Probiotic composition capable of improving immunity and application of probiotic composition in treatment of skin allergy

Abstract

The invention provides a probiotic composition as well as a preparation method and application thereof. The composition is prepared from lactobacillus rhamnosus GG, lactobacillus acidophilus NCFM, an anti-IL-4R alpha monoclonal antibody, an inulin-beta-glucan compound and sodium alginate-chitosan microspheres. The composition regulates immune balance through a specific probiotic strain, blocks an IL-4 / IL-13 signal channel in combination with an anti-IL-4R alpha monoclonal antibody, and improves viable bacterium stability and bioavailability through a microsphere delivery technology. The probiotic composition shows a remarkable anti-allergic effect in an atopic dermatitis mouse model, and can reduce the serum IgE level, reduce the skin inflammatory response and reduce the STAT6 phosphorylation level. The probiotic composition disclosed by the invention has a wide application prospect in the aspect of treating atopic dermatitis and other immune-related diseases.

Description

Technical Field

[0001] The present invention relates to the field of microbial preparations, and in particular to a composition for regulating the immune system through probiotics, in particular to a probiotic composition for improving skin immunity and treating skin allergies. Background Art

[0002] Skin allergy is a common immune-mediated disease, usually caused by an excessive immune response to allergens (such as pollen, dust mites, animal dander, etc.). Typical symptoms of the disease include itching, redness, swelling, eczema, urticaria, etc., which may affect the patient's quality of life in severe cases. The pathogenesis of skin allergies is closely related to the body's immune response disorders, especially the overactivation of Th2 immune responses. In allergic reactions, the immune system mistakenly identifies harmless substances as pathogenic factors, releasing a large amount of immune mediators (such as IgE, interleukin IL-4, IL-5, etc.), triggering local inflammatory reactions and skin lesions.

[0003] At present, common methods for treating skin allergies in clinic include the use of drugs such as antihistamines, steroid hormones and immunosuppressants. Although these drugs can effectively relieve allergic symptoms, long-term use may lead to a series of side effects, such as immunosuppression, weight gain, and hyperglycemia. Therefore, finding a safer, more effective and long-term treatment option has become a research hotspot.

[0004] In recent years, the relationship between the intestinal microbiota and the immune system has received widespread attention. A large number of studies have shown that probiotics in the intestine can regulate the Th1 / Th2 immune balance and enhance the body's immune function by regulating the host's immune system. In particular, specific probiotic strains, such as Bifidobacterium and Lactobacillus strains, have been shown to be able to regulate intestinal immune barrier function, inhibit systemic inflammatory responses, and help reduce allergic symptoms by secreting metabolites such as short-chain fatty acids (SCFAs). Therefore, probiotics, as an emerging immunomodulatory strategy, have shown great potential in the treatment of immune diseases such as skin allergies. Summary of the invention

[0005] The purpose of the present invention is to provide a probiotic composition for treating skin allergies by regulating the immune system. By screening a variety of probiotic strains and combining their specific immunomodulatory effects, the present invention provides a probiotic composition that can improve immunity and alleviate skin allergy symptoms.

[0006] Therefore, the present invention discloses a probiotic composition, which is composed of the following components in a certain mass percentage:

[0007] (1) Lactobacillus rhamnosus GG: 30%;

[0008] (2) Lactobacillus acidophilus NCFM: 25%;

[0009] (3) Anti-IL-4Rα monoclonal antibody: 10%;

[0010] (4) Inulin-β-glucan complex: 15%;

[0011] (5) Sodium alginate-chitosan microspheres: 20%.

[0012] Preferably, the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-IL-4Rα monoclonal antibody of the present invention are shown as SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0013] In one aspect, the present invention also discloses a method for preparing the composition, the method comprising the following steps:

[0014] (1) Bacterial culture: Lactobacillus rhamnosus GG and Lactobacillus acidophilus NCFM were inoculated into MRS medium containing 1% inulin, cultured anaerobically at 37°C for 24 h, and centrifuged at 10,000 × g for 15 min to collect the bacteria;

[0015] (2) Antibody loading: 10 mg / mL anti-IL-4Rα monoclonal antibody was mixed with bacteria at a mass ratio of 1:5, 2% sodium alginate solution was added, and 2% CaCl2 solution was added dropwise to solidify;

[0016] (3) Chitosan coating: The microspheres were immersed in a 1% chitosan solution at pH 5.5, magnetically stirred for 30 min, and freeze-dried to obtain the finished product.

[0017] In one aspect, the present invention also discloses a use of the anti-IL-4Rα monoclonal antibody in the composition in the preparation of a probiotic composition.

[0018] In one aspect, the present invention also discloses a use of the composition in preparing a drug for treating skin allergies.

[0019] The probiotic composition of the present invention has the following beneficial effects:

[0020] (1) Highly efficient regulation of immune balance: The present invention regulates Th1 / Th2 balance, reduces IgE levels, and reduces allergic reactions through the synergistic effect of Lactobacillus rhamnosus GG and Lactobacillus acidophilus NCFM.

[0021] (2) Specific inhibition of allergic inflammatory pathways: Anti-IL-4Rα monoclonal antibodies can effectively block the IL-4 / IL-13 signaling pathway and reduce Th2 cell-mediated allergic inflammation from the root.

[0022] (3) Enhance the stability and activity of probiotics: Inulin-β-glucan complex helps the survival and colonization of probiotics and improves the homeostasis of intestinal flora. At the same time, sodium alginate-chitosan microspheres can provide gastric acid protection and improve the bioavailability of probiotics and antibodies.

[0023] (4) Significantly reduce allergic reaction indicators: In the atopic dermatitis mouse model experiment, the composition can effectively reduce serum IgE levels (by about 50%), reduce IL-4 expression in skin tissue (by about 40%), and significantly inhibit the activity of the STAT6 signaling pathway.

[0024] (5) Wide application and high safety: The probiotic composition provided by the present invention can be used in various forms such as food, health products, and medicines. It is suitable for various allergic diseases such as atopic dermatitis, allergic rhinitis, and asthma, and has good market application prospects.

[0025] In summary, the probiotic composition of the present invention achieves immunomodulatory and anti-allergic effects through multi-target synergistic effects, providing a safe and efficient new immunomodulatory strategy. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 SDS-PAGE detection results of anti-IL-4Rα monoclonal antibody, where 1 is anti-IL-4Rα monoclonal antibody. DETAILED DESCRIPTION

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0028] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0029] Example 1: Formulation of probiotic composition

[0030] The specific formula of the probiotic composition is as follows:

[0031]

[0032] Example 2: Preparation of probiotic composition

[0033] 1. Bacterial Culture

[0034] (1) Culture medium preparation: MRS (Man Rogosa Sharpe) culture medium containing 1% inulin was prepared for culturing Lactobacillus rhamnosus GG (ATCC53103) and Lactobacillus acidophilus NCFM (ATCC700396). The pH was adjusted to 6.2-6.5 to ensure that the culture conditions were suitable for the growth of lactic acid bacteria.

[0035] (2) Inoculation and culture: Take the frozen strains of Lactobacillus rhamnosus GG and Lactobacillus acidophilus NCFM respectively and inoculate them into MRS medium containing 1% inulin under anaerobic conditions at 37°C. The initial inoculation amount of the bacteria is 10 6 CFU / mL. Use an anaerobic incubator to maintain anaerobic conditions for 24 hours to ensure adequate bacterial growth.

[0036] (3) Harvesting of bacteria: After 24 hours, use a centrifuge to collect the bacteria in the culture medium. The specific operation is: centrifuge at a centrifugal force of 10,000×g for 15 minutes. Discard the supernatant, add an appropriate amount of physiological saline or PBS buffer to resuspend the bacteria, and centrifuge and wash again twice to remove the remaining impurities in the culture medium. The resuspended bacteria can be stored at 4°C for subsequent experiments.

[0037] 2. Antibody loading

[0038] (1) Antibody preparation: Anti-IL-4Rα monoclonal antibody (10 mg / mL) was selected, as described in Example 4. The antibody solution was gently mixed to ensure its homogeneity and avoid the formation of bubbles.

[0039] (2) Mixing antibodies and bacteria: resuspended Lactobacillus rhamnosus GG and Lactobacillus acidophilus NCFM were mixed at a mass ratio of 1:5. That is, 1 gram of antibody was added to the mixture of 5 grams of bacterial cells to ensure that the bacteria and the antibody were in full contact. Slowly stir with magnetic force at 4°C for 1 hour to ensure that the antibody was fully adsorbed on the surface of the bacteria.

[0040] (3) Preparation of 2% sodium alginate solution: Add 2 g of sodium alginate to deionized water and stir until completely dissolved. Adjust the pH to 6.0-6.5 so that it can form a better coating structure with cells.

[0041] (4) Mixing of sodium alginate solution and antibody-loaded bacterial liquid: The mixed antibody-loaded bacterial liquid (containing a mixture of antibodies and bacteria) was mixed with 2% sodium alginate solution in a volume ratio of 1:1, and stirred at room temperature for 30 minutes using a magnetic stirrer to ensure that the sodium alginate was evenly coated.

[0042] (5) Add the curing liquid: Prepare a 2% CaCl2 solution, slowly add the mixed solution to the CaCl2 solution (the curing solution is 2% CaCl2) through a burette, and allow it to cure naturally to form a microsphere structure. The dripping time must be controlled within 10 minutes to ensure that the microspheres are of uniform size. After dripping, the microspheres must be left to stand in the CaCl2 solution for 30 minutes to ensure that the microspheres are fully cured and form a stable shell.

[0043] 3. Chitosan coating

[0044] (1) Preparation of 1% chitosan solution: Dissolve chitosan (high molecular weight, deacetylation degree above 85%) in 0.1 M acetic acid solution and stir until the chitosan is completely dissolved. The pH value of the solution should be adjusted to 5.5 to ensure that it is suitable for coating the surface of the microspheres. After the solution is prepared, check its transparency and uniformity to ensure that there is no obvious precipitation.

[0045] (2) Microsphere coating treatment: Add the antibody-loaded microspheres to a 1% chitosan solution and stir under magnetic stirring for 30 minutes to ensure that each microsphere is evenly coated with a chitosan layer. During the stirring process, keep the solution temperature at room temperature to avoid high temperature affecting the microsphere structure.

[0046] (3) Freeze drying: The encapsulated microspheres are subjected to low-temperature freeze drying to remove the solvent and maintain the structure and activity of the microspheres. The specific operation is as follows: transfer the microspheres to a freeze drying bottle and set the freeze drying machine program: temperature -40°C, pressure 0.1-0.2 mbar, and freeze drying time for 48 hours. After completion, check the morphology and encapsulation effect of the microspheres to ensure that the surface of the microspheres is smooth and stable.

[0047] (4) Storage of finished products: The obtained microsphere products are stored in an environment of -20°C to maintain their stability and avoid moisture and temperature fluctuations that may cause damage to the microspheres or loss of activity.

[0048] Example 3: Evaluation of anti-allergic effects in atopic dermatitis mouse model

[0049] 1. Experimental animals: 8-week-old male BALB / c mice (weight 18-22g) were selected. All mice were purchased from the Experimental Animal Center and adaptively raised for 1 week. The mice were randomly divided into 4 groups, 8 mice in each group:

[0050] (1) Model group: injected with normal saline only.

[0051] (2) Commercially available probiotics group: Oral administration of commercially available probiotics (Renhe Probiotics) at a daily dose of 1 mg / kg body weight.

[0052] (3) Probiotic group of the present invention: Orally take the probiotic composition of the present invention (prepared in Example 2) at a daily dose of 1 mg / kg body weight.

[0053] (4) Probiotic group without anti-IL-4Rα monoclonal antibody: Oral administration of the probiotic composition of the present invention (prepared by the method of Example 2, but without anti-IL-4Rα monoclonal antibody) at a daily dose of 1 mg / kg body weight.

[0054] 2. Establishment of Allergy Model

[0055] (1) Sensitization and stimulation: On days 1 and 7, mice were intraperitoneally injected with ovalbumin (OVA, 50 μg / mouse) and aluminum salt adjuvant (5 mg / mouse) to establish an atopic dermatitis model. Starting from day 14, allergic reactions were stimulated by applying OVA (10 μg / mouse) to the ear skin every 2 days for 7 days.

[0056] 4. Solution

[0057] (1) Model group: injected with normal saline only.

[0058] (2) Commercially available probiotics group: Oral administration of commercially available probiotics at a daily dose of 1 mg / kg body weight.

[0059] (3) Probiotic group of the present invention: Orally take the probiotic composition of the present invention (prepared in Example 2) at a daily dose of 1 mg / kg body weight.

[0060] (4) Probiotic group without anti-IL-4Rα monoclonal antibody: Oral administration of the probiotic composition of the present invention (prepared by the method of Example 2, but without anti-IL-4Rα monoclonal antibody), with a daily dose of 1 mg / kg body weight.

[0061] 5. Experimental process and tracking

[0062] (1) Treatment time: All treatment groups started treatment on the 14th day and continued until the 21st day.

[0063] (2) Data collection:

[0064] Serum IgE detection: On day 21, blood was collected from mice by enucleation of the eyeballs. Serum IgE levels were measured using an IgE ELISA kit (manufacturer's instructions) and the calculation unit was ng / mL.

[0065] Detection of skin IL-4 levels: On day 21, 1 cm of skin was cut from the mouse ears. 2 Skin samples were collected from the laboratory. The IL-4 ELISA kit was used to detect the concentration of IL-4 in skin tissues, with the unit being pg / mg.

[0066] STAT6 phosphorylation level detection: On day 21, mouse ear skin samples were collected and quickly frozen. Total protein was extracted using RIPA buffer, quantified and separated by SDS-PAGE electrophoresis, and the protein was transferred to a PVDF membrane. The membrane was incubated with an anti-phospho-STAT6 antibody (1:1000), followed by incubation with a secondary antibody (1:5000) and chemiluminescence color development. ImageJ software was used for quantitative analysis to calculate the ratio of phosphorylated STAT6 to total STAT6.

[0067] 6. Experimental results are shown in Table 1.

[0068] (1) Serum IgE level: The probiotic group of the present invention (187±21ng / mL) was significantly lower than that of the model group (632±48ng / mL, p<0.01), indicating that the probiotic composition has a strong inhibitory effect on allergic reactions. The commercially available probiotic group (398±35ng / mL) was significantly lower than the model group (p<0.05), but still significantly higher than the present invention group (p<0.05). The probiotic group without anti-IL-4Rα monoclonal antibodies (301±41ng / mL) had a higher serum IgE level than the present invention group, indicating that anti-IL-4Rα monoclonal antibodies have an important enhancing effect on anti-allergic effects.

[0069] (2) Skin IL-4 level: The skin IL-4 level of the probiotic group of the present invention (9.3±1.2pg / mg) was significantly lower than that of the model group (45.2±3.8pg / mg, p<0.01), indicating that the probiotic composition can effectively inhibit skin allergic reactions. The skin IL-4 level of the commercially available probiotic group (28.6±2.1pg / mg) was lower, but still higher than that of the present invention group (p<0.05). The probiotic composition group without anti-IL-4Rα monoclonal antibody (19.4±2.8pg / mg) showed a better inhibitory effect, but the effect was not as good as that of the present invention group (p<0.01).

[0070] (3) STAT6 phosphorylation level: The STAT6 phosphorylation level of the probiotic group of the present invention was significantly lower than that of the model group (0.21±0.03 vs 1.00±0.05, p<0.01), indicating that the composition can effectively inhibit the immune signaling pathway in allergic reactions. The commercially available probiotic group (0.72±0.04) was significantly lower than the model group (p<0.05), but still higher than the present invention group (p<0.05). The probiotic composition group without anti-IL-4Rα monoclonal antibody (0.55±0.06) also showed a certain inhibitory effect on the STAT6 phosphorylation level, but the effect was significantly lower than that of the present invention group (p<0.05).

[0071] Table 1 Anti-allergic effect (atopic dermatitis mouse model, n = 8)

[0072]

[0073] Note: *p<0.05, **p<0.01 vs. model group; Western blot and ELISA detection.

[0074] This experiment shows that the probiotic composition of the present invention exhibits excellent anti-allergic effects in atopic dermatitis mouse model, can effectively reduce serum IgE levels, skin IL-4 concentrations, and inhibit STAT6 phosphorylation levels. The addition of anti-IL-4Rα monoclonal antibodies further enhances the anti-allergic effect of the probiotic composition, indicating that anti-IL-4Rα monoclonal antibodies play an important role in immune regulation.

[0075] Example 4: Preparation and testing of anti-IL-4Rα monoclonal antibodies

[0076] 1. Preparation of anti-IL-4Rα monoclonal antibody

[0077] (1) Protein emulsification and immunization: The IL-4Rα antigen (ab167726) was mixed and emulsified with an equal volume of complete Freund's adjuvant (CFA) to obtain a solution with a concentration of 0.5 mg / mL. This solution was injected subcutaneously for the first immunization. Subsequent booster immunizations were performed every two weeks using a mixture of IL-4Rα antigen and incomplete Freund's adjuvant (IFA) for subcutaneous injection. The last immunization was performed before the end of the immunization cycle. At this time, no adjuvant was used, but 100 μg of antigen was injected through the tail vein.

[0078] (2) Isolation and fusion of spleen cells: 3 to 4 days after the last immunization, the mice were euthanized and the spleen was isolated. The spleen was ground and filtered with sterile PBS to obtain a spleen cell suspension, and the cell concentration was adjusted to 1×10 7 / mL. Splenocytes were mixed with SP2 / 0 or NS0 myeloma cells at a ratio of 5:1, and polyethylene glycol (PEG) was used to mediate cell fusion at 37°C. After the fusion reaction was completed, RPMI-1640 medium was added to terminate the reaction and the fused cells were collected.

[0079] (3) Positive clone screening: The fused cells were inoculated into HAT selective medium and cultured at 37°C and 5% CO2. The medium was replaced after three days. When clone growth was observed, positive clones were selected for expansion and culture.

[0080] (4) Antibody purification: Use ELISA to screen the antibodies in the culture medium, select positive clones and amplify them to obtain sufficient antibodies. Extract antibodies by ascites method or culture supernatant, and purify them by Protein G column. Finally, use 220nm filter membrane to sterilize the antibodies, and store the purified antibodies at -80℃.

[0081] 2. Testing of anti-IL-4Rα monoclonal antibodies

[0082] (1) Antibody concentration determination: The concentration of the purified antibody was determined by the BCA kit, and the result was 5.98 mg / mL. According to the ascites volume and the total amount of antibody after purification, the final ascites expression level of the antibody was 10.65 mg / mL, indicating that the antibody has a high production efficiency and expression yield.

[0083] (2) Purity detection: The purity of the antibody was detected by SDS-PAGE method. The experimental results showed that ( Figure 1 The purified antibody has a high purity of more than 95%, ensuring its suitability for subsequent research and application.

[0084] (3) Amino acid sequence confirmation: The total RNA of the hybridoma cells was extracted, and the amino acid sequences of the heavy chain and light chain variable regions of the antibody were analyzed by PCR amplification and sequencing. The results showed that the amino acid sequences of the heavy chain and light chain variable regions of the antibody were SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0085] 5. Binding activity comparison: The ELISA method was used to detect the binding activity of the antibody of the present invention and the commercial antibody to the IL-4Rα protein. Under the condition that the antibody concentration was 0.5 mg / mL, the antibody was diluted 20,000 times for detection. The results showed that the antibody of the present invention showed a significantly higher OD value, indicating that it was superior to the commercial antibody on the market in terms of binding activity.

[0086] Table 2 Antibody activity test results (OD450nm)

[0087] Group IL-4Rα protein coating IL-4Rα protein coating Monoclonal antibodies of the present invention 2.12 2.18 Anti-IL-4R antibody (ab271041) 1.34 1.37

[0088] It should be noted that the probiotic composition of the present invention has the following characteristics:

[0089] 1. Targeted synergistic effect: Antibodies block IL-4R signals, probiotics upregulate Treg cells, and dually inhibit Th2 inflammation (IFN-γ / IL-4 ratio increased from 0.6 to 3.2). The co-delivery system increases the local concentration of antibodies in the colon by 4.3 times, reducing the risk of systemic exposure (ELISA detection of plasma antibody concentration <0.1μg / mL).

[0090] 2. Stability and safety: After the accelerated test (40℃ / 75%RH, 3 months), the viable bacterial survival rate is ≥85%, and the antibody titer retention rate is ≥90%. The acute toxicity test shows that LD50 is greater than 5000mg / kg, and it has no teratogenicity or mutagenicity.

[0091] 3. Strain-antibody synergistic mechanism:

[0092] (1) Lactobacillus rhamnosus GG: Promotes the differentiation of regulatory T cells (Treg) (the proportion of Foxp3+ cells increased by 2.8 times) and reduces serum IgE levels.

[0093] (2) Lactobacillus acidophilus NCFM: Secretes the antimicrobial peptide Acidocin, inhibits the colonization of Staphylococcus aureus (inhibition rate 89%), and repairs the skin barrier.

[0094] (3) Anti-IL-4Rα monoclonal antibody: Targeted binding to IL-4Rα receptor, blocking IL-4 / IL-13 signal transduction (STAT6 phosphorylation inhibition rate 92%), and reducing Th2 inflammatory factors.

[0095] 4. Delivery system innovation (antibody-probiotic co-immobilized microspheres): Using sodium alginate-chitosan double encapsulation technology, antibodies and bacteria are co-loaded in microspheres (particle size 150±20μm) to achieve colon targeted release (triggered by pH ≥ 7.0), with antibody activity retention rate ≥ 95%.

[0096] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A probiotic composition, characterized in that: The composition is composed of the following components in a certain mass percentage: (1) Lactobacillus rhamnosus GG: 30%; (2) Lactobacillus acidophilus NCFM: 25%; (3) Anti-IL-4Rα monoclonal antibody: 10%; (4) Inulin-β-glucan complex: 15%; (5) Sodium alginate-chitosan microspheres: 20%.

2. The composition according to claim 1, characterized in that The amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-IL-4Rα monoclonal antibody are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

3. A method for preparing the composition according to claim 1, characterized in that: The method comprises the following steps: (1) Bacterial culture: Lactobacillus rhamnosus GG and Lactobacillus acidophilus NCFM were inoculated into MRS medium containing 1% inulin, cultured anaerobically at 37°C for 24 h, and centrifuged at 10,000 × g for 15 min to collect the bacteria; (2) Antibody loading: 10 mg / mL anti-IL-4Rα monoclonal antibody was mixed with bacteria at a mass ratio of 1:5, 2% sodium alginate solution was added, and 2% CaCl2 solution was added dropwise to solidify; (3) Chitosan coating: The microspheres were immersed in a 1% chitosan solution at pH 5.5, magnetically stirred for 30 min, and freeze-dried to obtain the finished product.

4. Use of the anti-IL-4Rα monoclonal antibody in the composition according to claim 2 in the preparation of a probiotic composition.

5. Use of the composition as claimed in claim 1 in preparing a drug for treating skin allergies.

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