Application of a strain of Lactobacillus rhamnosus B2-1 derived from breast milk in the preparation of a product for relieving atopic dermatitis

Abstract

The present invention provides an application of a strain of Lactobacillus rhamnosus B2-1 derived from breast milk in the preparation of a product for relieving atopic dermatitis. Lactobacillus rhamnosus B2-1 has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on December 8, 2022, with a deposit number of CGMCC NO. 26167. The Lactobacillus rhamnosus B2-1 of the present invention has a good effect of relieving atopic dermatitis and has potential functional development potential.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to application of a strain of Lactobacillus rhamnosus B2-1 derived from breast milk in the preparation of a product for alleviating atopic dermatitis. Background Art

[0002] Atopic dermatitis (AD), also known as eczema, is a chronic inflammatory skin disease. AD is characterized by recurrent eczematous skin lesions, often accompanied by blisters and crusted red patches, which can lead to scaling, cracking and thickening of the skin as well as intense itching and discomfort. The etiology of atopic dermatitis is complex and multifactorial. It involves a complex interaction between epidermal barrier dysfunction, abnormal skin microbiota and immune imbalance. Current treatment of the disease focuses on systemic or topical use of immunomodulators, which can cause side effects such as conjunctivitis or lymphocytopenia with long-term use.

[0003] Probiotics are essential for human growth and development, and immune regulation. A large number of research papers have proven that probiotics can regulate the immune system and alleviate immune-related diseases. Probiotics regulate the body's immune system by improving the composition of intestinal flora. Due to limited sample sources and low probiotic content, commercialized probiotics derived from breast milk account for a small proportion. Probiotics derived from breast milk have natural advantages over probiotics derived from the intestine in terms of safety and probiotic properties, and are psychologically more acceptable to the public.

[0004] Most of the probiotics studied so far come from food, such as dairy products and sauerkraut, while there are fewer studies on lactic acid bacteria from breast milk, and even fewer studies on their potential functions. It is of great significance to explore more functions of probiotics from breast milk. Summary of the invention

[0005] The purpose of the present invention is to provide a use of a strain of Lactobacillus rhamnosus B2-1 derived from breast milk in the preparation of a product for alleviating atopic dermatitis, which has a good effect of alleviating atopic dermatitis and has good application prospects in the fields of medicine and food.

[0006] Solution

[0007] In order to achieve the above object, the present invention provides a strain of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) B2-1 is used in the preparation of a product for relieving atopic dermatitis. The rhamnosus Lactobacillus B2-1 was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on December 8, 2022, with the deposit number CGMCC No.26167.

[0008] Furthermore, the product is a medicine, a food or a functional food.

[0009] Furthermore, the product comprises Lactobacillus rhamnosus B2-1 and a pharmaceutically acceptable carrier.

[0010] Furthermore, the product is a probiotic.

[0011] Furthermore, the dosage form of the product is one or more of a solution, a suspension, an emulsion, a powder, a lozenge, a pill, a syrup, a tablet and a capsule.

[0012] Furthermore, the tablet comprises an oral tablet.

[0013] Furthermore, the product is an oral medicine.

[0014] Furthermore, in the product, the number of viable bacteria is 0.1~100×10 8 cfu / mL.

[0015] Furthermore, in the product, the number of viable bacteria is 1~100×10 8 cfu / mL.

[0016] Furthermore, in the product, the number of viable bacteria is 2.5~15×10 8 cfu / mL.

[0017] Beneficial Effects

[0018] The breast milk-derived Lactobacillus rhamnosus B2-1 of the present invention has good acid and bile salt resistance, has the characteristics of a probiotic, is safe from breast milk, and has good effects on relieving atopic dermatitis after in vivo and in vitro verification. The bacteria has potential for probiotic development and fills the research gap of lactic acid bacteria derived from breast milk. The breast milk-derived Lactobacillus rhamnosus B2-1 of the present invention has a bright prospect for product development and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] One or more embodiments are exemplarily described by the pictures in the accompanying drawings, and these exemplary descriptions do not constitute limitations on the embodiments. The special word "exemplary" here means "used as an example, embodiment or illustrative". Any embodiment described as "exemplary" here is not necessarily interpreted as being superior or better than other embodiments.

[0020] Figure 1 The statistical results of the number of scratching of mice in each group on the 7th day of the atopic dermatitis mouse model in Test Example 1, among which the low, medium and high dose groups represent the low, medium and high dose groups of probiotics, respectively.

[0021] Figure 2The statistical results of the number of scratching of mice in each group on the 14th day of the atopic dermatitis mouse model in Test Example 1, among which the low, medium and high dose groups represent the low, medium and high dose groups of probiotics, respectively.

[0022] Figure 3 HE staining images (200×) of the mouse back skin tissue pathological sections of each group of mice on the 15th day of the atopic dermatitis mouse modeling in Test Example 2; wherein A is the blank group, B is the model group, C is the low-dose probiotic group, D is the medium-dose probiotic group, and E is the high-dose probiotic group.

[0023] Figure 4 The serum IgE level of mice on the 15th day of the atopic dermatitis mouse modeling in Test Example 3. Indicates P < 0.05, and the low, medium, and high dose groups represent the low, medium, and high dose groups of probiotics, respectively.

[0024] Figure 5 The serum IL-4 level of mice on the 15th day of the atopic dermatitis mouse model in Test Example 3. Indicates P < 0.05, and the low, medium, and high dose groups represent the low, medium, and high dose groups of probiotics, respectively. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] In addition, in order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In some embodiments, raw materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail, so as to highlight the main purpose of the present invention.

[0027] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0028] Unless explicitly stated otherwise, throughout the specification and claims, the term “comprise” or variations such as “include” or “comprising”, etc., will be understood to include the stated elements or components but not to exclude other elements or components.

[0029] The present invention is lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) B2-1, classified as Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ), was deposited on December 8, 2022 in the General Microbiology Center of China Microorganism Culture Collection (CGMCC for short, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101), with the deposit number: CGMCC NO.26167.

[0030] Among them, each reagent used in the embodiments of the present invention can be obtained commercially, for example:

[0031] The IgE test kit was purchased from Wuhan Huamei Bioengineering Co., Ltd., batch number: 202307.

[0032] The IL-4 test kit was purchased from Wuhan Huamei Bioengineering Co., Ltd., batch number: 202307.

[0033] Formaldehyde solution was purchased from Xilong Chemical Co., Ltd., No. 210326.

[0034] Paraffin sections were purchased from Leica, 1804040.

[0035] MRS broth medium was purchased from Beijing Luqiao Technology Co., Ltd. with the catalog number CM187.

[0036] Nutrient agar was purchased from Beijing Luqiao Technology Co., Ltd. with the product number CM107.

[0037] The instruments used in the following examples include: SPARK microplate reader, T1000 electronic balance, BS2202S electronic balance, pathology slide making system (KD-TSQ1 fully automatic closed tissue dehydrator, EG1150H / C paraffin embedding machine hot / cold stage, RM2245 semi-automatic rotary slicer, etc.), XSP-12CA biological microscope.

[0038] Example 1: Isolation and identification of Lactobacillus rhamnosus B2-1 from breast milk

[0039] Select fresh breast milk samples, perform gradient dilution with sterile PBS buffer solution, select appropriate dilution concentration, spread it on MRS solid culture medium containing 1% CaCO3, and culture it under anaerobic conditions at 37°C for 48h. Pick white or milky white single colonies with obvious transparent circles for Gram staining and microscopic examination, select rod-shaped and Gram-positive bacterial strains, select strains with better growth from many strains, and carry out enrichment culture in a 37°C anaerobic incubator.

[0040] The enriched strain was sequenced by 16S sequencing to confirm that it was Lactobacillus rhamnosus, and the whole genome sequencing analysis was performed using Pacbio (10Kb SMRT Bell library) and Illumina PE150 (350bp small fragment library) sequencing platforms for whole genome sequencing. The sequencing results were annotated with the NR (Non-Redundant Protein Database) database (for species information identification and can be used for species classification), and the strain was amplified and preserved in the laboratory, and sent to the General Microbiology Center of the China Microbiological Culture Collection Administration for preservation, with the preservation number CGMCC No.26167.

[0041] Example 2: Construction of atopic dermatitis mouse model

[0042] Experimental animals: 100 SPF-grade BALB / c male mice, weighing 20g-22g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. [License No.: SCXK (Beijing) 2021-0006]. Laboratory Animal Quality Certificate: No.110011231106845421. The experimental animals were kept in the SPF-grade animal room of the Health Food Function Testing Center of the School of Applied Arts and Sciences, Beijing Union University, and the experimental animal use license number is SYXK (Beijing) 2023-0013. After a 7-day adaptation period, the mice were randomly divided into five groups, each with 20 mice, namely, blank group, model group, probiotic low-dose group, probiotic medium-dose group, and probiotic high-dose group. The administration method was:

[0043] Mice in the blank group and model group were gavaged with 200 μL sterile PBS / mouse / day for 28 days;

[0044] The low-dose probiotic group was given 200 μL of PBS containing rhamnosus casei B2-1 per rat per day by gavage, where the dose of rhamnosus casei B2-1 was 0.5×10 8 CFU / bird / day, for 28 days;

[0045] The medium-dose probiotic group was given 200 μL of PBS containing rhamnosus casei B2-1 per rat per day by gavage, where the dose of rhamnosus casei B2-1 was 1×10 8 CFU / bird / day, for 28 days;

[0046] The high-dose probiotic group was given 200 μL of PBS containing rhamnosus casei B2-1 per rat per day by gavage, where the dose of rhamnosus casei B2-1 was 3×10 8 CFU / animal / day for 28 days.

[0047] Atopic dermatitis mouse modeling: The modeling was started on the 15th day. One day before modeling, 6% sodium sulfide was used to remove the hair on the back of the mouse, with an area of ​​about 2×2 cm. 2 On the first day of modeling, except for the blank group, all other groups (model group, probiotic low, medium and high dose groups) were sensitized by applying 100μL of 0.5% 2,4-dinitrofluorobenzene (DNFB) on the hair removal area. On the third day, 100μL of 0.5% DNFB was applied again to strengthen sensitization. On the 7th and 14th days of modeling, 50μL of 0.5% DNFB solution was applied on the back of mice to induce dermatitis, and the blank group was smeared with an equal volume of acetone olive oil matrix on the back skin. Except for the blank group and the model control group, which were given PBS, each dose group was given the corresponding test substance every day according to the above-mentioned administration method, and was gavaged for 28 consecutive days (that is, the corresponding drug was continued to be gavaged for 14 days after modeling). Related tests:

[0048] In the following test cases, SPSS23.0 software was used for statistical analysis. If the variance of the measurement data is homogeneous, or the variance is unequal but becomes homogeneous after transformation, one-way ANOVA is used, and the LSD method is used for inter-group comparison. If the variance of the data is still unequal after transformation, the rank sum test is used for statistical analysis.

[0049] Test Example 1: Skin Lesion Score and Itch Frequency Determination

[0050] On the 7th and 14th days of the atopic dermatitis mouse modeling in Example 2, the mice were sensitized with DNFB for 30 minutes, and then placed alone. The number of times the mice scratched their trunks and backs with their hind paws and bit various parts of the body with their mouths was observed within 10 minutes. Continuous scratching was counted as one time.

[0051] The number of scratching of mice on the 7th day of modeling was as follows Figure 1 As shown, the results showed that on the 7th day of modeling, the number of scratching times of mice in the blank group was 0, and there was no significant difference in the number of scratching times of mice in other groups.

[0052] The number of scratching of mice on the 14th day of modeling was as follows Figure 2 As shown in the figure, the scratching frequency of mice in the blank group was 0 on the 14th day of modeling. The scratching frequency of mice in the high-dose probiotic group was significantly lower than that of the model group. The scratching frequency of mice in the medium-dose probiotic group also tended to decrease compared with the model group ( P =0.068).

[0053] Test Example 2: Observation of mouse skin tissue pathological morphology

[0054] On the 15th day after the establishment of the atopic dermatitis mouse model in Example 2, the mice in each group were killed by cervical dislocation, and the skin on the back of the mice was cut. Part of the skin tissue was fixed with 4% formaldehyde solution, dehydrated with gradient ethanol, transparentized with xylene, embedded in paraffin, and sliced ​​at 5 μm to obtain paraffin sections of the mouse skin. The paraffin sections of the mouse skin were stained with HE for observation. Some of the results are shown in the figure. Figure 3 .

[0055] Figure 3 The results of A showed that the epidermis, appendages, dermis and subcutaneous tissue of the blank group mice were structurally intact, and no damaging changes or inflammatory cell infiltration were observed. Figure 3 The results of B showed that the pathological sections of the skin of the mice in the model group showed epidermal erosion, partial loss of the superficial epidermal cell layer, and accumulation of a large number of inflammatory cells, including segmented leukocytes, in the dermis. Figure 3 The results of C, D, and E showed that low, medium, and high doses of probiotics intervention resulted in a certain degree of improvement in skin pathological sections, especially high doses of probiotics intervention, which had the most obvious improvement.

[0056] Test Example 3: ELISA method for detecting the content of IgE and IL-4 in serum

[0057] On the 15th day after the modeling of atopic dermatitis mice in Example 2, blood was collected from the medial canthal venous plexus of the animals, centrifuged at 3000 r / min for 10 min, and serum was collected for the determination of IgE and IL-4 (because the amount of serum was small, the serum of two mice was used as one sample). The detection method was carried out according to the instructions of the IgE test kit and the IL-4 test kit. The results are shown in the figure. Figure 4 , Figure 5 .

[0058] Figure 4 The results showed that IgE can cause type I allergic reactions (allergies), and the serum IgE of mice with atopic dermatitis was significantly higher than that of the blank group. After intervention with low, medium and high doses of probiotics, IgE decreased to varying degrees, and there was a significant statistical difference between the low-dose group and the model group.

[0059] In mice, IL-4 is produced by Th2 cells. In the pathogenesis of atopic dermatitis (AD), IL-4 can induce immunoglobulin class conversion in B cells to IgE and stimulate afferent neurons through the IL-4 receptor subunit α, promoting itch. Figure 5 The results showed that low, medium and high doses of probiotics intervention of the present invention can significantly reduce the level of serum IL-4 in mice and alleviate symptoms.

[0060] The breast milk-derived Lactobacillus rhamnosus B2-1 of the present invention has good acid and bile salt resistance, has the characteristics of a probiotic, is safe from breast milk, and has good effects on relieving atopic dermatitis after in vivo and in vitro verification. The bacteria has potential functional and probiotic development potentials such as medicines and functional foods, and fills the research gap of lactic acid bacteria derived from breast milk. The breast milk-derived Lactobacillus rhamnosus B2-1 of the present invention has a bright prospect for product development and application.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of Lactobacillus rhamnosus from breast milk ( Lacticaseibacillus rhamnosus ) B2-1 is used in the preparation of drugs for relieving atopic dermatitis. The rhamnosus Lactobacillus B2-1 has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on December 8, 2022, with the deposit number CGMCC No.26167.

2. The use according to claim 1, characterized in that: The medicine comprises Lactobacillus rhamnosus B2-1 and a pharmaceutically acceptable carrier.

3. The use according to claim 1, characterized in that: The medicine is a probiotic.

4. The use according to any one of claims 1 to 3, characterized in that: The dosage form of the drug is one or more of solution, suspension, emulsion, powder, lozenge, pill, syrup, tablet and capsule.

5. The use according to claim 4, characterized in that: The lozenges comprise oral lozenges.

6. The use according to claim 4, characterized in that: The medicine is an oral medicine.

7. The use according to any one of claims 1 to 3, characterized in that: In the drug, the number of viable bacteria is 0.1~100×10 8 cfu / mL.

8. The use according to any one of claims 1 to 3, characterized in that: In the drug, the number of viable bacteria is 1~100×10 8 cfu / mL.

9. The use according to claim 7, characterized in that: The viable bacterial count in the drug is 2.5~15×10 8 cfu / mL.

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