Bifidobacterium animalis lactis for alleviating respiratory allergy caused by ovalbumin

Abstract

The application discloses an animal bifidobacterium lactis strain capable of producing indole-3-carboxaldehyde to relieve respiratory allergic diseases caused by ovalbumin, and belongs to the technical field of microorganisms and the technical field of medicine. The application provides an animal bifidobacterium lactis strain CCFM1274 capable of relieving the pathological characteristics of asthma mice and further relieving asthma. After the animal bifidobacterium lactis strain CCFM1274 is used on the asthma mice, allergic symptoms of the mice can be significantly relieved. Therefore, the animal bifidobacterium lactis strain CCFM1274 has great application prospect in the preparation of products for preventing and / or treating respiratory allergic diseases such as asthma.

Description

TECHNICAL FIELD

[0001] The present application relates to an animal bifidobacterium lactis with high yield of indole-3-carboxaldehyde to relieve respiratory allergy caused by ovalbumin, belonging to the technical field of microorganisms and the technical field of medicine. BACKGROUND

[0002] Bronchial asthma is a respiratory allergic disease, and its cause has been studied for a long time, but it has not been completely elucidated. It can be summarized as airway immune-inflammation mechanism, neural regulation mechanism and their interaction, and airway inflammation is considered to be the essence of respiratory allergy. In recent years, in-depth study of its pathology and physiology has found that it is a continuous inflammatory reaction throughout the upper and lower airways. When the body is stimulated by allergens, specific immunoglobulins (sIgE) are produced in the local respiratory tract, which can bind to the high-affinity IgE receptor (FcεRI) on the surface of mast cells and basophils, and the body forms a sensitized state. When the body contacts the same allergen again, it crosslinks with IgE on the surface of mast cells and basophils, activates sensitized cells, and causes cell degranulation, thereby releasing histamine and leukotrienes and other inflammatory mediators, and further triggering a series of inflammatory reactions. In addition, the release of inflammatory mediators such as histamine can further induce cells to express and secrete more inflammatory factors, thus forming a complex network of interaction, recruiting and activating immune cells such as eosinophils, basophils and helper T cells type 2 (T helper type 2, Th2), and further causing sensitized cells to release inflammatory mediators, so that type 2 pro-inflammatory response continues to dominate and worsens. In addition, more and more studies have confirmed that CD4+ helper T lymphocytes 17 play a key role in the immune mechanism of asthma, and the imbalance between Th17 and regulatory T cells is the main mechanism of respiratory allergic diseases such as asthma. In normal body, initial T cells differentiate into Th1 and Th2, and maintain a relative balance according to a certain proportion, but in the body of asthma patients, they are more biased towards Th2 and Th17, and a large amount of IL-4, IL-5, IL-13 and IL-17 and other pro-inflammatory factors are secreted, which increases the airway reactivity, airway contraction, and increases the secretion of mucus, and induces the occurrence of respiratory allergy.

[0003] There is no complete treatment method and drug for asthma at present, and the repeated attack of allergy causes the patient to need long-term drug dependence, and there are certain side effects, so the treatment of asthma is still a medical problem. In the immune mechanism of the pathogenesis of asthma, the role of intestinal flora and tryptophan metabolism has attracted widespread attention. Microbial disorder occurs in the intestine of patients with asthma, leading to imbalance of tryptophan metabolism. It can be seen that there is a pathological relationship between intestinal tryptophan metabolites and respiratory allergic diseases such as asthma. Studies have also confirmed that probiotics can effectively alleviate the allergic symptoms of mice with dermatitis by producing tryptophan metabolite indole 3-carboxaldehyde, and the probiotic strain improves the quality of life of patients with dermatitis and increases the content of indole 3-carboxaldehyde in feces. However, the effect of indole 3-carboxaldehyde on asthma has not been reported, so it is of great significance to explore the ability of probiotics to produce tryptophan metabolites indole 3-carboxaldehyde to effectively alleviate the allergic symptoms of asthma. In summary, it may be possible to alleviate the allergic symptoms of OVA-induced bronchial asthma by adjusting the content of small molecule metabolites in tryptophan metabolism. SUMMARY

[0004] The present application provides a new use of animal Bifidobacterium lactis subsp. Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis ) CCFM1274 in improving the pathological characteristics of asthma mice and alleviating the allergic symptoms of asthma.

[0005] The present application provides a new use of animal Bifidobacterium lactis subsp. Bifidobacterium animalis lactis Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis ) CCFM1274 in producing tryptophan metabolite indole 3-carboxaldehyde, improving the pathological characteristics of bronchial asthma mice, and alleviating the allergic symptoms of asthma. Bifidobacterium animalis lactis Bifidobacterium animalis lactis subsp Bifidobacterium animalis lactis ) CCFM1274 was deposited with the Guangdong Microbial Culture Collection Center on September 15, 2022, and the deposit number is GDMCC No: 62798, and the deposit address is No. 59, Building 5, 100, Martyrs Road, Guangzhou.

[0006] The present application provides a new use of animal Bifidobacterium lactis subsp. Bifidobacterium animalis lactis Bifidobacterium animalis lactis subsp Bifidobacterium animalis lactis ) CCFM1274 and / or the fermentation broth of animal Bifidobacterium lactis subsp. CCFM1274 in the preparation of products for alleviating the symptoms of asthma. The animal Bifidobacterium lactis subsp. CCFM1274 has been deposited with the Guangdong Microbial Culture Collection Center on September 15, 2022, and the deposit number is GDMCC No: 62798.

[0007] In an embodiment of the present application, the product contains live Bifidobacterium animalis subsp. lactis CCFM1274 in an amount of not less than 1 x 10 Bifidobacterium animalis lactis Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis ) CCFM1274 in an amount of not less than 1 x 10 6 CFU / mL.

[0008] In an embodiment of the present application, the product is a pharmaceutical product or a feed additive.

[0009] In an embodiment of the present application, the alleviating of the allergic symptoms of asthma includes alleviating the nasal allergic symptoms, or reducing the content of specific immunoglobulin in serum, or reducing lung inflammation, or inhibiting the secretion of pro-inflammatory factors in the alveolar lavage fluid.

[0010] In an embodiment of the present application, the alleviating of the allergic symptoms of asthma includes alleviating the nasal allergic symptoms such as sneezing, rhinitis, runny nose, or reducing the content of specific immunoglobulin in serum, or reducing lung inflammation, or inhibiting the secretion of pro-inflammatory factors in the alveolar lavage fluid.

[0011] In an embodiment of the present application, the asthma is the bronchial allergic reaction caused by OVA. In clinic, one of the indicators for diagnosing asthma is the total IgE in serum.

[0012] In an embodiment of the present application, the asthma is the bronchial allergic reaction caused by OVA. In clinic, one of the indicators for diagnosing asthma is the OVA-sIgE in serum.

[0013] In an embodiment of the present application, the pharmaceutical product contains Bifidobacterium animalis subsp. lactis CCFM1274, a pharmaceutical carrier and / or a pharmaceutical excipient.

[0014] In an embodiment of the present application, the dosage form of the pharmaceutical product includes granules, capsules, tablets, pills or oral liquids.

[0015] In an embodiment of the present application, the pharmaceutical excipient includes excipients and additional agents.

[0016] In an embodiment of the present application, the pharmaceutical excipient includes anti-adhesion agents, penetration enhancers, buffering agents, plasticizers, surfactants, antifoaming agents, thickening agents, inclusion agents, absorbents, humectants, solvents, propellants, solubilizers, cosolvents, emulsifiers, coloring agents, pH adjusters, adhesion agents, disintegrants, fillers, lubricants, wetting agents, integrating agents, osmotic pressure adjusters, stabilizers, flow aids, flavoring agents, preservatives, foaming agents, suspending agents, coating materials, fragrances, diluents, flocculants and deflocculants, filtration aids and release retardants.

[0017] In an embodiment of the present application, the additional agent comprises microcrystalline cellulose, hydroxypropyl methylcellulose, and refined lecithin.

[0018] In an embodiment of the present application, the drug carrier comprises microcapsules, microspheres, nanoparticles, and liposomes.

[0019] The present application also provides a method for producing a tryptophan metabolite, which comprises fermentation by Bifidobacterium animalis lactis CCFM1274.

[0020] The present application also provides the use of Bifidobacterium animalis lactis CCFM1274 in the preparation of a tryptophan metabolite or a product containing a tryptophan metabolite, which has been deposited with the Guangdong Microbial Culture Collection Center on September 15, 2022, and has the accession number GDMCC No: 62798.

[0021] In an embodiment of the present application, the product is a chemical.

[0022] The present application also provides a food product containing Bifidobacterium animalis lactis CCFM1274.

[0023] In an embodiment of the present application, the food product is a food product containing Bifidobacterium animalis lactis CCFM1274 or a fermentation metabolite thereof. Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis .

[0024] In an embodiment of the present application, the food product is a dairy product, a legume product, or a fruit and vegetable product produced using Bifidobacterium animalis lactis CCFM1274 or a fermentation agent containing Bifidobacterium animalis lactis CCFM1274. Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis . Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis .

[0025] In an embodiment of the present application, the food product is a solid beverage containing Bifidobacterium animalis lactis CCFM1274. Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis .

[0026] In an embodiment of the present application, the fermentation agent is prepared by culturing Bifidobacterium animalis lactis CCFM1274. Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactisCCFM1274 is inoculated into the culture medium at an inoculation amount of 2-4% of the total mass of the culture medium, and cultured at 37°C for 30h to obtain a culture solution; the culture solution is centrifuged to collect bacterial cells; the bacterial cells are washed with a phosphate buffer solution with a pH of 7.2 for 3 times, then resuspended with a freeze-drying protective agent to obtain a resuspension; the resuspension is freeze-dried by a vacuum freezing method to obtain the Bifidobacterium animalis subsp. lactis (Bifidobacterium animalis subsp. lactis) CCFM1274. Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis The fermentation agent of CCFM1274.

[0027] In an embodiment of the present application, the mass ratio of the freeze-drying protective agent to the bacterial cells is 2:1.

[0028] In an embodiment of the present application, the freeze-drying protective agent contains skimmed milk powder, malt dextrin and L-glutamic acid sodium; wherein the mass ratio of skimmed milk powder, malt dextrin and L-glutamic acid sodium is 8-10:8-10:1.

[0029] In an embodiment of the present application, the culture medium is prepared by dissolving 10% of skimmed milk, 0.5% of glucose, 1.5% of tryptone and 0.3% of yeast extract in water in the total mass of the culture medium.

[0030] In an embodiment of the present application, the pH of the culture medium is 6.8.

[0031] The present application also provides a method for producing tryptophan metabolites, which is prepared by fermentation using Bifidobacterium animalis subsp. lactis CCFM1274.

[0032] In an embodiment of the present application, the tryptophan metabolites include, but are not limited to, kynurenine, indolelactic acid, indole-3-carboxaldehyde and indoleacetic acid.

[0033] The present application also provides the use of the above-mentioned Bifidobacterium animalis subsp. lactis CCFM1274 in the preparation of tryptophan metabolites or products containing tryptophan metabolites, which has been preserved in the Guangdong Microbial Culture Collection Center on September 15, 2022, with the preservation number of GDMCC No:62798.

[0034] In an embodiment of the present application, the tryptophan metabolites include, but are not limited to, kynurenine, indolelactic acid, indole-3-carboxaldehyde and indoleacetic acid.

[0035] In an embodiment of the present application, the product is a chemical.

[0036] In an embodiment of the present application, the product is a food, a medicine or a health product.

[0037] Beneficial effects

[0038] The application provides a Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis ) CCFM1274 which can relieve the pathological characteristics of an asthma mouse and further relieve asthma. Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis ) CCFM1274 can significantly relieve the allergic symptoms of the mouse, and the specific manifestations are as follows (compared with the model group):

[0039] (1) The Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis ) CCFM1274 has high indole-3-carboxaldehyde production, and the yield is 598.513 ppb.

[0040] (2) The weight loss of the asthma mouse is relieved, and the weight is increased by 16.40%.

[0041] (3) The total IgE content in the serum of the asthma mouse is significantly reduced, and is reduced by 63.88%.

[0042] (4) The OVA-sIgE content in the serum of the asthma mouse is significantly reduced, and is reduced by 24.81%.

[0043] (5) The inflammation damage of the lung tissue of the asthma mouse is obviously relieved.

[0044] (6) The IL-4 level in the bronchoalveolar lavage fluid of the asthma mouse is reduced, and is reduced by 44.27%.

[0045] (7) The IL-5 level in the bronchoalveolar lavage fluid of the asthma mouse is significantly reduced, and is reduced by 22.61%.

[0046] (8) The IL-13 level in the bronchoalveolar lavage fluid of the asthma mouse is reduced, and is reduced by 13.31%.

[0047] (9) The IL-17 level in the bronchoalveolar lavage fluid of the asthma mouse is reduced, and is reduced by 27.58%.

[0048] (10) The TNF-α level in the bronchoalveolar lavage fluid of the asthma mouse is significantly increased, and is increased by 53.96%.

[0049] (11) The indole propionic acid level of tryptophan metabolites in the feces of the asthma mouse is increased by 51.86%.

[0050] (12) The indole lactic acid level of tryptophan metabolites in the feces of the asthma mouse is increased by 37.84%.

[0051] Therefore, the Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactisCCFM1274 has great potential for application in the preparation of products for the prevention and / or treatment of respiratory allergic diseases such as asthma.

[0052] Preservation of biological materials

[0053] A strain of Bifidobacterium animalis subspecies ( Bifidobacterium animalis lactis subsp Bifidobacterium animalis lactis CCFM1274, taxonomically named Bifidobacterium animalis lactis subsp Bifidobacterium animalis lactis It was deposited on September 15, 2022 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 62798. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Institute of Microbiology. Attached Figure Description

[0054] Bifidobacterium animalis lactis Changes in the percentage of body weight of mice in different groups.

[0055] Bifidobacterium animalis lactis Total IgE content in serum of mice in different groups of experiments.

[0056] Bifidobacterium animalis lactis Serum OVA-sIgE levels in mice from different experimental groups.

[0057] Bifidobacterium animalis lactis Pathological sections of lung tissue from mice in different experimental groups.

[0058] Bifidobacterium animalis lactis IL-4 content in bronchoalveolar lavage fluid of mice in different groups.

[0059] Bifidobacterium animalis lactis IL-5 content in bronchoalveolar lavage fluid of mice in different groups.

[0060] Bifidobacterium animalis lactis IL-13 content in bronchoalveolar lavage fluid of mice in different groups.

[0061] Bifidobacterium animalis lactis IL-17 content in bronchoalveolar lavage fluid of mice in different groups.

[0062] Bifidobacterium animalis lactis TNF-α content in bronchoalveolar lavage fluid of mice in different groups.

[0063] Bifidobacterium animalis lactis Content of indolepropionic acid, a metabolite of tryptophan, in feces of mice in different groups.

[0064] Bifidobacterium animalis lactis Content of indolelactate, a metabolite of tryptophan, in feces of mice in different groups.

[0065] Bifidobacterium animalis lactis Flow chart of animal experiment. DETAILED DESCRIPTION

[0066] BALB / c female mice involved in the following examples were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. OVA involved in the following examples was purchased from Sigma-Aldrich Company. Lactobacillus rhamnosus CCFM1228 involved in the following examples was described in the Chinese patent application with publication number CN114540245A; Lactobacillus salivarius FBJSY202, Bifidobacterium breve FHNXY26M4, Bifidobacterium breve CJ653, Bifidobacterium animalis lactis SHXXA4M1T78, Bifidobacterium animalis lactis ZJHZD20M6, Bifidobacterium animalis lactis FNMGHHHT2M2 involved in the following examples were preserved in the Jiangnan University College of Food Science and Biotechnology Center of Bacterial Preservation.

[0067] The preparation method of OVA solution involved in the following examples is as follows:

[0068] OVA sensitization solution: 1 mg of OVA was accurately weighed and dissolved in 1 mL of normal saline, and an equal amount of aluminum hydroxide adjuvant was added to mix; 0.2 mL of sensitization solution was injected intraperitoneally to each mouse.

[0069] OVA challenge solution: a certain amount of OVA was accurately weighed and dissolved in sterile normal saline to prepare a challenge solution with a concentration of 1 mg / mL; 20 μL of challenge solution was dropped into the nose of each mouse.

[0070] Detection of tryptophan metabolite content of probiotics involved in the following examples:

[0071] A tryptophan metabolite standard stock solution with a concentration of 1000 ppm was prepared under light-proof conditions, a certain amount of stock solution was prepared into a mixed standard, and after mixing, it was diluted with methanol water solution (methanol: water = 1 : 9) to a series of concentrations of 50 ppm~1.5625 ppb with a 2-fold gradient. An ultra-high performance liquid chromatography-high resolution mass spectrometry instrument was used for detection. An ACQUITY UPLC® BEHC18 1.7 µm (100 mm × 2.10 mm) chromatographic column was used, the column temperature was 36.5℃, and the automatic sampler temperature was 4℃. Each sample was detected in positive ion mode, and the mobile phase was (A) acetonitrile and (B) 0.1% formic acid aqueous solution. When gradient elution was used, A and B were mixed for elution, and finally balanced for 20 min. The injection amount was set to 2 μL, and the flow rate was 300 μL / min.

[0072] Detection of tryptophan metabolite content in mouse feces involved in the following examples:

[0073] Accurately weigh 50 mg of mouse feces, and 25 mg if the sample is insufficient, add 500 / 250 μL of extraction solution (methanol:acetonitrile:water = 2:2:1, pre-cooled at -40°C, containing 0.1% formic acid and isotope-labeled internal standard mixture) and mix for 30 s, then grind and crush, and ultrasonicate for 5 min under ice water bath conditions. Repeat the crushing and ultrasonication step twice. Then stand at -40°C for 1 h, centrifuge at 4°C at 12000 rpm for 15 min, transfer 320 / 200 μL of supernatant, dry under nitrogen, and add 80 / 50 μL of 0.1% formic acid aqueous solution for redissolution, then centrifuge at 4°C at 12000 rpm for 15 min, and take the supernatant for UHPLC-MS-MS analysis.

[0074] The target compound was separated by chromatography using an EXION LC System (SCIEX) ultra-high performance liquid chromatograph and a Waters ACQUITY UPLC HSS T3 (100 x 2.1 mm, 1.8 μm, Waters) liquid chromatography column. The liquid chromatography A phase was 0.1% formic acid aqueous solution, and the B phase was 0.1% formic acid acetonitrile. The column oven temperature was 40°C, the sample tray was set to 4°C, and the sample injection volume was 5 μL.

[0075] The culture media involved in the following examples are as follows:

[0076] The formula of M9 fermentation base medium (1 L): tryptophan 0.2042 g; glucose 4.0000 g; sodium chloride 0.5000 g; calcium chloride 0.0110 g; sodium phosphate dibasic 4.7840 g; potassium phosphate monobasic 2.9940 g; ammonium chloride 0.5001 g; magnesium sulfate 0.2407 g, pH 6.5-7.2.

[0077] The formula of LBS medium (1 L): peptone 10 g, yeast powder 5 g, glucose 20 g, sodium acetate anhydrous 17 g, Tween 80 1 mL, potassium phosphate monobasic 6 g, ammonium citrate 2 g, ferrous sulfate 0.034 g, magnesium sulfate 0.575 g, manganese sulfate 0.12 g, pH 5.4±0.2.

[0078] The formula of LBS solid medium (1 L): peptone 10 g, yeast powder 5 g, glucose 20 g, sodium acetate anhydrous 17 g, Tween 80 1 mL, potassium phosphate monobasic 6 g, ammonium citrate 2 g, ferrous sulfate 0.034 g, magnesium sulfate 0.575 g, manganese sulfate 0.12 g, agar 15 g, pH 5.4±0.2.

[0079] MRS medium formula (1 L): 10 g of proteose peptone, 10 g of beef extract, 5 g of yeast powder, 20 g of glucose, 2 g of K2HPO4, 2 g of diammonium citrate, 2 g of sodium acetate, 1 mL of Tween 80, 0.5 g of MgSO4·7H2O, 0.25 g of MnSO4·4H2O, 20 g of agar, pH 7.2-7.4.

[0080] MRS medium formula (1 L): 10 g of proteose peptone, 10 g of beef extract, 5 g of yeast powder, 20 g of glucose, 2 g of K2HPO4, 2 g of diammonium citrate, 2 g of sodium acetate, 1 mL of Tween 80, 0.5 g of MgSO4·7H2O, 0.25 g of MnSO4·4H2O, 20 g of agar, pH 7.2-7.4.

[0081] The detection method involved in the following examples is as follows:

[0082] The detection method of viable bacteria count: the national standard “GB 4789.35-2016 Food Safety National Standard Food Microbiology Detection Lactic Acid Bacteria Detection” is adopted.

[0083] The detection method of acidity: the national standard GB 431334-2010 is adopted.

[0084] Example 1: Obtaining and preservation of animal Bifidobacterium lactis (Bifidobacterium animalis subsp. lactis) CCFM1274 Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis

[0085] (1) Screening and identification of animal Bifidobacterium lactis (Bifidobacterium animalis subsp. lactis) CCFM1274 Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis

[0086] A 31-year-old male (Uygur nationality) stool sample from Shawan was used as a sample (sample number FXJSW62T82), and physiological saline was used for gradient dilution of the sample. 100 μL of the diluent was spread on a plate (MRS solid medium, 0.05% L-cysteine hydrochloride was added), and placed in a 37°C anaerobic incubator for inverted culture for 48 h. According to the colony morphology classification, a single colony was picked and plated on MRS solid medium, and then placed in a 37°C anaerobic incubator for inverted culture for 48 h. A single colony was inoculated into 5 mL of liquid MRS medium, and then placed in a 37°C anaerobic incubator for culture for 18-24 h. 1.5 mL of the bacterial body was centrifuged at 6000 r / min for 3 min to remove the supernatant, 1 mL of 30% sterile glycerol was added for preservation, and at the same time, 1.5 mL of the bacterial body was centrifuged, the supernatant was removed, and then resuspended with sterile water for strain identification.

[0087] ​​Sequencing analysis of this strain and nucleic acid sequence alignment in GenBank showed a 98.55% similarity to *Bifidobacterium animalis* subsp. *lactamella*, leading to its naming as *Bifidobacterium animalis* subsp. *lactamella*. Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis (CCFM1274)

[0088] Bifidobacterium animalis subsp. lactis CCFM1274 was inoculated into MRS solid medium supplemented with L-cysteine ​​hydrochloride and cultured anaerobicly at 37°C for 48 h. The colonies were observed and the cells were examined under a microscope. The colonies were found to be milky white, smooth, convex in the middle, and with neat edges. The cells were straight, slightly curved, rod-shaped, Y-shaped, with swollen tips and slightly irregular shapes. They were usually found singly, in pairs, or in small clusters, and sometimes in chains. The cells were parallel and formed a palisade.

[0089] (2) Preservation of Bifidobacterium animalis subsp. lactis

[0090] The bacterial strain was cultured in MRS basal medium, and *Bifidobacterium animalis* subsp. *lactamella* was cultured for one generation at 37°C for 15-24 hours. Single colonies from the second generation on solid medium were selected and cultured for the third generation under the same conditions. The culture tubes containing the bacterial culture were centrifuged at 8000 g for 2 min at 4°C, the supernatant was discarded, and the precipitate was added with 1 mL of sterile 30% glycerol solution. After shaking, the precipitate was stored at -80°C.

[0091] Example 2: Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis Cultivation of CCFM1274

[0092] The specific steps are as follows:

[0093] Bifidobacterium lactis subsp. animalis ( Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis CCFM1274 was inoculated into MRS liquid medium and cultured at 37℃ for 16 h. The bacterial culture was then inoculated into fresh MRS liquid medium at an inoculation rate of 4% (v / v) and cultured under the same conditions for 12 h. The bacterial cells were centrifuged at 8000 r / min for 15 min, washed with 0.9% physiological saline, and centrifuged again at 8000 r / min for 10 min. The bacterial cells were collected and resuspended in 30% (v / v) glycerol solution. The resuspensions were prepared and stored at -80℃ for later use.

[0094] Preparation of bacterial suspension for gavage: Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalis lactis Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis) CCFM1274 for gavage in mice, the prepared resuspension was taken out from -80℃, centrifuged at 4℃, 8000 r / min for 10 min, the supernatant was discarded, the bacterial solution was washed with 0.9% normal saline and resuspended to obtain the bacterial suspension for gavage, the bacterial concentration was: 5 × 10 9 CFU / mL.

[0095] Example 3: Bifidobacterium animalis lactis (B. animalis lactis) Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis ) CCFM1274 high tryptophan metabolite indole-3-carboxaldehyde

[0096] The specific steps are as follows:

[0097] (1) M9 fermentation medium (g / L): tryptophan, 0.2042; glucose, 4.0000; sodium chloride, 0.5000; calcium chloride, 0.0110; sodium phosphate dibasic, 4.7840; potassium phosphate monobasic, 2.9940; ammonium chloride, 0.5001; magnesium sulfate, 0.2407. MRS base medium was configured, and 0.05% L-cysteine hydrochloride was added to the MRS medium of Bifidobacterium.

[0098] (2) First, the strains were activated for two generations using MRS liquid medium, specifically: Lactobacillus rhamnosus CCFM1228, Lactobacillus salivarius FBJSY202, Bifidobacterium breve FHNXY26M4, Bifidobacterium breve CJ653, Bifidobacterium animalis lactis SHXXA4M1T78, Bifidobacterium animalis lactis ZJHZD20M6, and Bifidobacterium animalis lactis FNMGHHHT2M2 were inoculated into MRS liquid medium with 0.05% L-cysteine hydrochloride, and cultured at 37℃ under anaerobic conditions (N2:CO2:H2=8:1:1) for 24 h, then the above operation and culture conditions were repeated for another generation; the culture solution was prepared.

[0099] (3) The obtained culture solution was centrifuged at 2000 r / min for 10 min, the supernatant was discarded, 1 mL of M9 fermentation medium was added, and it was placed in an anaerobic workstation (37℃, N2:CO2:H2=8:1:1) for 24 h, after the end, it was taken out and centrifuged at 8000 r / min for 10 min, the obtained supernatant was pretreated and the tryptophan metabolite was detected.

[0100] The content of tryptophan metabolite is shown in Table 1.

[0101] Table 1: Content of tryptophan metabolite in fermentation supernatant of probiotics

[0102]

[0103] From Table 1, it can be seen that Lactobacillus rhamnosus CCFM1228 and Lactobacillus salivarius FBJSY202 have relatively high KYN production, Bifidobacterium breve FHNXY26M4 and CJ653 have relatively high indolelactic acid production, and Bifidobacterium animalis subsp. lactis CCFM1274, SHXXA4M1T78, ZJHZD20M6 and FNMGHHHT2M2 have relatively high indole-3-carboxaldehyde and indoleacetic acid production, and CCFM1274 and SHXXA4M1T78 have higher indole-3-carboxaldehyde and indoleacetic acid production, and Bifidobacterium animalis subsp. lactis (Bifidobacterium animalis subsp. lactis) Bifidobacterium animalis lactis Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis ) CCFM1274 has the highest ability to produce indole-3-carboxaldehyde, a tryptophan metabolite.

[0104] The above results show that Bifidobacterium animalis subsp. lactis (Bifidobacterium animalis subsp. lactis) Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis ) CCFM1274 has higher production of indole-3-carboxaldehyde, a tryptophan metabolite, than other probiotic strains.

[0105] Example 4: Remission effect of Bifidobacterium animalis subsp. lactis (Bifidobacterium animalis subsp. lactis) Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis ) CCFM1274 on weight loss in asthmatic mice

[0106] 6-8 week-old SPF female BALB / c mice were raised in the Experimental Animal Center of Jiangnan University, fed with normal feed, constant temperature 20-26℃, humidity 40%-70%, noise less than or equal to 60 dB, animal illumination 15-20 LX, 12 h light, 12 h dark (all animal experiment procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangnan University).

[0107] The experimental design lasted for 5 weeks, and the specific process was as follows Bifidobacterium animalis lactis The mouse body weight was recorded at the same time every week, a total of 5 times. The first week was the adaptation period of the mice, and the modeling started from the second week. After the adaptation period, the mice were randomly divided into blank group, model group and CCFM1274 group. There were 10 mice in each group.

[0108] Specifically as follows:

[0109] Blank group: After the newly arrived mice were adapted for one week, 200 μL of 0.9% normal saline was intraperitoneally injected on the 7th, 14th and 21st days, 20 μg of 0.9% normal saline was dripped into the nose on the 28th-32nd day, 200 μL of 0.9% normal saline was orally administered to the mice from the 7th to the 32nd day, and the mice were allowed to freely eat and drink.

[0110] Model group: After the new mice adapt for a week, intraperitoneal injection of 200 μL OVA sensitizing solution (1 mg of OVA is dissolved in 1 mL of 0.9% saline, and an equal amount of aluminum hydroxide adjuvant is added) is performed on the 7th, 14th and 21st day, and 20 μg of OVA solution (1 mg of OVA is dissolved in 1 mL of 0.9% saline) is dripped into the nose of the mice on the 28th-32nd day; 200 μL of 0.9% saline is administered to the mice by gavage every day from the 7th to the 32nd day; and the mice are allowed to eat and drink freely.

[0111] Experimental group: After the new mice adapt for a week, intraperitoneal injection of 200 μL OVA sensitizing solution (1 mg of OVA is dissolved in 1 mL of 0.9% saline, and an equal amount of aluminum hydroxide adjuvant is added) is performed on the 7th, 14th and 21st day, and 20 μg of OVA solution (1 mg of OVA is dissolved in 1 mL of 0.9% saline) is dripped into the nose of the mice on the 28th-32nd day; 200 μL of animal Bifidobacterium lactis CCFM1274 bacterial suspension (bacterial concentration: 5 × 10 9 CFU / mL) is administered to the mice by gavage from the 7th to the 32nd day; and the mice are allowed to eat and drink freely.

[0112] Gavage is performed 2 hours before the OVA solution is dripped into the nose to trigger an allergic reaction. The last trigger is performed on the 32nd day, and the mice are weighed every other week to calculate the percentage (%). Bifidobacterium animalis lactis .

[0113] Table 2: Changes in the body weight of mice in different groups

[0114]

[0115] As can be seen from Table 2 and Bifidobacterium animalis lactis , compared with the blank group, OVA modeling causes the mice to gradually lose weight during the sensitization and trigger periods, especially after five nose drops, on the 32nd day, the body weight of the mice in the model group decreases dramatically (93.32% ± 6.92%), and during this period, the mice have disheveled hair and poor condition. Compared with the model group, the body weight of the mice in the CCFM1274 group decreases slightly (99.52% ± 2.50%) after one week of intraperitoneal injection of OVA, but as the number of days of gavage with animal Bifidobacterium lactis CCFM1274 increases, the body weight of the mice gradually increases and the mice have a weight gain phenomenon (107.533 ± 3.14%), and the nose-dropped OVA solution during the trigger period does not cause the body weight of the mice in this group to decrease dramatically (107.69% ± 6.13%).

[0116] The above experimental results show that animal Bifidobacterium lactis CCFM1274 can effectively prevent the allergic reaction of mice caused by OVA, and can effectively prevent the weight loss of mice caused by OVA. Bifidobacterium animalis lactisBifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis CCFM1274 can effectively alleviate the degree of weight loss in food allergy mice, and maintain the weight of mice.

[0117] Example 5: Bifidobacterium animalis lactis (Bifidobacterium animalis subsp. lactis) CCFM1274 can effectively alleviate the degree of weight loss in food allergy mice, and maintain the weight of mice. Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis Inhibition effect of CCFM1274 on total IgE in serum of asthmatic mice

[0118] 6-8 week-old SPF BALB / c female mice were raised in the Experimental Animal Center of Jiangnan University, fed with ordinary feed, constant temperature 20-26℃, humidity 40%-70%, noise less than or equal to 60 dB, animal illumination 15-20 LX, 12 h light, 12 h dark (all animal experiment procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangnan University).

[0119] The experimental design lasted for 5 weeks, and the body weight of the mice was recorded at the same time every week, a total of 5 times. The first week was the adaptation period of the mice, and the second week was the modeling period. After the adaptation period, the mice were randomly divided into blank group, model group and CCFM1274 group. There were 10 mice in each group.

[0120] Specifically as follows:

[0121] Blank group: After the newly arrived mice were adapted for one week, 200 μL of 0.9% normal saline was intraperitoneally injected on the 7th, 14th and 21st days, 20 μg of 0.9% normal saline was dripped into the nose on the 28th-32nd days; 200 μL of 0.9% normal saline was administered to the mice by gavage from the 7th to the 32nd day; free feeding and drinking.

[0122] Model group: After the newly arrived mice were adapted for one week, 200 μL of OVA sensitizing solution (1 mg of OVA dissolved in 1 mL of 0.9% normal saline, and an equal amount of aluminum hydroxide adjuvant was added) was intraperitoneally injected on the 7th, 14th and 21st days, 20 μg of OVA solution (1 mg of OVA dissolved in 1 mL of 0.9% normal saline) was dripped into the nose on the 28th-32nd days; 200 μL of 0.9% normal saline was administered to the mice by gavage every day from the 7th to the 32nd day; free feeding and drinking.

[0123] Experimental group: After the new mice adapted for one week, intraperitoneal injection of 200 μL of OVA sensitizing solution (1 mg of OVA was dissolved in 1 mL of 0.9% normal saline, and an equal amount of aluminum hydroxide adjuvant was added) was performed on the 7th, 14th and 21st days, and 20 μg of OVA solution (1 mg of OVA was dissolved in 1 mL of 0.9% normal saline) was dripped into the nose of the mice on the 28th-32nd day; 200 μL of Bifidobacterium animalis lactis CCFM1274 bacterial suspension (bacterial concentration: 5 × 10 9 CFU / mL) was administered to the mice by gavage from the 7th to the 32nd day; and the mice were allowed to freely eat and drink.

[0124] In the last week of the challenge period, the mice were administered OVA solution by nose drop 2 hours after gavage. The last challenge was performed on the 32nd day, and the mice were enucleated and blood was collected 3 hours later. The blood was allowed to stand for 2 hours and then centrifuged at 4°C and 3000 r / min for 15 min. The serum was collected and stored in clean PCR tubes at -20°C. The total IgE content in the mouse serum was detected according to the operation instruction of the immunoglobulin ELISA kit. The results are shown in Bifidobacterium animalis lactis .

[0125] The results show that:

[0126] As can be seen from the above results, the total IgE content in the serum of the blank group was the lowest (3688.27 ng / mL), the sensitization and challenge of the allergen OVA caused a significant increase in the total IgE in the serum of the mice (48857.09 ng / mL), and after the mice were administered Bifidobacterium animalis lactis (subsp. Bifidobacterium animalis lactis ) CCFM1274, the total IgE content in the serum was reduced (17647.20 ng / mL) compared with the model group, which was significantly reduced by 63.88% (P<0.01). It shows that the Bifidobacterium animalis lactis (subsp. Bifidobacterium animalis lactis ) CCFM1274 in the present application can inhibit the production of total IgE in the serum of asthmatic mice. Bifidobacterium animalis lactis P Bifidobacterium animalis lactis Bifidobacterium animalis lactis The above experimental results show that the Bifidobacterium animalis lactis (subsp.

[0127] ) CCFM1274 can reduce the total IgE content in the serum of asthmatic mice, which is beneficial to the relief of allergic symptoms. Bifidobacterium ​ ​ Example 6: Bifidobacterium animalis lactis (subsp.

[0128] subsp. Bifidobacterium animalis lactis ) CCFM1274 can reduce the total IgE content in the serum of asthmatic mice, which is beneficial to the relief of allergic symptoms. Figure 3 ​​​​The inhibitory effect of CCFM1274 on serum OVA-sIgE in asthmatic mice

[0129] SPF-grade female BALB / c mice aged 6-8 weeks were housed at the Experimental Animal Center of Jiangnan University. They were fed with ordinary feed, kept at a constant temperature of 20-26℃, humidity of 40%-70%, noise level of less than or equal to 60 dB, and animal illumination of 15-20 LX, with 12 hours of light and 12 hours of darkness (all animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangnan University).

[0130] The experiment lasted for 5 weeks, with mouse body weight recorded at the same time each week for a total of 5 weeks. The first week was the acclimatization period for mice, and modeling began in the second week. After the acclimatization period, mice were randomly divided into a control group, a model group, and a CCFM1274 group. Each group consisted of 10 mice.

[0131] The experimental grouping and treatment methods are the same as in Example 5;

[0132] Blood was collected from the eyeballs of mice 3 hours after nasal drop stimulation on day 37.

[0133] After allowing the blood to stand for 2 hours, centrifuge at 4°C and 3000 r / min for 15 min. Collect the supernatant and detect the OVA-sIgE content in mouse serum. The results are as follows. Figure 3 As shown.

[0134] The results show:

[0135] Depend on Bifidobacterium animalis lactis It was found that the OVA-sIgE level in the serum of the blank group mice was extremely low (0.70 ng / mL), and the sensitization challenge with the allergen OVA led to a significant increase in the OVA-sIgE level in the serum of asthmatic mice (37.88 ng / mL) (P<0.001). However, it was observed that *Bifidobacterium animalis* subsp. *lactam* (… Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis Continuous gavage administration of CCFM1274 reduced the OVA-sIgE level in the serum of asthmatic mice by 24.81% (to 28.48 ng / mL), significantly inhibiting the production of specific immunoglobulins compared to the model group (P<0.001) and suppressing allergic reactions.

[0136] The above results indicate that *Bifidobacterium animalis* subsp. *lactum* (… Bifidobacterium animalis lactis subsp. Figure 4 CCFM1274 significantly reduced the OVA-sIgE content in the serum of asthmatic mice and effectively inhibited allergic symptoms in asthmatic mice.

[0137] Example 7: Bifidobacterium animalis subsp. lactis ( Figure 4 subsp.Bifidobacterium animalis lactis CCFM1274 alleviates lung tissue inflammation in mice.

[0138] SPF-grade female BALB / c mice aged 6-8 weeks were housed at the Experimental Animal Center of Jiangnan University. They were fed with ordinary feed, kept at a constant temperature of 20-26℃, humidity of 40%-70%, noise level of less than or equal to 60 dB, and animal illumination of 15-20 LX, with 12 hours of light and 12 hours of darkness (all animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangnan University).

[0139] The experiment lasted for 5 weeks, with mouse body weight recorded at the same time each week for a total of 5 weeks. The first week was the acclimatization period for mice, and the modeling process began in the second week. After the acclimatization period, mice were randomly divided into a control group, a model group, and a CCFM1274 group. Each group contained 10 mice.

[0140] The experimental grouping and treatment methods are the same as in Example 5;

[0141] Two hours after gavage, an allergic reaction was induced by nasal drops of OVA solution. A final challenge was performed on day 32, followed by enucleation of the eyeballs for blood collection three hours later. After dissection, the left lung was removed and fixed in 4% paraformaldehyde for 24 hours. The lung tissue underwent resting, dehydration, clearing, paraffin embedding, embedding, and sectioning to prepare paraffin sections. Hematoxylin-eosin staining was used, and tissue damage was observed using a digital slide scanner. The results of the HE pathological sections of the lung tissue are shown below. Bifidobacterium animalis lactis

[0142] Depend on Bifidobacterium animalis lactis It was found that the control group mice did not show alveolar wall thickening or inflammatory cell infiltration in their lungs, while the model group mice showed severe lung damage, alveolar collapse, and an increase in inflammatory cells in the lungs. Compared to the model group, *Bifidobacterium animalis* subsp. *lactamase* (… Bifidobacterium animalis lactis subsp. Figure 5 CCFM1274 can improve lung tissue inflammation in mice, reduce the degree of inflammatory cell infiltration around the bronchial and alveolar walls, and alleviate allergic respiratory inflammation in asthmatic mice.

[0143] The above results indicate that *Bifidobacterium animalis* subsp. *lactum* (… Figure 5 subsp. Bifidobacterium animalis lactis CCFM1274 can alleviate lung inflammation in asthmatic mice and inhibit respiratory allergies.

[0144] Example 8: Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis Effects of CCFM1274 on IL-4 levels in bronchoalveolar lavage fluid of asthmatic mice

[0145] SPF grade 6-8 weeks old BALB / c female mice were raised in the experimental animal center of Jiangnan University, fed with ordinary feed, constant temperature 20-26℃, humidity 40%-70%, noise less than or equal to 60 dB, animal illumination 15-20 LX, 12 h light, 12 h dark (all animal experiment procedures were reviewed and approved by the animal welfare and ethics management committee of Jiangnan University).

[0146] The experimental design was 5 weeks, and the body weight of the mice was recorded at the same time every week, a total of 5 times. The first week was the adaptation period of the mice, and the second week was the modeling. After the adaptation period, the mice were randomly divided into blank group, model group and CCFM1274 group. Each group had 10 mice.

[0147] The experimental grouping and treatment method were the same as in Example 5;

[0148] After 2 hours of gavage, the mice were challenged with OVA solution by nasal drops to induce allergic reaction. The last challenge was performed on the 32nd day, and the mice were enucleated and bled 3 hours later. The lung lavage was performed using a retention needle, repeated 2 times, and the lung lavage fluid was obtained. The supernatant was collected by centrifugation at 4℃, 1500 rpm for 5 min, and stored in a-20℃ refrigerator for inflammation factor detection. The IL-4 content in the lung lavage fluid of the mice is shown in Bifidobacterium animalis lactis .

[0149] As can be seen from Figure 6 , the contents of the pro-inflammatory factor IL-4 in the lung lavage fluid of the mice in the blank group, the model group and the CCFM1274 group were 86.44 pg / mL, 300.18 pg / mL and 167.28 pg / mL respectively. Compared with the model group, the content of IL-4 in the lung lavage fluid of the mice gavaged with Bifidobacterium animalis lactis (Bifidobacterium animalis lactis) Figure 6 subsp. Bifidobacterium animalis lactis ) CCFM1274 was reduced by 44.27%, indicating that Bifidobacterium animalis lactis (Bifidobacterium animalis lactis) Bifidobacterium animalis lactis Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis ) CCFM1274 in the present application can inhibit Th2 response.

[0150] The above results show that Bifidobacterium animalis lactis (Bifidobacterium animalis lactis) Figure 7 subsp. Figure 7 ) CCFM1274 can reduce the IL-4 level in the lung lavage fluid of asthmatic mice, alleviate the degree of lung inflammation in asthmatic mice, and inhibit respiratory allergy.

[0151] Example 9: Effect of Bifidobacterium animalis lactis (Bifidobacterium animalis lactis) Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis ) CCFM1274 on the IL-5 level in the lung lavage fluid of asthmatic mice

[0152] SPF-grade female BALB / c mice aged 6-8 weeks were housed at the Experimental Animal Center of Jiangnan University. They were fed with ordinary feed, kept at a constant temperature of 20-26℃, humidity of 40%-70%, noise level of less than or equal to 60 dB, and animal illumination of 15-20 LX, with 12 hours of light and 12 hours of darkness (all animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangnan University).

[0153] The experiment was designed to last 5 weeks, with mouse weight recorded at the same time each week for a total of 5 weeks. The first week was the acclimatization period for mice, and modeling began in the second week. After the acclimatization period, mice were randomly divided into a control group, a model group, and a CCFM1274 group. Each group contained 10 mice.

[0154] The experimental grouping and treatment methods are the same as in Example 5;

[0155] Two hours after gavage, an allergic reaction was induced by nasal drops of OVA solution. A final provocation was performed on day 32, and blood was collected from the eyes 3 hours later. Bronchoalveolar lavage was performed using an indwelling needle, repeated twice, yielding bronchoalveolar lavage fluid. The fluid was centrifuged at 1500 rpm for 5 minutes at 4°C, and the supernatant was collected and temporarily stored at -20°C for inflammatory factor detection. The IL-5 content in the mouse bronchoalveolar lavage fluid is shown in […]. Bifidobacterium animalis lactis .

[0156] Depend on Bifidobacterium animalis lactis The levels of the pro-inflammatory factor IL-5 in the bronchoalveolar lavage fluid of mice in the blank group, model group, and CCFM1274 group were 32.76 pg / mL, 48.87 pg / mL, and 37.82 pg / mL, respectively. Compared with the model group, mice administered Bifidobacterium lactis subsp. *Lactobacillus* via gavage... Figure 8 subsp. Figure 8 After CCFM1274, the IL-5 content in the bronchoalveolar lavage fluid decreased significantly by 22.61% (P<0.05); indicating that the *Bifidobacterium lactis* subsp. *animal* in this invention (… Bifidobacterium animalis lactis Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis CCFM1274 can inhibit the Th2 response.

[0157] The above results indicate that *Bifidobacterium animalis* subsp. *lactum* (… Bifidobacterium animalis lactis subsp. Figure 9 CCFM1274 can reduce the level of IL-5 in the bronchoalveolar lavage fluid of asthmatic mice, alleviate the degree of lung inflammation in asthmatic mice, and inhibit respiratory allergies.

[0158] Example 10: Bifidobacterium animalis subsp. lactis ( Figure 9 subsp. Bifidobacterium animalis lactis Effect of CCFM1274 on IL-13 levels in bronchoalveolar lavage fluid of asthmatic mice

[0159] SPF-grade female BALB / c mice aged 6-8 weeks were housed at the Experimental Animal Center of Jiangnan University. They were fed with ordinary feed, kept at a constant temperature of 20-26℃, humidity of 40%-70%, noise level of less than or equal to 60 dB, and animal illumination of 15-20 LX, with 12 hours of light and 12 hours of darkness (all animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangnan University).

[0160] The experiment was designed to last 5 weeks, with mouse weight recorded at the same time each week for a total of 5 weeks. The first week was the acclimatization period for mice, and modeling began in the second week. After the acclimatization period, mice were randomly divided into a control group, a model group, and a CCFM1274 group. Each group contained 10 mice.

[0161] The experimental grouping and treatment methods are the same as in Example 5.

[0162] Two hours after gavage, mice were given OVA solution via nasal instillation to elicit an allergic reaction. A final provocation was performed on day 32, followed by enucleation of the eyeballs for blood collection three hours later. Bronchoalveolar lavage was performed using an indwelling needle, repeated twice, yielding bronchoalveolar lavage fluid. This fluid was centrifuged at 1500 rpm for 5 minutes at 4°C, and the supernatant was collected and temporarily stored at -20°C for inflammatory factor detection. The IL-13 content in the mouse bronchoalveolar lavage fluid is shown in [reference needed]. Bifidobacterium animalis lactis .

[0163] Depend on Bifidobacterium animalis lactis The levels of the pro-inflammatory factor IL-13 in the bronchoalveolar lavage fluid of mice in the blank group, model group, and CCFM1274 group were 11.91 pg / mL, 16.34 pg / mL, and 14.17 pg / mL, respectively. Compared with the model group mice, the levels of Bifidobacterium lactis subsp. *Lactobacillus* administered via gavage were significantly higher. Bifidobacterium animalis lactis subsp. Figure 10 The IL-13 content in the bronchoalveolar lavage fluid of mice (CCFM1274) decreased by 13.31%, indicating that the *Bifidobacterium lactis* subsp. *animal* in this invention (…) Figure 10 Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis CCFM1274 can inhibit the Th2 response.

[0164] The above results indicate that *Bifidobacterium animalis* subsp. *lactum* (… Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis CCFM1274 can reduce the level of IL-13 in the bronchoalveolar lavage fluid of asthmatic mice, alleviate the degree of lung inflammation in asthmatic mice, and inhibit respiratory allergies.

[0165] Example 11: Bifidobacterium animalis subsp. lactis ( Figure 11 subsp. Figure 11Effect of CCFM1274 on IL-17 level in bronchoalveolar lavage fluid of asthmatic mice

[0166] The 6-8 week old SPF level BALB / c female mice were raised in the experimental animal center of Jiangnan University, fed with ordinary feed, constant temperature 20-26℃, humidity 40%-70%, noise less than or equal to 60 dB, animal illumination 15-20 LX, 12 h light, 12 h dark (all animal experiment procedures were reviewed and approved by the animal welfare and ethics management committee of Jiangnan University).

[0167] The experimental design was 5 weeks, and the body weight of the mice was recorded at the same time every week, a total of 5 times. The first week was the adaptation period of the mice, and the second week was the modeling. After the adaptation period, the mice were randomly divided into blank group, model group and CCFM1274 group. Each group had 10 mice.

[0168] The experimental grouping and treatment method were the same as in Example 5.

[0169] After 2 h of gavage, the mice were challenged with OVA solution by nasal drops to induce allergic reaction. The last challenge was performed on the 32nd day, and the mice were enucleated and bled 3 h later. Bronchoalveolar lavage was performed using a retention needle, repeated 2 times, and lung lavage fluid was obtained. Centrifugation was performed at 4℃, 1500 rpm for 5 min, and the supernatant was collected and temporarily stored in a-20℃ refrigerator for inflammation factor detection. The IL-17 content in the bronchoalveolar lavage fluid of the mice is shown in Bifidobacterium animalis lactis .

[0170] As can be seen from ​ , the contents of pro-inflammatory factor IL-17 in the bronchoalveolar lavage fluid of the mice in the blank group, the model group and the CCFM1274 group were 75.89 pg / mL, 223.25 pg / mL and 161.68 pg / mL respectively. Compared with the model group of mice, the mice gavaged with Bifidobacterium animalis lactis (Bifidobacterium animalis lactis) ​ subsp. ​ ) CCFM1274 reduced the content of IL-17 in the bronchoalveolar lavage fluid of the mice by 27.58%, indicating that Bifidobacterium animalis lactis (Bifidobacterium animalis lactis) ​ ​ subsp. ​ ) CCFM1274 can inhibit Th17 response.

[0171] The above results show that Bifidobacterium animalis lactis (Bifidobacterium animalis lactis) ​ subsp. ​ ) CCFM1274 can reduce the IL-17 level in the bronchoalveolar lavage fluid of asthmatic mice, relieve the degree of lung inflammation in asthmatic mice, and inhibit respiratory allergy.

[0172] Example 12: Bifidobacterium animalis lactis (Bifidobacterium animalis lactis) ​ subsp.​ ) Effect of CCFM1274 on TNF-α level in the bronchoalveolar lavage fluid of asthmatic mice

[0173] The 6-8 week old SPF level BALB / c female mice were raised in the experimental animal center of Jiangnan University, fed with ordinary feed, constant temperature 20-26℃, humidity 40%-70%, noise less than or equal to 60 dB, animal illumination 15-20 LX, 12 h light, 12 h dark (all animal experiment procedures were reviewed and approved by the animal welfare and ethics management committee of Jiangnan University).

[0174] The experimental grouping and treatment method were the same as in Example 5.

[0175] After 2 h of gavage, the allergic reaction was stimulated by nasal dripping of OVA solution. The last stimulation was performed on the 32nd day, and the mice were enucleated for blood collection 3 h later. The bronchoalveolar lavage was performed using a retention needle, repeated 2 times, and the lung lavage fluid was obtained, centrifuged at 4℃, 1500 rpm for 5 min, and the supernatant was collected and temporarily stored in a-20℃ refrigerator for inflammation factor detection. The TNF-α content in the bronchoalveolar lavage fluid of mice is shown in ​ .

[0176] It can be seen that the contents of pro-inflammatory factor TNF-α in the bronchoalveolar lavage fluid of mice in the blank group, the model group and the CCFM1274 group were 44.82 pg / mL, 38.16 pg / mL and 58.74 pg / mL respectively. Compared with the model group of mice, the mice gavaged with Bifidobacterium animalis lactis (Bifidobacterium animalis lactis) ​ subsp. ​ ) CCFM1274, the content of TNF-α in the bronchoalveolar lavage fluid of mice increased by 53.96% (P<0.05); it was proved that Bifidobacterium animalis lactis (Bifidobacterium animalis lactis) ​ subsp. ​ ) CCFM1274 in the application could promote Th1 response. ​ ​ The above results showed that Bifidobacterium animalis lactis (Bifidobacterium animalis lactis) subsp.

[0177] ) CCFM1274 could further increase the level of TNF-α in the bronchoalveolar lavage fluid of asthmatic mice, relieve the degree of lung inflammation of asthmatic mice, and improve the respiratory allergy. ​ ​ Example 13: Bifidobacterium animalis lactis (Bifidobacterium animalis lactis) subsp.

[0178] ) CCFM1274 increased tryptophan metabolite indolepropionic acid in asthmatic mice ​ ​

[0179] ​SPF-grade female BALB / c mice aged 6-8 weeks were housed at the Experimental Animal Center of Jiangnan University. They were fed with ordinary feed, kept at a constant temperature of 20-26℃, humidity of 40%-70%, noise level of less than or equal to 60 dB, and animal illumination of 15-20 LX, with 12 hours of light and 12 hours of darkness (all animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangnan University).

[0180] The experimental grouping and treatment methods are the same as in Example 5.

[0181] The day before the end of the experiment, mice were placed in clean cages and their feces were collected after a period of time and placed in centrifuge tubes. The tubes were then placed on ice and brought back to the laboratory, where they were stored at -80°C. Subsequently, tryptophan-targeted metabolomics was used to detect the target compounds in the mouse feces. The indolepropionic acid content in the feces is shown in [see figure]. ​ .

[0182] Depend on ​ The absolute contents of tryptophan metabolite indolepropionic acid in the feces of mice in the blank group, model group, and CCFM1274 group were 21.40 nmol / kg, 18.02 nmol / kg, and 27.37 nmol / kg, respectively. Compared with the model group mice, the levels of Bifidobacterium lactis subsp. *bifidobacterium* (Bifidobacterium lactis) administered via gavage to animals... ​ subsp. ​ CCFM1274 increased the content of indolepropionic acid, a metabolite of tryptophan, in mouse feces by 51.86%; indicating that the animal Bifidobacterium lactis subsp. (Bifidobacterium lactis) of this invention ( ​ subsp. ​ CCFM1274 can improve the gut-derived tryptophan metabolism process in asthmatic mice.

[0183] The above results indicate that *Bifidobacterium animalis* subsp. *lactum* (… ​ subsp. ​ CCFM1274 can increase the indolepropionic acid content in the feces of asthmatic mice and improve the tryptophan metabolism process under the action of intestinal flora.

[0184] Example 14: Bifidobacterium animalis subsp. lactis ( ​ subsp. ​ CCFM1274 increases the production of indolelactone, a tryptophan metabolite, in asthmatic mice.

[0185] SPF-grade female BALB / c mice aged 6-8 weeks were housed at the Experimental Animal Center of Jiangnan University. They were fed with ordinary feed, kept at a constant temperature of 20-26℃, humidity of 40%-70%, noise level of less than or equal to 60 dB, and animal illumination of 15-20 LX, with 12 hours of light and 12 hours of darkness (all animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangnan University).

[0186] The experiment lasted for 5 weeks, with mouse body weight recorded at the same time each week for a total of 5 weeks. The first week was the acclimatization period for mice, and the modeling process began in the second week. After the acclimatization period, mice were randomly divided into a control group, a model group, and a CCFM1274 group. Each group contained 10 mice.

[0187] The experimental grouping and treatment methods are the same as in Example 5.

[0188] The day before the end of the experiment, mice were placed in clean cages and their feces were collected after a period of time and placed in centrifuge tubes. The tubes were then placed on ice and brought back to the laboratory, where they were stored at -80°C. Subsequently, tryptophan-targeted metabolomics was used to detect the target compounds in the mouse feces. The indolelactone content in the feces is shown in [see figure]. ​ .

[0189] Depend on ​ The absolute contents of tryptophan metabolite indolelactone in the feces of mice in the blank group, model group, and CCFM1274 group were 2267.66 nmol / kg, 2130.77 nmol / kg, and 2936.97 nmol / kg, respectively. Compared with the model group mice, the levels of Bifidobacterium lactis subsp. *bifidobacterium* (Bifidobacterium lactis) administered via gavage were significantly higher. ​ subsp. ​ CCFM1274 upregulated indolelactone, a metabolite of tryptophan, in mouse feces, increasing it by 37.84%, indicating that the animal *Bifidobacterium lactis* subsp. *animal* in this invention (… Bifidobacterium animalis lactis subsp. ​ CCFM1274 can improve the gut-derived tryptophan metabolism process in asthmatic mice.

[0190] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Use of Bifidobacterium animalis subsp. lactis CCFM1274 and / or fermentation broth of Bifidobacterium animalis subsp. lactis CCFM1274 in the preparation of a medicament for alleviating asthma symptoms, said Bifidobacterium animalis subsp. lactis CCFM1274 has been deposited at Guangdong Microbial Culture Collection Center on September 15, 2022, and the accession number is GDMCC No: 62798.

2. Use according to claim 1, characterized in that, The number of viable cells of Bifidobacterium animalis subsp. lactis CCFM1274 in the pharmaceutical product is not less than 1 x 10 6 CFU / mL.

3. Use according to claim 2, characterized in that, The alleviating asthma symptoms includes alleviating nasal allergy symptoms, or reducing the content of specific immunoglobulin in serum, or reducing lung inflammation, or inhibiting the secretion of pro-inflammatory factors in alveolar lavage fluid.

4. Use according to claim 3, characterized in that, The medicament contains Bifidobacterium animalis subsp. lactis CCFM1274, a pharmaceutical carrier and / or a pharmaceutical excipient.

5. Use according to claim 4, characterized in that, The dosage form of the medicament includes granules, capsules, tablets, pills or oral liquids.

6. Use according to claim 4, characterized in that, The pharmaceutical excipients include anti-adhesion agents, penetration enhancers, buffers, plasticizers, surfactants, antifoaming agents, thickening agents, inclusion agents, absorbents, humectants, solvents, propellants, solubilizers, cosolvents, emulsifiers, coloring agents, pH regulators, adhesives, disintegrants, fillers, lubricants, wetting agents, integrating agents, osmotic pressure regulators, stabilizers, flow aids, flavorings, preservatives, foaming agents, suspending agents, coating materials, fragrances, diluents, flocculants and deflocculants, filter aids and release retardants.

7. Use of Bifidobacterium animalis subsp. lactis CCFM1274 in the preparation of a medicament for improving the pathological characteristics of asthma and alleviating asthma allergy symptoms, said Bifidobacterium animalis subsp. lactis CCFM1274 has been deposited at Guangdong Microbial Culture Collection Center on September 15, 2022, and the accession number is GDMCC No: 62798.

8. A method of producing a tryptophan metabolite, characterized by, The method is prepared by fermentation of Bifidobacterium animalis subsp. lactis CCFM1274, said Bifidobacterium animalis subsp. lactis CCFM1274 has been deposited at Guangdong Microbial Culture Collection Center on September 15, 2022, and the accession number is GDMCC No: 62798.

9. Use of Bifidobacterium animalis subsp. lactis CCFM1274 in the preparation of tryptophan metabolites or medicaments containing tryptophan metabolites, said Bifidobacterium animalis subsp. lactis CCFM1274 has been deposited at Guangdong Microbial Culture Collection Center on September 15, 2022, and the accession number is GDMCC No: 62798.

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