A strain of Lactobacillus helveticus HC1253 beneficial to people with pollen allergies and its application

Abstract

The present invention relates to the field of microbial technology, and in particular to a strain of Lactobacillus helveticus HC1253 beneficial to people with pollen allergies and its application. Lactobacillus helveticus ) HC1253 has been deposited in the General Microbiology Center of China National Committee for the Administration of Microbiological Culture Collection on February 20, 2023, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 26588. The Lactobacillus helveticus HC1253 provided by the present invention can be prepared as a medicine for improving pollen allergy, and has a significant improvement effect on single pollen allergy or mixed pollen allergy caused by juniper pollen, birch pollen, poplar pollen, wormwood pollen, humulus pollen or / and ragweed pollen, with strong universality and no side effects.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Lactobacillus helveticus HC1253 beneficial to people allergic to pollen and an application thereof. Background Art

[0002] Allergic diseases, also known as allergic diseases, refer to a type of disease that causes different forms of functional disorders or tissue damage after the body comes into contact with the same allergen again. Among the many allergic diseases, allergic reactions caused by the body's inhalation or contact with allergenic pollens are called pollen allergies. Pollen allergy has seasonal characteristics. Generally speaking, spring allergenic pollens mostly come from woody plants, including Cupressaceae, Pinaceae, Platanaceae, Salicaceae, Moraceae, Ulmaceae and Betulaceae; autumn allergenic pollens mostly come from herbaceous plants, including Artemisia, Humulus, Ambrosia, Chenopodiaceae, etc. The allergenic pollens of people with pollen allergies are often different. Some people with pollen allergies only have allergic reactions to a certain type of pollen, and some people with pollen allergies have allergic reactions to multiple different pollens at the same time. Because of the diversity of allergenic pollens, it is more difficult to treat people with pollen allergies. If pollen allergy is not treated in time, it is likely to develop into allergic rhinitis, allergic conjunctivitis, allergic asthma or allergic dermatitis, which will bring greater pain to people with pollen allergies. In addition, in addition to the typical symptoms of pollen allergy, some people with pollen allergy may also suffer from food allergy. This condition is called pollen-food allergy syndrome, which is not uncommon among people with pollen allergy.

[0003] At present, there are many ways to improve pollen allergy, but each has its own shortcomings in clinical practice. Western medicine treatment is one of the main treatments for pollen allergy. The most commonly used drugs are M receptor blockers, leukotriene antagonists, glucocorticoids, antihistamines, etc. The treatment effect is good, but there are disadvantages such as high price, long treatment cycle, inconvenience of purchasing with prescriptions, large side effects, and easy harm to the patient's body; Chinese medicine treatment has disadvantages such as different prescriptions for different people and poor universality; physical blocking to avoid contact with allergens (wearing masks, glasses and other physical protective equipment) has disadvantages such as affecting normal life and difficult to persist. Summary of the invention

[0004] In view of the problems that the means for improving pollen allergy in the prior art have great side effects, affect the health of patients, have poor universality or / and affect normal life, the present invention provides a strain of Lactobacillus helveticus HC1253 beneficial to people with pollen allergies and an application thereof. Lactobacillus helveticus HC1253 has obvious improvement effects on single pollen allergy or mixed pollen allergy caused by juniper pollen, birch pollen, poplar pollen, wormwood pollen, humulus pollen or / and ragweed pollen, has strong universality and no side effects.

[0005] In a first aspect, the present invention provides a strain of Lactobacillus helveticus (Lactobacillus helveticus ) HC1253 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on February 20, 2023. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.26588.

[0006] In a second aspect, the present invention provides an application of Lactobacillus helveticus HC1253 in the preparation of a medicine for improving pollen allergy, wherein pollen allergy includes single pollen allergy and mixed pollen allergy; the allergenic pollen source of single pollen allergy is Juniperus, Betula, Populus, Artemisia, Humulus or Ambrosia; the allergenic pollen source of mixed pollen allergy is selected from at least two of Juniperus, Betula, Populus, Artemisia, Humulus and Ambrosia.

[0007] Furthermore, the allergenic pollen for single pollen allergy is juniper pollen, birch pollen, poplar pollen, wormwood pollen, humulum pollen or ragweed pollen; the allergenic pollen for mixed pollen allergy is selected from at least two of juniper pollen, birch pollen, poplar pollen, wormwood pollen, humulum pollen and ragweed pollen.

[0008] Furthermore, improving pollen allergy includes reducing the allergenicity of pollen proteins in allergenic pollen.

[0009] Furthermore, improving pollen allergy includes improving respiratory allergy symptoms and skin allergy symptoms.

[0010] Furthermore, respiratory allergy symptoms include nasal allergy symptoms, eye allergy symptoms, and trachea and bronchial allergy symptoms; nasal allergy symptoms include sneezing, nasal congestion and runny nose; eye allergy symptoms include conjunctival congestion, conjunctival edema, eyelid edema and increased secretions; trachea and bronchial allergy symptoms include shortness of breath, chest tightness, breath holding and difficulty breathing; skin allergy symptoms include erythema, itching, edema and wheals.

[0011] Furthermore, improving pollen allergy includes reducing the level of allergic factors during allergic stimulation and increasing the level of anti-allergic factors during allergic stimulation; allergic factors include IL-4, histamine and protein-specific IgE of allergenic pollen; anti-allergic factors include gamma interferon.

[0012] Furthermore, improving pollen allergy includes alleviating pollen allergy symptoms by regulating the intestinal flora structure; regulating the intestinal flora structure includes increasing the number of Lactobacillus and Bifidobacterium in the intestine and reducing the number of Escherichia coli in the intestine.

[0013] Furthermore, the drug for improving pollen allergy includes Lactobacillus helveticus HC1253 bacterial agent; the Lactobacillus helveticus HC1253 bacterial agent has a live cell count of 2.0×10 10 CFU / g freeze-dried single bacterial powder.

[0014] The beneficial effects of the present invention are:

[0015] The invention provides a strain of Lactobacillus helveticus HC1253 which is beneficial to pollen allergy crowd. The strain can reduce the content of pollen-specific IgE in pollen allergy crowd and reduce the allergy level of the crowd, thereby reducing the probability of pollen allergy in the allergic crowd, blocking the disease process of pollen allergy and achieving a certain desensitization effect. Lactobacillus helveticus HC1253 can reduce the content of pollen-specific IgE in the crowd without stimulating allergy, indicating that the strain has a certain application potential in the crowd with severe pollen allergy or the crowd with severe allergy symptoms, and expands the application scope of the strain in the crowd with pollen allergy. Moreover, when the allergic crowd has a pollen allergy reaction, the Lactobacillus helveticus HC1253 entering the crowd can balance the Th1 / Th2 cell immune response by reducing the content of allergic factors such as IL-4 and histamine and increasing the content of anti-allergic factors such as gamma interferon, thereby reducing the reaction intensity of pollen allergy symptoms and shortening their duration, and maximally alleviating the suffering of the crowd with pollen allergy. In addition, Lactobacillus helveticus HC1253 also has the function of regulating the balance of lactobacillus, bifidobacteria and Escherichia coli in the intestines, and in this way helps relieve pollen allergy symptoms. During the experiment, neither BN rats nor volunteers experienced adverse reactions other than pollen allergy symptoms, which shows that Lactobacillus helveticus HC1253 has no toxic side effects, and it has been verified that the bacteria has a strong tolerance to gastric acid and bile salts, indicating that Lactobacillus helveticus HC1253 has great market application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 This is the verification result of the allergenicity of pollen protein solution.

[0018] Figure 2 The effect of different strains on the allergenicity of juniper pollen protein.

[0019] Figure 3 The effect of different strain treatments on the allergenicity of birch pollen protein.

[0020] Figure 4 The effect of different strains on the allergenicity of Populus tomentosa pollen protein.

[0021] Figure 5 The effect of different strains on the allergenicity of Artemisia ordosica pollen protein.

[0022] Figure 6 The effect of different strain treatments on the allergenicity of Humulus japonicus pollen protein.

[0023] Figure 7 The effect of different strain treatments on the allergenicity of ragweed pollen protein.

[0024] Figure 8 is the concentration of Sabina pollen-specific IgE in the serum of BN rats.

[0025] Fig. 9 are the scoring results of allergic symptoms of BN rats in different treatment groups.

[0026] Fig.10 These are the results of detection of IgE concentrations of juniper pollen protein in the serum of rats in different treatment groups.

[0027] Fig.11 These are the results of IL-4 content detection in serum of BN rats in different treatment groups.

[0028] Fig.12 These are the results of the detection of histamine levels in the serum of BN rats in different treatment groups.

[0029] Fig.13 These are the results of detection of interferon-γ content in serum of BN rats in different treatment groups.

[0030] Fig.14 It is the result of the changes in the number of Lactobacillus flora in the intestines of BN rats in different treatment groups.

[0031] Fig.15 It is the result of the changes in the number of Bifidobacterium flora in the intestines of BN rats in different treatment groups.

[0032] Fig.16 It is the result of the changes in the number of Escherichia coli flora in the intestines of BN rats in different treatment groups. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only 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 should fall within the scope of protection of the present invention.

[0034] Example 1 Bacterial strain isolation

[0035] (1) Source: dry cheese, collected in Xining City, Qinghai Province, China in July 2022.

[0036] (2) Isolation of strains:

[0037] Weigh 1g of dry cheese sample and put it into a triangular flask containing 99mL of sterile saline. Stir with a magnetic stirrer to fully dissolve and mix the sample. Then dilute the solution 10 times with sterile saline, take the appropriate dilution gradient and apply it on the MRS solid culture medium plate. After culturing at 37℃ for 48h, select single colonies with different morphology, size and color on the MRS solid culture medium plate for purification. The purification is carried out by three-zone streaking of MRS solid culture medium. After streaking, it is placed at 37℃ for 48h. After each streaking, observe whether the morphology, size and color of the single colonies on the culture medium are consistent. If not, the single colonies with different characteristics need to be streaked separately until the characteristics of the colonies on the plate are consistent. When the characteristics of the colonies on the culture medium are consistent, streak purification is carried out twice, and then the strains are preserved and numbered according to the common lactic acid bacteria preservation method, and one of them is identified.

[0038] Example 2 Identification of bacterial species

[0039] The identification sequence is as follows:

[0040]

[0041] The strain was identified as Lactobacillus ( Lactobacillus ), presumably Lactobacillus helveticus ( Lactobacillus helveticus ), the strain was named Lactobacillus helveticus HC1253, and sent to the General Microbiological Center of China Microbiological Culture Collection Administration for preservation. The preservation information is as follows:

[0042] This Lactobacillus helveticus ( Lactobacillus helveticus ) HC1253, deposited on February 20, 2023 in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.26588.

[0043] Experimental Example 1: Obtaining Pollen Protein

[0044] 1. Extraction of Pollen Protein

[0045] Research has found that most substances that cause pollen allergies are proteins. Therefore, in order to study pollen allergies, this study first extracted proteins from the pollens of selected representative plants. The extraction process, method and results are as follows:

[0046] 1. Pollen collection and processing

[0047] The male cones, male inflorescences or stamens of juniper, birch, poplar, wormwood, humulus and ragweed were collected in the flowering season, respectively, and crushed after vacuum freeze drying, and then passed through a 200-mesh nylon sieve to remove impurities and obtain their respective coarse pollen.

[0048] Add 75% alcohol to the crude pollen obtained above, stir for 10-15 minutes, centrifuge at 2000 rpm for 3 minutes, collect the precipitate, then add sterile water to the precipitate for washing, stir for 2-3 minutes, collect the precipitate by centrifugation, repeat the washing 2-3 times, collect the precipitate by centrifugation, vacuum freeze-dry under sterile conditions, weigh and mark them separately, and put them in a 4°C refrigerator for use.

[0049] 2. Extraction of pollen protein

[0050] The proteins in the pollens of the above six plants were extracted respectively, and the extraction process is as follows:

[0051] Weigh a certain mass of pollen and transfer it to a sterile mortar, then add liquid nitrogen and grind it. After grinding for 10 minutes, transfer all the materials in the mortar to a beaker, and add pre-cooled tris / HCl buffer solution (20mmol / L tris / HCl, 1mmol / L EDTA, pH8.0) to the beaker at a mass-to-volume ratio of 1:25 (W / V). After stirring at 4°C for 1 hour, the resulting solution was centrifuged at 10000rpm at 4°C for 5 minutes, and the supernatant was collected to obtain a crude extract containing the corresponding pollen protein.

[0052] At 4°C, add ammonium sulfate to the crude extract of pollen protein, stirring slowly while adding, and make the final concentration of ammonium sulfate in the extract reach 70% within 5-10 minutes, then continue to stir slowly at 4°C, avoid bubbling of the solution during stirring, after 20 minutes, centrifuge at 3000rpm for 10 minutes at 4°C, collect the precipitate, and then add pre-cooled sterile 1× PBS buffer according to the weight of the weighed pollen at a mass volume ratio of 2:1 (W / V), centrifuge at 10000rpm for 15 minutes at 4°C, collect the supernatant, and filter and sterilize with a 0.22µm filter membrane to obtain the pollen protein solution of the corresponding plant. After determining the protein content in each pollen protein solution with a BCA protein concentration determination kit, place it at -20°C for use.

[0053] 3. Experimental results

[0054] After testing, the protein content in the six plant pollen protein extracts used in the experiment is shown in Table 1:

[0055] Table 1 Protein content in pollen protein extracts of six plants

[0056]

[0057] Experimental Example 2 Verification of the allergenicity of pollen protein

[0058] The ELISA method was used to verify the allergenicity of the pollen protein solutions of the six plants extracted above. The method steps and results are as follows:

[0059] 1. Collect serum

[0060] 10 mL of venous blood was drawn from patients who were diagnosed as allergic to juniper pollen, birch pollen, poplar pollen, wormwood pollen, humulus pollen, and ragweed pollen by the hospital. After being placed at room temperature for 4 hours, the blood was centrifuged at 3000 rpm for 10 minutes at 4°C to separate the serum, which was numbered and stored at -20°C for later use.

[0061] 2. Preparation of ELISA solution

[0062] (1) Sodium bicarbonate coating solution: Weigh 0.795 g of sodium carbonate and 1.465 g of sodium bicarbonate, dissolve in 500 mL of deionized water, and store at 4°C until use.

[0063] (2) Pollen protein test solution: The protein concentrations of the pollen protein solutions of the six plants obtained in Experimental Example 1 were adjusted to 5 µg / mL using the sodium bicarbonate coating solution, and the solutions were used as pollen protein test solutions and stored at 4°C for later use.

[0064] (3) Sterile 1× PBS buffer: Weigh 8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of disodium hydrogen phosphate, and 0.24 g of potassium dihydrogen phosphate, and dissolve in 800 mL of distilled water. Adjust the pH of the solution to 7.4 with hydrochloric acid, then add distilled water to make up to 1 L. Sterilize at 121°C for 20 min and store at 4°C until use.

[0065] (4) 1× PBST washing solution: Add 0.5 mL of Tween-20 to 1 L of 1× PBS buffer and mix thoroughly to obtain 1× PBST washing solution. Store at 4°C for later use.

[0066] (5) Blocking solution: Weigh 1 g of bovine serum albumin (BSA) and dissolve it in 100 mL of 1× PBST solution. Store at 4°C until use.

[0067] (6) Stop solution: Measure 50 mL of concentrated sulfuric acid and add it to a beaker containing 500 mL of deionized water. Take care to drain slowly along the glass rod and stir continuously. After cooling to room temperature, store for later use.

[0068] 3. ELISA method to verify the allergenicity of each pollen protein

[0069] (1) Coating: 100 µL of the above pollen protein test solution was added to a 96-well ELISA plate, sealed with plastic film, and incubated overnight at 4°C. The protein content in the commercially available skin prick solution of the above different plant pollens was adjusted to 5 µg / mL and then subjected to the same embedding operation and used as the positive control group of this experiment; blank sodium bicarbonate coating solution was used as the blank control group of this experiment.

[0070] (2) Washing: The next day, remove the ELISA plate, discard the coating solution of each treatment group and pat it dry on absorbent paper. Wash the plate three times with 1× PBST solution, each time for about 1 min. Discard the last wash solution and pat it dry on absorbent paper.

[0071] (3) Blocking: Discard the 1× PBST washing solution and pat dry. Add 200 µL of blocking solution to each well and block at 37°C for 1 h.

[0072] (4) Washing: Discard the blocking solution and pat dry on absorbent paper. Wash the plate three times with 1× PBST, each time for about 1 min. Discard the last wash solution and pat dry on absorbent paper.

[0073] (5) Primary antibody incubation: The primary antibody used in this experiment was the serum of pollen allergy patients corresponding to the pollen protein to be tested. After diluting it to 1000 times with blocking solution, 100 μL per well was added to the corresponding wells of the 96-well ELISA plate and incubated at 37°C for 2 hours. Three replicate wells were set for each treatment group. The blank control group and the positive control group also used the serum of the corresponding pollen allergy patients as the primary antibody, and three replicate wells were set for each treatment group.

[0074] (6) Washing: Discard the primary antibody solution and pat dry on absorbent paper. Wash the plate three times with 1× PBST, each time for about 1 min. Discard the last wash solution and pat dry on absorbent paper.

[0075] (7) Secondary antibody incubation: The secondary antibody used in this experiment was mouse anti-human IgE antibody (Mouse Anti-human IgE-HRP) labeled with horseradish peroxidase. It was diluted to 2500 times with blocking solution and 100 µL was added to each well of a 96-well plate and incubated at 37°C for 1 h.

[0076] (8) Washing: Discard the secondary antibody solution and pat dry on absorbent paper. Wash the plate three times with 1× PBST, each time for about 1 min. Discard the last wash solution and pat dry on absorbent paper.

[0077] (9) Color development and termination: Add 150 µL of TMB color development solution to each well, incubate at room temperature in the dark, and observe the color development reaction. After sufficient color development, add 50 µL of stop solution to each well to terminate the reaction.

[0078] (10) Place the 96-well ELISA plate into an ELISA reader and read and record the OD value of each treatment group at 450 nm.

[0079] 4. Experimental results and conclusions

[0080] Depend on Figure 1 It can be seen that the OD450 in the blank control group is close to 0, indicating that there is no interference from foreign proteins, false positives, etc. in the reaction system of this experiment, and the experimental results are reliable. In addition, the serum of patients with pollen allergies contains specific immunoglobulin E (sIgE), which can specifically recognize and bind to the pollen allergens corresponding to the patients, and the main component of pollen skin prick solution is pollen allergen protein.

[0081] Depend on Figure 1It can be seen that the OD values ​​of the positive control group and the pollen protein test solution group are comparable, indicating that the sera of different pollen allergic subjects used in the experiment can specifically bind to the corresponding skin prick solution, and can also specifically bind to the pollen protein extracted in Experimental Example 1, thereby indicating that the pollen protein solution extracted in Experimental Example 1 contains components similar to the pollen allergen protein in the skin prick solution, that is, this experiment proves that the pollen protein extracted in Experimental Example 1 has certain allergenicity.

[0082] Experimental Example 3 Screening of specific strains

[0083] 1. Preparation of bacterial suspension

[0084] The strains of different genera preserved in this unit (among which Lactobacillus helveticus was Lactobacillus helveticus HC1253) were inoculated into MRS liquid culture medium respectively, and cultured at 37°C for 48 h (Note: Bifidobacterium needs to be cultured under anaerobic conditions). The culture fluid of each strain was centrifuged at 5000 rpm for 5 min, the bacteria were collected, washed twice with sterile water, and the OD600 value was adjusted to about 50 with sterile 1×PBS buffer, as the bacterial suspension of the corresponding strain, which was preserved at 4°C for later use.

[0085] 2. Digestion of bacterial suspension and pollen protein

[0086] The protein concentrations of the six plant pollen protein solutions prepared in Experimental Example 1 were adjusted to 1.0 mg / mL using sterile 1×PBS buffer, and then 1 mL was taken and placed in a 5 mL centrifuge tube. 1 mL of the bacterial suspension prepared above was added and incubated at 37°C for 0 h, 12 h, and 24 h, respectively. Sterile 1×PBS buffer was used as a blank control during the experiment.

[0087] 3. Evaluation of the allergenicity of different pollen proteins after treatment

[0088] The ELISA method was used to evaluate the allergenicity of the pollen proteins after the co-digestion treatment. The ELISA experimental process was the same as the above-mentioned Experimental Example 2. The difference was that before the start of this ELISA experiment, the solutions in the above-mentioned groups of treatments were diluted 100 times with sterile 1×PBS buffer and filtered with a 0.22µm filter membrane for sterilization before the coating operation.

[0089] 4. Experimental results and conclusions

[0090] The sensitization effects of different pollen proteins treated with different bacterial suspensions are shown in Figure 2~Figure 7 .like Figure 2 As shown in the figure, the allergenicity of juniper pollen protein can be significantly reduced after being digested by Lactobacillus helveticus HC1253, Lactobacillus rhamnosus and Lactobacillus crispatus. Figure 3As shown in the figure, birch pollen protein can significantly reduce its allergenicity after being digested by Lactobacillus helveticus HC1253, Lactobacillus salivarius and Lactobacillus casei; Figure 4 As shown in the figure, the allergenicity of Populus tomentosa pollen protein can be significantly reduced after being digested with Lactobacillus helveticus HC1253, Lactobacillus johnsonii and Lactobacillus casei; Figure 5 As shown in the figure, the allergenicity of Artemisia ordosica pollen protein can be significantly reduced after being digested with Lactobacillus helveticus HC1253, Lactobacillus reuteri and Lactobacillus crispatus. Figure 6 As shown in the figure, the allergenicity of Humulus pollen protein can be significantly reduced after being digested with Lactobacillus helveticus HC1253, Lactobacillus casei and Lactobacillus salivarius; Figure 7 As shown, the allergenicity of ragweed pollen protein can be significantly reduced after being digested by Lactobacillus helveticus HC1253, Bifidobacterium lactis and Streptococcus thermophilus.

[0091] In summary, the same pollen protein has different effects on its allergenicity after being digested by different bacterial suspensions; at the same time, the same bacterial suspension has certain differences in the effect of treating different plant pollen proteins. Among them, compared with other strains, Lactobacillus helveticus HC1253 has a more significant effect on the allergenicity of the above six plant pollen proteins. For pollen allergies to juniper pollen, birch pollen, poplar pollen, wormwood pollen, humulus pollen or ragweed pollen, or for mixed pollen allergies to at least two of the pollens of juniper pollen, birch pollen, poplar pollen, wormwood pollen, humulus pollen and ragweed pollen, Lactobacillus helveticus HC1253 has shown a significant effect of reducing its allergenicity.

[0092] In order to further verify the actual effect of Lactobacillus helveticus HC1253 in pollen allergy, the present invention conducted the following experiments.

[0093] Experimental Example 4 Establishment of pollen allergy animal model

[0094] The experimental process is described using juniper pollen as an example. The other five types of pollen were tested using the same experimental method. The experimental process and results are as follows:

[0095] 1. Sensitization of BN rats

[0096] First, the juniper pollen protein solution obtained in the above Experimental Example 1 was prepared into a 2 mg / mL dilution with 1×PBS buffer, filtered and sterilized with a 0.22 µm filter membrane, and stored at 4°C for later use.

[0097] After one week of adaptive training, the purchased 5-month-old BN rats were randomly divided into a sensitized group and a blank control group. 100 µL of a 2 mg / mL sterile juniper pollen protein solution was subcutaneously injected between the two forearms behind the neck of the BN rats in the sensitized group, and the BN rats were observed for 30 minutes after the injection. If the BN rats showed reactions such as excitement, tremor or shock, the concentration of the juniper pollen protein solution used for sensitization should be appropriately reduced, and the sensitization operation should be repeated. The BN rats in the blank control group were subcutaneously injected with an equal amount of sterile 1× PBS buffer. After the first injection, the BN rats were injected once every 5 days using the same method, for a total of 4 injections.

[0098] 2. Allergic stimulation of BN rats

[0099] First, the juniper pollen protein solution obtained in the above Experimental Example 1 was prepared into a 4 mg / mL dilution with 1×PBS buffer, filtered through a 0.22 µm filter membrane for sterilization, and stored at 4°C for later use.

[0100] After the last subcutaneous injection of BN rats in the blank control group and the sensitized group, 5 μL of 4 mg / mL juniper pollen protein solution was used to stimulate the BN rats’ eyes on the next day, and 25 μL of the same solution was used to stimulate the rats’ noses at 24-hour intervals for a total of three times. The blank control group and the sensitized group were stimulated in the same way.

[0101] Three BN rats were randomly selected from the sensitized group and the blank control group 12 h after the last subcutaneous injection, 12 h after the first stimulation, 12 h after the second stimulation, and 12 h after the third stimulation, respectively. They were killed by dislocating the neck and then inverted with their heads facing downward. After removing one eyeball, the blood of the BN rats in each group was collected in 5 mL centrifuge tubes. After the blood samples were placed at room temperature for 4 h, they were centrifuged at 3000 rpm for 10 min at 4°C. The upper serum was carefully aspirated and transferred to new 5 mL centrifuge tubes and stored at -80°C for later use.

[0102] 3. Measurement of the content of sabinosus pollen-specific IgE in serum of BN rats

[0103] In this experiment, the ELISA method was used to detect the content of specific IgE (sIgE) in the serum of each group of BN rats, and this was used as an indicator to determine whether the juniper pollen allergy model of BN rats had been successfully established.

[0104] The operation method of ELISA is the same as that of Experiment 2. The differences are: (1) the primary antibody used in this ELISA experiment is the serum of BN rats in different treatment groups; (2) the secondary antibody used in this ELISA experiment is mouse anti-rat IgE antibody labeled with horseradish peroxidase (Mouse anti-rat IgE-HRP).

[0105] 4. Experimental results and conclusions

[0106] Depend on Figure 8 It can be seen that the OD values ​​of each sampling point in the sensitized group were significantly higher than those in the blank control group, and the OD value measured after the second stimulation reached a higher level, and was not much different from the OD value measured after the third stimulation. According to the experimental principle of the ELISA method, the higher the OD value, the more specific IgE in the serum of BN rats that can bind to juniper pollen protein, which also proves that the juniper pollen allergy model of BN rats has been successfully established.

[0107] During the modeling process, no severe allergic reaction occurred in any group of rats, and there was no shock or death. In addition, judging from the OD values ​​of each sampling point in the blank control group, they did not change much during the entire experiment, indicating that the successful establishment of the BN rat allergy model was caused by juniper pollen protein and had nothing to do with the PBS buffer solution and operation method used. The solution concentration and the number of sensitization and stimulation times used in the modeling process were reasonable. This method can be used to establish the BN rat juniper pollen allergy model.

[0108] At the same time, this method can be used to establish pollen allergy models of birch pollen protein, poplar pollen protein, Artemisia ordosica pollen protein, Humulus japonicus pollen protein and ragweed pollen protein in BN rats in turn (i.e., the juniper pollen protein solution used in the experiment is replaced by the corresponding pollen protein solution), and other pollen allergy models have been successfully established.

[0109] Experimental Example 5 Verification of the beneficial effects of Lactobacillus helveticus HC1253 on pollen allergy model

[0110] It can be seen from the above Experimental Example 3 that the Lactobacillus helveticus HC1253 used in the experiment has a good effect in reducing the allergenicity of plant pollen proteins, but whether it is beneficial for pollen allergies caused by plant pollen in vivo has not yet been verified.

[0111] Therefore, this experiment explored the role of Lactobacillus helveticus HC1253 suspension in plant pollen allergy by gavage into BN rats. The experimental process was described using juniper pollen as an example, and the other five types of pollen were tested using the same experimental method. The experimental process and results are as follows:

[0112] 1. Preparation of Lactobacillus helveticus HC1253 bacterial suspension

[0113] A single colony of Lactobacillus helveticus HC1253 was inoculated into MRS liquid medium and cultured at 37°C for 48 h. The cells were collected by centrifugation at 5000 rpm for 5 min, washed twice with sterile water, and resuspended with an appropriate amount of sterile 1× PBS buffer to a viable count of 1.0×10 10 CFU / mL, stored at 4℃ for future use. The bacterial count was measured according to the method in the national standard "GB4789.35-2016 National Food Safety Standard Food Microbiology Inspection Lactic Acid Bacteria Inspection".

[0114] 2. Grouping and treatment of BN rats

[0115] It can be seen from Experimental Example 4 that although the sterile 1×PBS buffer and operating method used in the experiment will not cause an increase in the concentration of juniper pollen-specific IgE in the serum of BN rats, it is still unknown whether the PBS treatment will cause other inflammatory reactions or affect the experimental indicators to be measured. Therefore, this experiment not only set up a blank control group treated with PBS buffer, but also added a natural group that was naturally raised and not treated in any way.

[0116] After one week of adaptive culture, the normally cultured 5-month-old BN rats were randomly divided into a natural group, a blank control group, a positive control group, a bacterial agent group A, and a bacterial agent group B, with 21 rats in each group. The BN rats in the positive control group, the bacterial agent group A, and the bacterial agent group B were sensitized and stimulated according to the method in Experimental Example 4 to establish a BN rat juniper pollen allergy model. The establishment method of the blank control group in this experiment is the same as that in Experimental Example 4. In this experiment, BN rats were gavage-treated before sensitization and after allergy stimulation, and the treatment methods are shown in Table 2:

[0117] Table 2 Oral gavage treatments of BN rats in different groups

[0118]

[0119] in:

[0120] (1) Pre-sensitization treatment: Before the experiment, except for the natural group, the blank control group, positive control group, bacterial agent A group, and bacterial agent B group were gavaged every day for a total of 14 times. As shown in Table 2, the blank control group and the positive control group were gavaged with 0.2 mL of sterile 1× PBS buffer, while the bacterial agent A group and the bacterial agent B group were gavaged with 0.2 mL of Lactobacillus helveticus HC1253 suspension (1.0×10 10 CFU / mL), the natural group was not treated.

[0121] (2) Sensitization treatment: After the pre-sensitization treatment was completed, the positive control group, bacterial agent group A, and bacterial agent group B were sensitized according to the method in Experimental Example 4; at the same time, the blank control group was treated with an equal amount of sterile 1× PBS buffer, and the natural group was not treated. 12 hours after the sensitization was completed, three BN rats were randomly selected from each group, and after being killed by cervical dislocation, the serum of each group of rats was collected according to the method in Experimental Example 4, and the relevant indicators were tested.

[0122] (3) Allergy stimulation treatment: Except for the natural group, the blank control group, positive control group, bacterial agent A group, and bacterial agent B group were stimulated with allergy according to the method in Experimental Example 4. The behavioral responses of BN rats in each treatment group were observed and recorded 12 hours after the first stimulation and 12 hours after the third stimulation. Three BN rats were randomly selected from each of the five treatment groups, and the rats were killed by cervical dislocation. The serum of the rats in each group was collected according to the method in Experimental Example 4, and the relevant indicators were tested.

[0123] (4) Post-challenge treatment: As shown in Table 2, 12 h after the third challenge, the BN rats in the bacterial agent B group were gavaged with 0.2 mL of the 1.0 × 10 10 CFU / mL bacterial suspension, and gavage once every 48 hours in the same way; the blank control group, positive control group, and bacterial agent A group were gavaged with an equal amount of sterile 1×PBS buffer at the same time point; the natural group was not treated. After gavage 5 times, 24 hours after the last gavage, three BN rats were randomly selected from each group, and after being killed by cervical dislocation, the serum of each group of rats was collected according to the method in the above experimental example 4, and the relevant indicators were detected.

[0124] (III) Testing methods and results of each indicator

[0125] 1. Behavioral observation of BN rats in different treatment groups

[0126] After the last sensitization, the first challenge, the third challenge and the last gavage, the behavioral responses of the remaining BN rats in each treatment group within 30 minutes, such as scratching the nose, runny nose, sneezing, tearing, eyelid swelling, conjunctival congestion, etc., were recorded and observed, and scored according to the standards in Tables 3 and 4. The scores of the 7 indicators in Tables 3 and 4 were added together and the average score of each rat in each treatment group was calculated as the symptom score of the BN rats in each treatment group in this experiment. The scoring results of each treatment group are shown in the figure below. Fig. 9 shown.

[0127] Table 3 Scoring criteria for nasal allergy symptoms

[0128]

[0129] Table 4 Scoring criteria for ocular allergy symptoms

[0130]

[0131] Depend on Fig. 9 It can be seen that the scores of BN rats in the blank control group are not much different from those in the natural group, indicating that the allergic symptoms of BN rats during the experiment have nothing to do with the PBS buffer and the operation method used. In addition, the allergic symptoms of BN rats in the positive control group, the bacterial agent group A and the bacterial agent group B became more and more serious as the sensitization and stimulation process progressed, and when the stimulation was stopped, the allergic symptoms all improved to a certain extent. The difference is that the scores of the bacterial agent group A and the bacterial agent group B after the third stimulation were lower than those of the positive control group, which shows that the treatment of BN rats with Lactobacillus helveticus HC1253 before the allergy occurs can alleviate the symptoms of juniper pollen allergy to a certain extent. On the other hand, by comparing the data of the bacterial agent group A and the bacterial agent group B, it can be seen that after the three stimulations are completed, continuing to treat the allergic BN rats with Lactobacillus helveticus HC1253 can further accelerate the disappearance of allergic symptoms, and after 5 intragastric treatments, the symptom score can be close to the normal level. This preliminarily shows that Lactobacillus helveticus HC1253 has a certain effect in alleviating the symptoms of juniper pollen allergy.

[0132] 2. Measurement of various indicators in serum of BN rats in different treatment groups

[0133] (1) Measurement of sIgE concentration in serum of BN rats in different treatment groups

[0134] In this experiment, the concentration of sabinosus pollen protein-specific immunoglobulin E (sIgE) in rat serum was measured by ELISA. The experimental method was referred to Experimental Example 4. The test results are shown in Fig.10 shown.

[0135] (2) Measurement of IL-4 levels in serum of BN rats in different treatment groups

[0136] The rat interleukin 4 (IL-4) ELISA kit was used to measure the IL-4 content in the serum of BN rats in different treatment groups. The experimental process was carried out according to the instructions of the kit. The test results are shown in Fig.11 shown.

[0137] (3) Measurement of histamine content in serum of BN rats in different treatment groups

[0138] The histamine (HIS) ELISA kit was used to measure the histamine (Histamine) content in the serum of BN rats in different treatment groups. The experimental process was carried out according to the instructions of the kit. The test results are shown in Fig.12 shown.

[0139] (4) Measurement of IFN-γ content in serum of BN rats in different treatment groups

[0140] The IFN-γ ELISA kit was used to measure the IFN-γ content in the serum of BN rats in different treatment groups. The experimental process was carried out according to the instructions of the kit. The test results are shown in Fig.13 shown.

[0141] Depend on Fig.10 , Fig.11 , Fig.12 It can be seen that with the progress of sensitization and stimulation, the levels of sabinoparasiticus pollen protein-specific IgE (sIgE), IL-4 and histamine in the serum of BN rats in the positive control group, inoculant group A and inoculant group B were significantly increased compared with the blank control group and the natural group. When the stimulation was stopped, the above three indicators all declined to a certain extent.

[0142] Combined with the principle of pollen allergy and compared Fig. 9 From the data, we can see that the above three indicators have the same changing trend with allergic symptoms. Therefore, it can be considered that there is a positive correlation between the levels of sabinopril pollen protein-specific IgE (sIgE), IL-4 and histamine in the serum of BN rats and the sabinopril pollen allergic symptoms of rats. When the levels of sabinopril pollen protein-specific IgE (sIgE), IL-4 and histamine in the serum of rats are higher, the sabinopril pollen allergic symptoms of rats will be more severe. Conversely, the reduction of sIgE, IL-4 and histamine levels in the serum of rats will also alleviate the allergic symptoms of BN rats. Fig.10 , Fig.11 , Fig.12 It can be seen that compared with the positive control group, the data of the bacterial agent group A and the bacterial agent group B can show that the intragastric administration of Lactobacillus helveticus HC1253 to BN rats before sensitization can reduce the synthesis or secretion of sIgE, IL-4 and histamine of juniper pollen protein when allergy occurs, thereby alleviating allergic symptoms. In addition, by comparing the data in the bacterial agent group A and the bacterial agent group B, it can be shown that the bacterial agent group B, which is continuously treated with Lactobacillus helveticus HC1253 intragastric administration after three allergic stimulations, can further reduce the content of sIgE, IL-4 and histamine of juniper pollen protein, and after 5 intragastric administrations, the content can be close to the normal level.

[0143] On the other hand, interferon-γ is an anti-allergic factor, and its content is negatively correlated with the intensity of allergic symptoms. Fig.13It can be seen that compared with the positive control group, the BN rats in the bacterial agent group A and the bacterial agent group B treated with Lactobacillus helveticus HC1253 before sensitization can maintain a higher level of interferon-γ when allergy occurs. At the same time, by comparing the data in the bacterial agent group A and the bacterial agent group B, it can be seen that the bacterial agent group B, which was continuously given oral gavage treatment with the bacterial agent after three allergic stimulations, can further increase the content of interferon-γ, and after 5 oral gavage treatments, its content can be close to the normal level.

[0144] This shows that BN rats treated with Lactobacillus helveticus HC1253 can not only alleviate their allergic symptoms when allergies occur, giving them a certain anti-allergic ability, but also continue to use Lactobacillus helveticus HC1253 to treat allergic BN rats after the allergy occurs, which can greatly accelerate the speed at which various indicators return to normal levels, thereby greatly accelerating the disappearance of allergic symptoms.

[0145] Based on the above indicators, it can be proved that after intragastric administration of Lactobacillus helveticus HC1253, not only can the level of allergic factors in BN rats during allergic stimulation be effectively reduced, but also the level of anti-allergic factors in the serum of BN rats can be increased. Moreover, after continuous treatment with this bacterium, the speed at which allergic factors return to normal levels can be accelerated, and the content of anti-allergic factors can be increased. After 5 intragastric administrations, each parameter can be close to or restored to normal levels. This is consistent with the behavioral response of BN rats in this experiment, and explains the behavioral response of BN rats to allergies during the experiment from the perspective of the occurrence and disappearance of allergic reactions.

[0146] In addition, similar results were obtained when experiments were conducted on BN rats with birch pollen allergy model, poplar pollen allergy model, Artemisia sphaerocephala pollen allergy model, Humulus japonicus pollen allergy model and ragweed pollen allergy model, indicating that Lactobacillus helveticus HC1253 has an improvement effect on pollen allergies caused by juniper pollen, birch pollen, poplar pollen, Artemisia sphaerocephala pollen, Humulus japonicus pollen and ragweed pollen.

[0147] 3. Changes in the number of intestinal flora in BN rats in different treatment groups

[0148] Studies in recent years have shown that the occurrence of allergic diseases is related to intestinal flora disorders. Intestinal flora disorders will increase the probability of allergic diseases or aggravate the patient's allergic symptoms. The main manifestation is that the number of aerobic bacteria in the intestines of allergic patients, especially Escherichia coli and Staphylococcus aureus, is large, while the number of Lactobacillus and Bifidobacterium is small.

[0149] Therefore, this experiment selected blank control group, positive control group, bacterial agent group A and bacterial agent group B as research objects, and collected the rat feces of these four groups of BN rats within 12 hours after the third stimulation and within 12 hours after the last gavage, respectively. After weighing, they were placed in a conical flask with glass beads and sterile saline, and shaken on a shaker at 4°C for 30 minutes. The supernatant was taken for gradient dilution, and the number of lactobacilli, bifidobacteria and Escherichia coli was detected. The measurement of lactobacilli and bifidobacteria was carried out in accordance with "GB4789.35-2016 National Food Safety Standard Food Microbiology Inspection Lactic Acid Bacteria Inspection", and the counting method of Escherichia coli was carried out in accordance with "GB4789.3-2016 National Food Safety Standard Food Microbiology Inspection Coliform Count" "Method 2 Coliform Plate Count Method". The measurement results are as follows Figure 14-16 shown.

[0150] Depend on Figure 14-16 It can be seen that compared with the blank control group, the number of Lactobacillus and Bifidobacterium in the intestines of the positive control group, bacterial agent group A and bacterial agent group B decreased, while the number of Escherichia coli increased significantly after three allergic stimulations, which shows that the occurrence of allergic diseases is indeed related to changes in intestinal flora.

[0151] In addition, compared with the positive control group, the number of Escherichia coli in the bacterial agent group A and the bacterial agent group B was relatively small, while the number of lactobacillus and bifidobacterium was relatively large, which indicated that after oral administration of Lactobacillus helveticus HC1253 to BN rats, the destructive effect of the allergic reaction to juniper pollen on the intestinal flora structure could be weakened. On the other hand, by comparing the data of lactobacillus, bifidobacterium and Escherichia coli in the bacterial agent group A and the bacterial agent group B, it can be seen that when BN rats are allergic, the bacterial agent group B, which is continuously treated with Lactobacillus helveticus HC1253, can accelerate the recovery rate of intestinal flora after allergy. After 5 oral administrations, the number of lactobacillus and bifidobacterium was higher than the normal level, and the number of Escherichia coli was lower than the normal level. This indicates that Lactobacillus helveticus HC1253 can not only relieve juniper pollen allergy by regulating the structure of intestinal flora, but also that the bacteria itself has the effect of improving intestinal flora.

[0152] Similarly, similar results were obtained when experiments were conducted on BN rats with birch pollen allergy model, poplar pollen allergy model, Artemisia pollen allergy model, Humulus pollen allergy model and ragweed pollen allergy model, indicating that Lactobacillus helveticus HC1253 can alleviate pollen allergies caused by birch pollen, poplar pollen, Artemisia pollen, Humulus pollen and ragweed pollen by regulating the structure of intestinal flora.

[0153] 4. Buehler Test (BT) test of BN rats in different treatment groups

[0154] In addition to causing allergic rhinitis, allergic conjunctivitis, and allergic asthma, pollen allergy can also cause allergic dermatitis. Therefore, this experiment used a closed patch test to study the effect of Lactobacillus helveticus HC1253 in dealing with allergic dermatitis caused by allergenic pollen.

[0155] Since the corresponding pollen allergy model of BN rats had been established in the early stage of the experiment, in this experiment, it was only necessary to conduct skin allergy stimulation tests on BN rats in different treatment groups to explore the role of Lactobacillus helveticus HC1253 in coping with allergic dermatitis caused by pollen.

[0156] In this experiment, BN rats in the blank control group, positive control group, inoculum A group and inoculum B group were selected as research subjects, and the experiments were carried out after the third stimulation and the last gavage, respectively.

[0157] The materials used in the experiment were 4 mg / mL different plant pollen protein solutions (the preparation method of which is shown in Experimental Example 4 above) that were sterilized by filtration with a 0.22µm filter membrane, and used as the experimental group of this experiment; at the same time, sterile 1× PBS buffer was used as the negative control of this experiment, and was marked as the PBS group. The experimental method was discussed using the juniper pollen allergy model of rats as an example, and the experimental methods of the other five pollen allergy models were the same.

[0158] First, according to the selected time period, about 2 hours before the start of the experiment, three BN rats were randomly selected from the blank control group, positive control group, inoculum A group, and inoculum B group, and the hair on the left side of their backs was removed, with a hair removal range of 4 cm. 2 ~6cm 2 , avoid damaging the skin during the operation. Then, 200 μL of 4 mg / mL juniper pollen protein solution was applied to the hairless skin of BN rats in each treatment group, covered with two layers of gauze and one layer of cellophane, and then sealed with non-irritating medical tape. After 6 hours, the sealed area was opened, and 24 hours after the tape was removed, the degree of skin erythema and edema reaction in the test area was evaluated according to the modified Magnusson and Kligman grading score standard (see Table 5), and the experimental group was scored accordingly; the PBS group did the same operation, except that the juniper pollen protein solution was replaced with sterile 1× PBS buffer. The experimental results are shown in Table 6 using the juniper pollen allergy model as an example. The data in the table are the total scores of three rats in each group.

[0159] Table 5 Modified Magnusson and Kligman grading criteria

[0160]

[0161] Table 6 Results of closed patch test on BN rat model of juniper pollen allergy

[0162]

[0163] From the data of the PBS group in Table 6, it can be seen that after being treated with sterile 1×PBS, the BN rats in different treatment groups had no obvious skin reaction, indicating that the PBS buffer and test method used in the test had no significant effect on the test results. On the other hand, after the third stimulation and the last intragastric treatment, the scores of the BN rats in the bacterial agent group A and the bacterial agent group B were lower than those in the positive control group, and by comparing the data of the bacterial agent group A and the bacterial agent group B after the last intragastric treatment, it can be seen that the bacterial agent group B treated with continuous intragastric treatment of Lactobacillus helveticus HC1253 had a significantly lower score of skin allergy symptoms after the last intragastric treatment than the bacterial agent group A, and approached the normal level. This shows that after being treated with Lactobacillus helveticus HC1253, not only can the skin allergic reaction caused by juniper pollen be reduced, but also continuous treatment with the bacteria can accelerate the disappearance of skin allergy symptoms. This suggests that Lactobacillus helveticus HC1253 has a certain positive effect in alleviating allergic dermatitis caused by juniper pollen.

[0164] Similarly, when experiments were conducted on birch pollen allergy models, poplar pollen allergy models, wormwood pollen allergy models, humulus pollen allergy models and ragweed pollen allergy models of BN rats, the experimental results were consistent with the results in the juniper pollen allergy model, indicating that Lactobacillus helveticus HC1253 has a certain positive effect in alleviating allergic dermatitis caused by juniper pollen, birch pollen, poplar pollen, wormwood pollen, humulus pollen or / and ragweed pollen, and also indicates that when faced with mixed pollen allergies caused by the above pollens, Lactobacillus helveticus HC1253 can show good improvement effects.

[0165] Experimental Example 6 Study on the application potential of probiotics

[0166] 1. Evaluation of bile salt tolerance of Lactobacillus helveticus HC1253

[0167] First, prepare MRS liquid culture medium containing 2 g / L sodium thioglycolate and divide it into portions, then add ox bile salt to make the final concentration of ox bile salt 0 g / L, 1.5 g / L, 3.0 g / L, and 4.5 g / L, respectively. Sterilize at 121°C for 20 min, cool and set aside, and add trypsin solution sterilized by 0.22 µm filter membrane to each treatment group before use to make the final concentration of 1.0 g / L.

[0168] According to the method in Experimental Example 5, a suspension of Lactobacillus helveticus HC1253 was prepared, and inoculated into the above-mentioned culture medium at an inoculum size of 5% (V / V), cultured at 37°C, and the initial bacterial count and the bacterial count after 12 hours of culture were detected, and the survival rate of the bacteria under different bile salt concentrations was calculated. The results are shown in Table 7.

[0169] Table 7 Results of bile salt tolerance test of Lactobacillus helveticus HC1253 bacterial suspension

[0170]

[0171] As shown in Table 7, Lactobacillus helveticus HC1253 can still maintain a survival rate of 92.03% after being treated at 37°C for 12 hours at a bile salt concentration of 4.5 g / L, indicating that the bacteria has a strong bile salt tolerance.

[0172] 2. Evaluation of gastric juice tolerance of Lactobacillus helveticus

[0173] This experiment used the method described in the group standard "T / CNHFA 435-2024 Test Method for Gastric Juice Tolerance of Probiotics" published by the China Nutrition and Health Food Association to evaluate the gastric juice tolerance of Lactobacillus helveticus HC1253. The sample to be tested in the experiment was 1×10 10 CFU / mL bacterial suspension (its preparation method is the same as that of Experimental Example 5), its experimental method and experimental results are shown in Table 8.

[0174] Table 8 Gastric juice tolerance test results of Lactobacillus helveticus HC1253

[0175]

[0176] As shown in Table 8, after being treated with different simulated gastric juices, the survival rate of Lactobacillus helveticus HC1253 was above 97%, indicating that the bacteria had a relatively strong tolerance to gastric juice.

[0177] In summary, the Lactobacillus helveticus HC1253 screened out in the present invention has a high tolerance to bile salts and gastric juice, indicating that the bacteria has great application potential.

[0178] Experimental Example 7

[0179] Pollen allergy has a certain seasonality. March to May and July to September of each year are the peak seasons for spring pollen allergy and autumn pollen allergy, respectively. In order to comprehensively evaluate the actual use effect of Lactobacillus helveticus HC1253 in the population, this experiment was conducted 1 to 2 months before the start of the two pollen seasons. First, the freeze-dried powdered single bacterial agent of Lactobacillus helveticus HC1253 was produced according to the common technology in the industry (lactic acid bacteria high-density culture technology and vacuum freeze-drying technology), and the number of live bacteria in it was tested in accordance with the "GB4789.35-2016 National Food Safety Standard Food Microbiology Inspection Lactic Acid Bacteria Inspection", and then diluted to 2.0×10 with food-grade maltodextrin. 10 CFU / g, prepare Lactobacillus helveticus HC1253 inoculum, and pack it into independent small packages at 2g / pack, and store it at -20℃ for future use. Then, recruit volunteers, and the selection criteria of volunteers are those who have been diagnosed with pollen allergy by the hospital (pollen is juniper pollen, birch pollen, poplar pollen, wormwood pollen, humulus pollen or / and ragweed pollen), and have shown symptoms of pollen allergy for two consecutive years.

[0180] 1. Skin prick test

[0181] Volunteers stopped taking antihistamines 3 days before the start of the experiment. If the volunteers had taken long-acting antihistamines orally, the corresponding experiment had to start 2 weeks after taking the medication.

[0182] During the experiment, the required pollen pricking solution was first diluted 1000 times with sterile saline. The type of pollen pricking solution should be consistent with the allergens of the volunteers. Then the skin on the palmar forearm of the volunteers was selected as the experimental site. After disinfection with 75% alcohol and drying, a small amount of the above diluted pollen pricking solution was placed on the skin surface using a disposable pricking needle, and then gently pierced into the epidermis in a vertical direction, taking care not to touch the dermis. Sterile saline was used as the negative control group; 0.01mg / mL histamine phosphate solution was sterilized by filtering with a 0.22µm filter membrane as the positive control of this experiment. The distance between each pricking site was >3cm to prevent the red blush after the reaction from blending with each other.

[0183] 2. SPT result determination

[0184] The wheal or red area of ​​the positive control solution was used as a reference, and the wheal or red area of ​​the negative control solution was used for correction. The reaction level was determined based on the ratio of the area of ​​the wheal or red area at the prick point to the area of ​​the wheal or red area of ​​the positive control. The diameter of the wheal or red area was observed and measured 10 minutes after the prick was completed, and the corresponding area was calculated. Then the time when the wheal or red area disappeared was observed and recorded. The criteria for judging the reaction level of the skin prick test are as follows: if the area of ​​the wheal or red area at the prick point of the prick solution is 0-25% of the positive control area or the same as the wheal or red area caused by the negative control, it is negative (-); if it is 26%-50%, it is positive (+), and the sensitization level is level I; if it is 51%-100%, it is positive (++), and the sensitization level is level II; if it is 101%-200%, it is positive (+++), and the sensitization level is level III; if it is >200%, it is positive (++++), and the sensitization level is level IV.

[0185] 3. Detection of pollen-specific IgE (sIgE) in the serum of volunteers

[0186] 5 mL of venous blood was drawn from the volunteers, placed at room temperature for 4 hours, and then centrifuged at 3000 rpm for 10 minutes at 4°C. The upper serum was carefully aspirated and transferred to new centrifuge tubes and stored at -80°C for later use. The pollen-specific IgE antibody detection kit corresponding to the pollen allergen of the volunteers was then used to detect the pollen-specific IgE (sIgE) content in the serum. The criteria for different levels are shown in Table 9.

[0187] Table 9 Levels corresponding to the content of pollen-specific IgE (sIgE)

[0188]

[0189] 4. Experimental design and measurement of corresponding indicators

[0190] First, at the beginning of the experiment, the recruited volunteers were numbered, and skin prick tests were carried out to detect the level of pollen-specific IgE (sIgE) in the volunteers' serum. The experimental results obtained were filed and preserved as the background allergy level of the volunteers.

[0191] Then, the volunteers were graded according to the skin prick test and the grading standards of pollen-specific IgE (sIgE) content, and those whose skin prick reaction was grade III and whose pollen-specific IgE (sIgE) content was grade 4 were selected from the recruited volunteers for subsequent experiments.

[0192] Once selected, the volunteers began to take the homemade Lactobacillus helveticus HC1253 agent, taking one packet (2 g / packet, with a bacterial content of 2.0 × 10 10CFU / g), and take it continuously for 30 days. If the volunteer does not have pollen allergy symptoms during this period, a skin prick test will be performed on him and the pollen-specific IgE (sIgE) content in his serum will be tested. If the volunteer has pollen allergy symptoms during this period, the volunteer needs to continue to take 1 pack of Lactobacillus helveticus HC1253 agent every day after the allergy, and keep corresponding records of the medication and symptoms after the allergy. After 30 days, fill out an experimental record form (as shown in Table 10), and then fill out the form again by reviewing the medication and allergic symptoms when pollen allergy occurred in previous years. After the experiment, the form will be collected and statistically analyzed. During the experiment, allergic people can receive corresponding medical treatment as needed according to their own conditions.

[0193] Table 10. Record of medication and allergic symptoms of volunteers

[0194]

[0195] 5. Experimental results

[0196] (1) A total of 205 volunteers were recruited for this experiment. After skin prick tests and detection of pollen-specific IgE (sIgE) in serum, their respective classifications are shown in Table 11.

[0197] Table 11 Background level allergy classification of volunteers

[0198]

[0199] It can be seen from Table 11 that the volunteers' grading according to the skin prick test and the grading according to the pollen-specific IgE content have certain similarities, indicating that these two grading methods can both reflect the severity of allergies in patients with pollen allergy, and it can be considered that there is a certain correlation between these two experimental indicators.

[0200] In addition, after grading by the two grading methods, according to the experimental needs, there were 45 volunteers who met the experimental conditions and were numbered as Volunteer 1 to Volunteer 45.

[0201] (2) Measurement results of various indicators of 45 volunteers after taking Lactobacillus helveticus HC1253 for 30 days

[0202] Although the allergy level of the volunteers selected for the experiment was relatively high, none of the 45 selected volunteers developed pollen allergy within 30 days after the start of the experiment. Ten volunteers were randomly selected from the 45 volunteers to measure various indicators, and the measurement results are shown in Table 12.

[0203] Table 12 Changes in experimental indicators after volunteers took Lactobacillus helveticus HC1253 for one month

[0204]

[0205] As shown in Table 12, since the volunteers selected in this experiment had a high level of allergy, the content of pollen-specific IgE in the volunteers was high, the area of ​​the wheal or redness formed by the skin prick test was relatively large, and the time taken for the wheal or redness to disappear was also long. However, after taking Lactobacillus helveticus HC1253 for 30 consecutive days, the above indicators all had obvious changes, mainly manifested as follows: 1) The area of ​​the wheal or redness formed by the skin prick test was significantly reduced, and according to the results of the skin prick test, the allergy level of the selected volunteers was reduced from grade III to grade II; 2) The content of pollen-specific IgE in the serum of the selected volunteers decreased significantly; 3) The disappearance time of the erythema formed by the skin prick test was greatly shortened. This shows that Lactobacillus helveticus HC1253 can not only reduce the level of pollen-specific IgE in the serum of volunteers, and has a certain desensitization effect, but also accelerate the disappearance of allergic symptoms and reduce the physical pain of patients with pollen allergy. In addition, since Lactobacillus helveticus HC1253 can reduce the level of pollen-specific IgE in the serum of volunteers, it may play a certain positive role in alleviating the pollen-food allergy syndrome caused by pollen allergy.

[0206] (3) Effect of Lactobacillus helveticus HC1253 on the medication use and allergic symptoms of volunteers

[0207] After the experiment, statistical analysis of the data in the collected "Volunteer Medication and Allergy Symptom Record Form" showed that taking Lactobacillus helveticus HC1253 can significantly reduce the severity of volunteers' pollen allergy symptoms and shorten the duration of pollen allergy symptoms, thereby greatly reducing the number of volunteers' medication and medication dosage and minimizing the side effects of drug treatment on the body.

[0208] In summary, this experiment proved that Lactobacillus helveticus HC1253 can reduce the content of pollen-specific IgE in pollen-allergic organisms and reduce the allergy level of the organism, thereby reducing the probability of pollen allergy in allergic organisms, blocking the disease process of pollen allergy, and achieving a certain desensitization effect through BN rat pollen allergy model experiments and human experiments. In addition, Lactobacillus helveticus HC1253 can reduce the content of pollen-specific IgE in the body without stimulating allergies, which shows that it has certain application potential in people with severe pollen allergies or pollen allergies with severe allergic symptoms, thereby expanding the scope of application of Lactobacillus helveticus HC1253 in pollen allergies.

[0209] When an allergic body has a pollen allergy reaction, the Lactobacillus helveticus HC1253 bacterial agent that enters the body can balance the Th1 / Th2 cell immune response by reducing the content of allergic factors such as IL-4 and histamine, while increasing the content of anti-allergic factors such as gamma interferon, thereby reducing the intensity of the reaction of pollen allergy symptoms and shortening their duration, thus relieving the pain of pollen allergy sufferers to the greatest extent. In addition, the bacteria also has the function of regulating the balance of lactobacillus, bifidobacteria and Escherichia coli flora in the intestines, and in this way helps relieve pollen allergy symptoms.

[0210] In addition, during the experiment, neither the BN rats nor the volunteers experienced any adverse reactions other than pollen allergy symptoms, which indicates that the bacteria has no toxic side effects. It has also been verified that the bacteria has a strong tolerance to gastric acid and bile salts, indicating that Lactobacillus helveticus HC1253 has great market application potential.

[0211] Although the present invention has been described in detail with reference to the accompanying drawings and in combination with the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily think of changes or substitutions within the technical scope disclosed by the present invention, and these shall be within the scope of protection of the present invention.

Claims

1. A strain of Lactobacillus helveticus HC1253 beneficial to people with pollen allergies, characterized in that: Lactobacillus helveticus ( Lactobacillus helveticus ) HC1253 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on February 20, 2023. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 26588.

2. A use of Lactobacillus helveticus HC1253 as claimed in claim 1 in the preparation of a medicine for improving pollen allergy, characterized in that: Pollen allergy includes single pollen allergy and mixed pollen allergy; the allergenic pollen source of single pollen allergy is Juniper, Birch, Poplar, Artemisia, Humulus or Ragweed; the allergenic pollen source of mixed pollen allergy is selected from at least two of the genera Juniper, Birch, Poplar, Artemisia, Humulus and Ragweed.

3. The use according to claim 2, characterized in that The allergenic pollens for single pollen allergy are juniper pollen, birch pollen, poplar pollen, wormwood pollen, humulum pollen or ragweed pollen; the allergenic pollens for mixed pollen allergy are selected from at least two of juniper pollen, birch pollen, poplar pollen, wormwood pollen, humulum pollen and ragweed pollen.

4. The use according to claim 2, characterized in that Improving pollen allergy includes improving respiratory allergy symptoms and skin allergy symptoms.

5. The use according to claim 4, characterized in that Respiratory allergy symptoms include nasal allergy symptoms and trachea and bronchial allergy symptoms; nasal allergy symptoms include sneezing, nasal congestion and runny nose; trachea and bronchial allergy symptoms include shortness of breath, chest tightness, shortness of breath and difficulty breathing; skin allergy symptoms include erythema, itching, edema and wheals.

6. The use according to claim 2, characterized in that Improving pollen allergy also includes improving eye allergy symptoms; eye allergy symptoms include conjunctival congestion, conjunctival edema, eyelid edema and increased secretions.

7. The use according to claim 2, characterized in that Drugs for improving pollen allergies include Lactobacillus helveticus HC1253 bacterial agent; the number of live bacteria of Lactobacillus helveticus HC1253 is 2.0×10 10 CFU / g freeze-dried single bacterial powder.

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