A strain of Lactobacillus oris and its application

Abstract

The present invention mainly relates to the field of microbial technology, and specifically relates to a strain of Lactobacillus oris and its application. The strain ProfMIC-203 is deposited in the China Center for Type Culture Collection, and the deposit number is CCTCC NO:M20211438. This strain ProfMIC-203 has the functions of maintaining and repairing the skin barrier, promoting cell repair, and reducing skin inflammation, and can be used in medicines, cosmetics, etc.

Description

Technical Field

[0001] The invention mainly relates to the technical field of microorganisms, and in particular to a strain of Lactobacillus oris and application thereof. Background Art

[0002] The skin is the largest organ in the human body, accounting for about 16% of the body weight. On the one hand, it maintains the stability of the body, and on the other hand, it is also the first line of defense against the invasion of adverse external factors. Studies have shown that if the external environment causes abnormalities in the relevant genes in the skin barrier, it will induce skin diseases.

[0003] The skin barrier is a structural barrier formed by the lipids between the epidermal cells and the keratin in the stratum corneum. The skin barrier prevents the body from releasing too much water and prevents harmful substances such as chemicals or microorganisms from entering our body. The keratinocyte epithelium that constitutes the surface of dead keratinocytes plays an important role in the stability of intercellular lipids. Damage to the skin barrier will cause dry skin, skin aging, pigmentation atopic dermatitis, eczema, psoriasis, ichthyosis, photodermatitis and other skin sensitivity, irritant dermatitis, hormone-dependent dermatitis and other greasy skin, seborrhea diseases such as acne, rosacea, and seborrheic dermatitis.

[0004] The content of ceramide, a structural lipid in the stratum corneum, gradually increases during the differentiation process from the basal layer to the keratin, and reaches the stratum corneum and is discharged into the intercellular space, forming a barrier to prevent water loss. The water content in keratinocytes is high. As the cells metabolize and differentiate upward, the shape of keratinocytes gradually becomes flat, and the nucleus and organelles begin to degenerate and shrink, forming dead cells without nuclei and organelles in the stratum corneum. The hydrophilicity and barrier function of the stratum corneum itself, as well as the natural moisturizing factors contained in the stratum corneum, namely amino acids, lactates and sugars, make the stratum corneum usually contain 10-30% water. This environment has become a cradle for the growth of the skin's own microbial colonies. However, with age, the water content of the stratum corneum will gradually decrease. When the water content is less than 10%, it will cause various skin problems.

[0005] Microecological preparations are used in medicines and cosmetics to balance the epidermal flora of the skin and repair the skin barrier. As reported in the literature, topical probiotics can treat atopic dermatitis, that is, inhibit the proliferation of other bacteria, balance the microbial flora on the surface of the skin, and promote the repair of the skin barrier. At the same time, microecological preparations play an important role in maintaining the homeostasis of the host body, activating the immune system, and maintaining the body's immune balance. Studies have found that probiotics can prevent and treat various inflammatory diseases by regulating the immune function and phagocytic ability of macrophages, and by controlling the systemic immune state, including the release of regulatory cytokines.

[0006] The present invention aims to develop relevant microbial strains to achieve corresponding functions. Summary of the invention

[0007] To achieve the above technical objectives, the present invention separates and identifies Lactobacillus oralis ProfMIC-203 from the skin of a healthy boy, which has both skin barrier repair and anti-inflammatory functions and has great application potential in the fields of medicines, cosmetics, etc.

[0008] The above objectives are achieved through the following technical solutions:

[0009] The present invention first provides a strain of Lactobacillus oris, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO:M 20211438.

[0010] The oral lactobacillus was derived from the facial skin of a healthy boy and was numbered ProfMIC-203. It was identified as Lactobacillus oris by 16SrRNA. The strain is Gram-positive and rod-shaped under a microscope. When grown on an MRS plate, it forms round colonies with smooth, opaque surfaces, white in color, and neat edges. It grows evenly and turbidly in MRS liquid culture medium, and the bacteria form white precipitates after long-term storage. The optimal growth temperature is 37°C.

[0011] The present invention further provides the use of the Lactobacillus oris for skin barrier repair, anti-inflammation and the like.

[0012] According to the functions of Lactobacillus oris, the present invention further provides another use of the Lactobacillus oris, which can be used to prepare medicines, cosmetics and other products related to skin barrier repair and anti-inflammatory.

[0013] Furthermore, when used for preparing related medicines, cosmetics and the like, the Lactobacillus oris may be its bacterial body or / and its derivatives or / and its metabolites.

[0014] Wherein, the bacteria may be live bacteria or / and inactivated bacteria;

[0015] The derivative is a lysate or / and extract of the above-mentioned Lactobacillus oris, including but not limited to cell wall, intracellular protein, intracellular polysaccharide, etc.;

[0016] The metabolites should be understood as the primary metabolites or secondary metabolites of the above-mentioned Lactobacillus oris, such as probiotics, compounds containing immunogenic components, extracellular polysaccharides, etc., and can also be directly the supernatant of the fermentation broth.

[0017] ProfMIC-203 beneficial effects of the present invention:

[0018] In vitro cell experiments show that the Lactobacillus oris ProfMIC-203 of the present invention has the effect of promoting epidermal cell repair, and ProfMIC-203 increases the survival rate of HaCaT cells damaged by sodium dodecyl sulfate (SDS) by 112.11% to 157.93%.

[0019] In vitro cell experiments show that the Lactobacillus oris ProfMIC-203 of the present invention has the effect of up-regulating the expression of the cell repair-related factors filaggrin gene FLG and loricrin gene LOR, and the gene expression amount is up-regulated by 1.34 to 6.94 times.

[0020] In vitro cell experiments show that the Lactobacillus oris ProfMIC-203 of the present invention has the effect of reducing the release of inflammatory factors, and can reduce the nitric oxide (NO) production of mouse macrophage Raw264.7 induced by lipopolysaccharide (LPS) by 24.70% to 28.11%.

[0021] In vitro cell experiments show that the Lactobacillus oris ProfMIC-203 of the present invention has the effect of down-regulating the expression of genes related to inflammatory factors in HaCaT cells induced by Staphylococcus aureus, and the gene expression amount is down-regulated by 30% to 66%.

[0022] Collection Information

[0023] Deposit date: November 18, 2021;

[0024] Name of depository: China Center for Type Culture Collection;

[0025] Deposit number: CCTCC NO:M20211438;

[0026] Address of depository: Wuhan University, Wuhan, China;

[0027] Classification name: Lactobacillus oris ProfMIC-203. DETAILED DESCRIPTION

[0028] The following is a further detailed description of the above contents of the present invention through specific implementation methods in the form of embodiments, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies implemented based on the above contents of the present invention belong to the scope of the present invention, and unless otherwise specified, the following embodiments are all completed using conventional existing technologies.

[0029] In the following examples, ProfMIC-203 refers to Lactobacillus oris, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M20211438.

[0030] Example 1: Isolation of ProfMIC-203

[0031] The samples were collected from the facial skin of a 2-year-old healthy boy. After the samples were properly treated, they were shaken and mixed in physiological saline. The supernatant was streaked on an MRS solid plate and cultured at 37°C for 24 to 48 hours. White colonies were picked and repeatedly inoculated and screened until a uniform single colony was obtained, which was named ProfMIC-203.

[0032] Gram staining microscopy: strain ProfMIC-203 is Gram-stain positive and rod-shaped under a microscope; it grows on MRS plates to form white, smooth, round, opaque small colonies with neat edges; it grows evenly turbid in MRS liquid culture medium, and the bacteria form white precipitates after long-term storage.

[0033] Example 2: Nucleic acid identification of ProfMIC-203

[0034] 1. 16s rRNA gene sequence analysis:

[0035] Pick a single colony and place it in MRS liquid medium. After culturing overnight at 37°C, collect the bacteria by centrifugation and follow the steps of the DNA extraction kit. Use bacterial universal primers 27F, 1492R as primers, and the PCR amplification system is 50μL system, 95°C pre-denaturation for 5min; 94°C for 15s, 57°C for 15s, 72°C for 40s, 35 cycles; 72°C extension for 10min.

[0036] 2. Results

[0037] The sequencing results of the PCR product were compared with the standard sequences published in GenBank by BLASTN, and it was concluded that the ProfMIC-203 strain was Lactobacillus oris.

[0038] Example 3: ProfMIC-203 promotes SDS-induced HaCaT cell damage repair experiment

[0039] 1. Preparation of ProfMIC-203 bacterial solution:

[0040] The activated Lactobacillus oris ProfMIC-203 bacterial suspension was cultured in MRS liquid medium in a 37°C incubator for 16-18 h, and the OD was adjusted with PBS buffer. 600=2.0, after centrifugation, the supernatant was filtered with a 0.22 μm filter membrane, the cells were washed twice with PBS and resuspended in PBS to OD 600 =0.2, and the supernatant and bacterial samples were finally inactivated by high pressure sterilization at 121℃ for 30min.

[0041] 2. Promote HaCaT cell manipulation and repair experiments

[0042] HaCaT cells (5×10 4 cell / well) to a 96-well plate and culture overnight until the cells adhere to the wall. Prepare 50μg / ml SDS, add 100μl to each well, incubate for 8h, add 5% strain supernatant (V / V, the corresponding control group 1 is the same volume of PBS), add 10% inactivated bacteria (V / V, the corresponding control group 2 is the same volume of PBS) to the experimental group 1, set up 3 parallels for each group, and incubate for 24h. Add 10μl CCK-8 solution, incubate for 4h, and detect the absorbance at 450nm.

[0043] Cell survival rate = A 实验 / A 对照 ×100%.

[0044] Among them, A 实验 The absorbance value of the experimental group at 450 nm, A 对照 The absorbance value of the control group was detected at 450 nm.

[0045] result:

[0046] ProfMIC-203 promotes repair of HaCaT cells

[0047]

[0048] Both the inactivated supernatant and inactivated bacteria of ProfMIC-203 have a repairing effect on SDS-damaged HaCaT cells.

[0049] Example 4: Experiment on ProfMIC-203 promoting HaCaT barrier repair-related gene expression

[0050] 1. Preparation of ProfMIC-203 bacterial solution (same as Example 3).

[0051] 2. Inoculation of human immortalized keratinocytes HaCaT (5×10 5cell / well) to a 6-well plate and cultured overnight until the cells adhered to the wall. Experimental group 1 added 5% strain supernatant (V / V, the corresponding control group was an equal volume of PBS), and experimental group 2 added 10% inactivated bacteria (V / V, the corresponding control group was an equal volume of PBS). Each group was set up in 3 parallels. After culturing for 24 hours, lysis buffer was added, total cell RNA was extracted, and RNA concentration and purity were detected. All samples were adjusted to 1 μg and reverse transcribed into cDNA. GAPDH was used as an internal reference gene, and qPCR was performed to detect the expression of FLG and LOR genes. The expression change factor was calculated according to the formula.

[0052] Formula: F = 2 -ΔΔCT

[0053] result:

[0054] Experimental group 1 (ProfMIC-203 supernatant) upregulated the expression of barrier repair-related genes

[0055]

[0056] Experimental group 2 (ProfMIC-203 inactivated bacteria) up-regulated the expression of barrier repair-related genes

[0057]

[0058] The results showed that the addition of ProfMIC-203 can promote skin barrier repair.

[0059] Example 5: ProfMIC-203 reduces NO production in Raw264.7 cells

[0060] 1. Preparation of ProfMIC-203 bacterial solution

[0061] ProfMIC-203 was cultured in MRS medium overnight and the OD 600 PBS buffer was used to adjust the bacterial concentration to OD 600 =0.2, after centrifugation, the supernatant was filtered with a 0.22 μm filter membrane, the bacteria were washed twice with PBS and resuspended in PBS to OD 600 =0.2, and the supernatant and bacterial samples were finally inactivated by high pressure sterilization at 121℃ for 30min.

[0062] 2. Raw264.7 Cell Preparation

[0063] Raw264.7 cells were digested and then cultured at 2×10 5 The cells / well were inoculated into a 24-well plate and cultured overnight at 37°C in a 5% carbon dioxide incubator.

[0064] 3. ProfMIC-203 addition and LPS stimulation

[0065] Raw264.7 cells were cultured overnight, and 5% supernatant (V / V, the corresponding control group was an equal volume of PBS) was added to experimental group 1, and 10% inactivated bacteria (V / V, the corresponding control group was an equal volume of PBS) was added to experimental group 2. After 2 hours, 0.5 ml of 0.2 μg / ml LPS solution was added to induce inflammation of Raw264.7 cells. After 20 hours, the cell culture supernatant was collected, and the standard curve was drawn according to the method described in the Bio-Tech NO Detection Kit to calculate the NO concentration and inhibition rate in the samples.

[0066] ProfMIC-203 reduces NO production in Raw264.7 cells

[0067]

[0068] The results showed that ProfMIC-203 had anti-inflammatory effects and could reduce the NO production in Raw264.7 cells induced by LPS. Compared with the LPS control group, the supernatant was reduced by 24.70% and the inactivated bacteria was reduced by 28.11%.

[0069] Example 6: ProfMIC-203 down-regulates the expression of inflammatory factors in HaCaT cells

[0070] 1. Preparation of ProfMIC-203 bacterial solution (same as Example 5);

[0071] 2. Preparation of HaCaT cells:

[0072] HaCaT cells were digested and then cultured at 2×10 5 The cells / well were inoculated into a 24-well plate and cultured overnight at 37°C in a 5% carbon dioxide incubator.

[0073] 3. Preparation and addition of Staphylococcus aureus

[0074] Staphylococcus aureus was inoculated into nutrient broth medium and cultured overnight at 37°C in a shaking incubator. The concentration of the bacterial solution was adjusted to OD 600 =6.0, 100 μl per well was added into HaCaT cells cultured overnight to stimulate the cells to produce inflammatory factors, the cell culture medium was discarded after 3 h, the cells were washed 5 times with PBS, and 1 ml of MEM serum-free culture medium was added to each well.

[0075] 4. ProfMIC-203 sample addition

[0076] The inactivated supernatant of ProfMIC-203 was added to the HaCaT cells stimulated with Staphylococcus aureus at 5% (V / V, the corresponding control group was an equal volume of PBS), with 3 replicate wells in each group, and cultured overnight.

[0077] 5. qPCR method to detect the relative expression of inflammatory factor mRNA

[0078] After discarding the culture medium from the above cells, RNA was extracted using an RNA extraction kit, and the RNA concentration and purity were tested. All samples were adjusted to 1 μg, and RT-PCR and qPCR were performed using a reverse transcription kit and a SYBRGreen qPCR kit, and the relative expression fold F of the inflammatory factor genes IL-8, IL-22, COX-2, and TRPV1 were calculated.

[0079] Formula: F = 2 -ΔΔCT

[0080] result:

[0081] ProfMIC-203 inactivated supernatant down-regulates the expression of inflammatory factor genes

[0082]

[0083] The results showed that ProfMIC-203 could down-regulate the expression of inflammatory factor-related genes in HaCaT cells induced by Staphylococcus aureus, and the expression level was reduced by 30-66%. Therefore, ProfMIC-203 has an anti-inflammatory effect.

[0084] In addition, the fermentation supernatant or inactivated bacteria involved in all the above embodiments have been verified to have corresponding efficacy when further processed or extracted to obtain derivatives (lysates or extracts) of ProfMIC-203, or primary metabolites or secondary metabolites of ProfMIC-203 in the fermentation supernatant.

Claims

1. A strain of Lactobacillus oralis ( Lactobacillus oris ), It is characterized in that It is deposited in the China Center for Type Culture Collection with the accession number CCTCC NO: M 20211438.

2. Use of a strain of Lactobacillus oris in the preparation of a medicine for promoting skin barrier repair, It is characterized in that The Lactobacillus oris is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20211438; the Lactobacillus oris is a live bacterium or / and an inactivated bacterium.

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