteria regulates HaCaT cell damage-related genes induced by Malassezia restricta

[0066] The results showed that NHNK-612 down-regulated the genes related to inflammatory factors in skin keratinocytes HaCaT induced by Malassezia restricta IL-1β, IL-6, and TNF-α Upregulation of cell matrix-related genes FLG and OCLN expression.

[0067] Example 9 NHNK-612 improves the physiological characteristics of atopic dermatitis mice 1. Preparation of NHNK-612 gel and bacterial suspension Pick a single colony of saliva-associated Lactobacillus NHNK-612 in MRS liquid medium and culture at 37°C for 24h. Centrifuge at 5000rpm for 10min and discard the supernatant. Wash the bacteria twice with sterile PBS to obtain live bacteria. Sterilize the live bacteria at 121°C for 15min to obtain inactivated bacteria. Weigh 1g of carbomer, add 99mL of ultrapure water, and stir quickly with a glass rod. Place at room temperature for 3h until the carbomer swells. Then add triethanolamine dropwise to adjust the pH to 7, add NHNK-612 inactivated bacteria, stir and mix, adjust the bacterial amount to 6×10^8cells / mL, and obtain NHNK-612 gel. Resuspend the NHNK-612 live bacteria with sterile PBS, adjust the bacterial amount to 6×10^8 cfu / mL, and obtain NHNK-612 bacterial suspension.

[0068] 2. Experimental grouping and treatment of mice Thirty-two 6-week-old SPF (specific pathogen free) male C57BL / 6 mice were randomly divided into 4 groups, 8 mice in each group, namely blank group, model group, NHNK-612 coating group and NHNK-612 gavage group. All mice were raised in SPF-grade barriers and had free access to food and water.

[0069] The experiment lasted for five weeks, with the first week being the adaptation period for mice, and the second to third weeks being the dermatitis modeling period. From the second to fifth weeks, the mice in the NHNK-612 live bacteria gavage group were gavaged with NHNK-612 live bacteria suspension, and the other groups were gavaged with an equal amount of PBS. From the fourth to fifth weeks, the ears of the mice were coated with NHNK-612 inactivated bacteria gel, and the other groups were coated with an equal amount of base gel without NHNK-612. Acetone and olive oil were mixed in a ratio of 3:1 (v / v) and used as a matrix for dissolving DNCB (2,4-dinitrochlorobenzene). On the first day of modeling, 10 μL of 0.5% (v / v) DNCB solution was used to smear the left and right ears of the mice in the model group, NHNK-612 inactivated bacteria coating group, and NHNK-612 live bacteria gavage group to stimulate dermatitis. On the 5th, 8th, 11th and 14th days, 10 μL of 0.2% (v / v) DNCB solution was applied to the left and right ears of the mice in the model group, NHNK-612 application group and NHNK-612 gavage group. When the mice showed dermatitis symptoms such as skin erythema, scaling and scab, the model was successfully established. The blank group mice were only smeared with an equal amount of acetone / olive oil matrix on the left and right ears as a control. Starting from the fourth week, the left and right ears of the mice in the NHNK-612 coating group were smeared with 0.05 mL of gel containing inactivated NHNK-612 bacteria (the total amount of inactivated bacteria in a single coating was 1×10^8 cells / mouse) daily, and the mice in the blank group, model group and NHNK-612 gavage group were smeared with an equal amount of gel without NHNK-612 as a control; the mice in the NHNK-612 gavage group were gavaged with 0.1 mL of NHNK-612 live bacteria solution daily (the total amount of live bacteria in a single gavage was 1×10^8 cfu / mouse), and the mice in the blank group, model group and NHNK-612 coating group were gavaged with an equal amount of PBS as a control.

[0070] After the NHNK-612 intervention period, the ears of mice were photographed. Figure 2 The thickness of the left and right ears of the mice was then measured using a vernier caliper. Figure 4 shown.

[0071] The thickness of the left and right ears of mice is shown in Tables 10 and 11 below: Table 10: Effect of NHNK-612 on the thickness of the left ear of mice with DNCB-induced dermatitis

[0072] Table 11: Effect of NHNK-612 on the thickness of the right ear of mice with DNCB-induced dermatitis

[0073] Depend on Figure 2 and Figure 3It can be seen that the ear tissues of the mice in the blank group were intact and had normal morphology; the ears of the mice in the model group showed exudate and swelling, and the ears were significantly thickened; the ear morphology and thickness of the mice in the NHNK-612 coating group were improved compared with those in the model group, and the ear morphology and thickness of the mice in the NHNK-612 gavage group were normal, without exudate and swelling. The p value between the left ear model group and the normal group of mice was 0.000, and the p value between the right ear model group and the normal group was 0.003. The p values ​​of the two groups were both less than 0.05, and the difference was statistically significant, indicating that the model was successful; the p values ​​between the left and right ear model groups and the NHNK-612 inactivated bacteria coating group were both 0.05, and the difference was statistically significant, indicating that the ear inflammation of mice in the NHNK-612 inactivated bacteria coating group was significantly alleviated compared with the model group; the p values ​​between the left and right ear model groups and the NHNK-612 live bacteria gavage group were 0.008 and 0.013, respectively, and the difference was statistically significant, indicating that the ear inflammation of mice in the NHNK-612 live bacteria gavage group was significantly alleviated compared with the model group.

[0074] The mice were killed and ear sections were prepared for hematoxylin-eosin (H&E) staining and toluidine blue staining. The results are shown in Figures 4 to 5 .Depend on Figure 4 It can be seen that the epidermal cell structure of the ear skin of the mice in the normal group was normal, and no inflammatory reaction was observed; the pathological changes of the ear skin lesions of the mice in the model group showed inflammatory proliferative changes, ear swelling, neutrophil infiltration (red arrow) and bruises (black arrow); Figure 5 It can be seen that the number of mast cells in the ear tissue of mice in the model group increased (red arrows). The swelling of the ear skin of mice in the NHNK-612 killed bacteria coating group and the NHNK-612 live bacteria gavage group was reduced, the inflammatory hyperplasia was alleviated, and the number of neutrophils and mast cells was significantly reduced.

[0075] The above experiments show that both application and oral administration of NHNK-612 can alleviate the swelling of the ears of atopic dermatitis model mice, reduce neutrophil infiltration and mast cell increase, and effectively alleviate the symptoms of atopic dermatitis.

[0076] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A saliva-based lactobacillus for improving seborrheic and atopic dermatitis ( Ligilactobacillus salivarius ), which is Lactobacillus salivarius ( Ligilactobacillus salivarius ) NHNK-612 was deposited in the China Center for Type Culture Collection on April 25, 2024, and its deposit number is CCTCC NO: M 2024799.

2. The saliva-associated lactobacillus as claimed in claim 1 ( Ligilactobacillus salivarius ) in the preparation of products for improving seborrheic dermatitis and atopic dermatitis.

3. The use according to claim 2, characterized in that: The improvement of seborrheic dermatitis and atopic dermatitis includes inhibiting the growth and biofilm formation of Malassezia, flocculating Malassezia, reducing Staphylococcus aureus ( Staphylococcus aureus ) at least one of the functions of damaging skin-related toxins, reducing skin keratin damage induced by Staphylococcus aureus, reducing skin keratin damage induced by Malassezia, and alleviating atopic dermatitis symptoms.

4. The use according to claim 3, characterized in that The method of inhibiting the growth of Malassezia and biofilm formation includes inhibiting the growth of Malassezia restricta ( Malassezia restricta )、Malassezia furfur Malassezia furfur ) growth and biofilm formation.

5. The use according to claim 3, characterized in that: The flocculant Malassezia includes at least one of Malassezia restricta and Malassezia furfur.

6. The use according to claim 3, characterized in that: The method for reducing the toxins associated with Staphylococcus aureus damaging skin comprises down-regulating the α-hemolysin gene of Staphylococcus aureus hla , Staphylococcus aureus protein A gene spa , phenol-soluble regulatory protein α gene psmα , phenol-soluble regulatory protein type β gene psmβ at least one of the expressions.

7. The use according to claim 3, characterized in that: The method for reducing the skin keratin damage induced by Staphylococcus aureus comprises up-regulating the cell matrix-related filaggrin gene of skin keratinocyte HaCaT damage induced by Staphylococcus aureus FLG , OVO-like protein family genes OVOL , tight junction protein gene OCLN , loricrin gene LOR , involucrin gene IVL , Moisture-related water channel protein gene AQP3 and glucocerebrosidase gene GBA down-regulation of interleukin-1β gene related to inflammatory factors IL-1β , interleukin-6 gene IL-6 , interleukin-8 gene IL-8 and tumor necrosis factor alpha gene TNF-α at least one of the expressions.

8. The use according to claim 3, characterized in that: The method for reducing the skin keratin damage induced by Malassezia comprises down-regulating the interleukin-1β gene related to the inflammatory factor of the skin keratinocyte HaCaT damage induced by Malassezia restricta IL-1β , interleukin-6 gene IL-6 , tumor necrosis factor alpha gene TNF-α Matrix-associated filaggrin gene FLG and tight junction protein genes OCLN at least one of the expressions.

9. The use according to claim 3, characterized in that: The function of alleviating the symptoms of atopic dermatitis includes improving the degree of swelling of atopic dermatitis skin.

10. A product for improving seborrheic dermatitis and atopic dermatitis, characterized in that: comprising the salivary lactobacillus as claimed in claim 1 ( Ligilactobacillus salivarius ), and acceptable excipients and / or adjuvants.

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