Bifidobacterium longum subsp. infantis capable of alleviating and preventing allergic rhinitis, and use thereof

Abstract

Provided are a bifidobacterium longum subsp. infantis capable of alleviating and preventing allergic rhinitis, and a use thereof, relating to the technical fields of microorganisms and medicine. The bifidobacterium longum subsp. infantis CCFM111 can alleviate allergic reactions in allergic mice, and alleviate and prevent allergic disorders in patients with perennial allergic rhinitis. Specifically: (1) the bifidobacterium longum subsp. infantis CCFM111 alleviates weight loss and symptoms caused by allergies in mice, reduces pulmonary inflammation infiltration, lowers serum OVA-sIgE and total IgE levels, and regulates the expression of pro-inflammatory and anti-inflammatory factors. (2) The bifidobacterium longum subsp. infantis CCFM111 alleviates allergic symptoms and reduces medication scores in patients, increases the proportion of symptom-free days of patients, lowers the serum sIgE levels against dermatophagoides farinae and dermatophagoides pteronyssinus in patients, and regulates the expression of cytokines in the serum and nasal lavage fluid of patients. Therefore, the bifidobacterium longum subsp. infantis CCFM111 has broad prospects for application in the preparation of a product for preventing and / or treating allergic rhinitis.

Description

A strain of Bifidobacterium longum infantis that can relieve and prevent allergic rhinitis and its application Technical Field

[0001] This invention relates to a strain of Bifidobacterium longum infantis that can relieve and prevent allergic rhinitis and its applications, belonging to the fields of microbial technology and pharmaceutical technology. Background Technology

[0002] Allergic rhinitis (AR) is a non-infectious chronic inflammatory disease of the nasal mucosa, primarily mediated by immunoglobulin E (IgE), following exposure to allergens in atopic individuals. Typical symptoms include paroxysmal sneezing, clear nasal discharge, nasal itching, and nasal congestion; ocular symptoms may also be present, including itchy eyes, tearing, redness, and burning sensation. Studies indicate that AR affects 10%-40% of adults and 2%-25% of children worldwide. In recent years, with the development of modern industrialization and increasing environmental pollution, the types of allergens have also increased, leading to a significant increase in the number of AR patients. AR has now become one of the most common chronic inflammatory respiratory diseases, and the World Health Organization has listed it as a global health problem, drawing significant attention from the public and society. Currently, there is no cure for allergic rhinitis (AR) in clinical practice. The "Guidelines for the Diagnosis and Treatment of Allergic Rhinitis in China (2022, Revised Edition)" points out that the main clinical treatment strategies currently include: (1) avoiding allergens, (2) conventional drug therapy, and (3) allergen-specific immunotherapy (AIT). However, firstly, some allergens, especially inhaled allergens (such as dust mites, pollen, and mold), are difficult to completely avoid. Secondly, due to the recurrent nature of AR, its treatment drugs usually need to be taken for life, and these drugs can only relieve the patient's immediate allergy symptoms without having any positive impact on the occurrence and development of AR. Moreover, the side effects of long-term use of chemical drugs are also worth noting. In addition, AIT has problems such as long treatment cycles (usually 2-3 years), unstable compliance, and high treatment costs. These factors make the current treatment of AR still quite challenging.

[0003] From an immunological perspective, allergic reactions (AR) are nasal mucosal inflammations primarily caused by an imbalance in the Th1 / Th2 and Treg / Th17 immune responses. Atopic individuals in a sensitized state produce more allergen-specific IgE (sIgE) and total IgE than healthy individuals. Upon re-exposure to the allergen, they recruit and activate immune cells such as mast cells, eosinophils, and basophils, thereby prompting Th2 and Th17 cells to release pro-inflammatory factors while inhibiting the secretion of anti-inflammatory factors by Th1 and Treg cells. The relationship between gut microbiota structure and immune function has attracted widespread attention in the pathogenesis of allergic diseases. Numerous epidemiological surveys have shown that gut microbiota dysbiosis influences the course of AR to some extent. Probiotics are a class of beneficial live microorganisms that colonize the human body and alter the composition of the host's microbiota in a specific area. Probiotics can enhance the intestinal barrier by influencing the composition and metabolism of the gut microbiota, and regulate the host immune system by downregulating the levels of pro-inflammatory factors (such as interleukin-4 (IL-4), IL-13, IL-6 and IL-17) and upregulating the levels of anti-inflammatory factors (such as IL-10) to restore the immune balance of Th1 / Th2 and Th17 / Treg, thereby alleviating AR symptoms.

[0004] Clinically, serum specific allergen-sIgE levels are the primary objective indicator for assessing allergic rhinitis (AR). However, AR patients themselves are more concerned with subjective experiences such as relieving AR symptoms, reducing the frequency of medication use, and preventing recurrent AR attacks. The Combined Symptom and Medication Score (CSMS) scale was used to subjectively score patients' nasal symptoms, ocular symptoms, and routine medication use. The scores were then used to evaluate the efficacy of probiotics in alleviating AR symptoms. Due to the persistent nature of flare-ups in patients with perennial AR (symptom attacks ≥4 days / week, and ≥4 consecutive weeks), the proportion of symptom-free days was compared to evaluate the preventative effect of probiotics on AR. Currently, no edible probiotics have been found that can efficiently regulate serum allergen-sIgE levels in allergic individuals with a regulatory effect superior to that of medications, nor have any probiotics been found that have achieved significant inhibitory effects on AR in clinical applications. Summary of the Invention

[0005] This invention screened a food-safe strain that can efficiently downregulate specific allergen-sIgE in animal and human serum and "effectively alleviate AR symptoms, reduce drug use, and reduce AR recurrence." This provides a new approach and solution to the current situation of allergic rhinitis being difficult to cure, prone to recurrence, and having significant side effects from drug treatment, and provides a powerful tool for clinical application.

[0006] This invention provides a strain of *Bifidobacterium longum* subsp. *infantis* CCFM111, classified as *Bifidobacterium longum* subsp. *infantis*, which was deposited on October 31, 2024, at the Guangdong Provincial Institute of Microbiology, Guangdong Province, with accession number GDMCC No: 65375. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] In one embodiment of the present invention, the *Bifidobacterium longum* subsp. *infantis* CCFM111 was isolated from the feces of exclusively breastfed healthy infants from Changsha, Hunan Province. Sequencing analysis of this strain revealed its 16S rDNA sequence as shown in SEQ ID NO.1. The sequenced sequence was then compared with the BLAST algorithm of NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The results showed a 99.86% similarity to the nucleic acid sequence of *Bifidobacterium longum* subsp. *infantis*, and it was named *Bifidobacterium longum* subsp. *infantis* CCFM111.

[0008] The present invention also provides a microbial preparation containing the above-mentioned Bifidobacterium longum subsp. infantis CCFM111.

[0009] In one embodiment of the present invention, the quantity of *Bifidobacterium longum subsp. infantis* CCFM111 in the microbial preparation is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0010] In one embodiment of the present invention, the microbial preparation contains, by weight percentage: 40-50% fructooligosaccharides, 30-40% maltodextrin, 5-10% xylitol, 5-10% strawberry fruit powder, and 2.5-5% (3.5 × 10⁻⁶) Bifidobacterium longum subsp. infantis CCFM111. 9 ~7×10 9 CFU).

[0011] In one embodiment of the present invention, the Bifidobacterium longum infantis CCFM111 bacterial powder also contains trehalose.

[0012] In one embodiment of the present invention, the Bifidobacterium longum infantis CCFM111 bacterial powder is obtained by resuspending the bacterial precipitate collected after culture with a 100g / L trehalose freeze-drying protectant and then freeze-drying it.

[0013] In one embodiment of the present invention, trehalose at a concentration of 100 g / L and bacterial precipitate are suspended at a mass ratio of 2:1.

[0014] The present invention also provides a product containing live cells, inactivated cells, supernatant, lysate and / or metabolites of Bifidobacterium longum subsp. infantis CCFM111.

[0015] In one embodiment of the present invention, the quantity of Bifidobacterium longum subsp. infantis CCFM111 in the product is not less than 1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0016] In one embodiment of the present invention, the product includes food or medicine.

[0017] In one embodiment of the present invention, the product includes, but is not limited to, ordinary food, special food, and pharmaceuticals.

[0018] In one embodiment of the present invention, the food is a food containing Bifidobacterium longum subsp. infantis CCFM111 or its fermented metabolites.

[0019] In one embodiment of the present invention, the food is a dairy product, soy product, meat product or fruit and vegetable product produced using Bifidobacterium longum subsp. infantis CCFM111 or a fermentation agent containing Bifidobacterium longum subsp. infantis CCFM111.

[0020] In one embodiment of the present invention, the food is a solid beverage containing Bifidobacterium longum subsp. infantis CCFM111.

[0021] In one embodiment of the present invention, the fermentation agent is prepared as follows: Bifidobacterium longum subsp. infantis CCFM111 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 30 h to obtain a culture solution; the culture solution is centrifuged and the bacterial cells are collected; the bacterial cells are washed three times with phosphate buffer solution at pH 7.2 and then resuspended with a lyophilization protectant to obtain a resuspension; the resuspension is lyophilized using a vacuum freeze-drying method to obtain the fermentation agent of Bifidobacterium longum subsp. infantis CCFM111.

[0022] In one embodiment of the present invention, the mass ratio of the freeze-drying protectant to the bacterial cells is 2:1.

[0023] In one embodiment of the present invention, the freeze-drying protectant contains skim milk powder, maltodextrin and monosodium glutamate; wherein the ratio of skim milk powder: maltodextrin: monosodium glutamate is (8-10):(8-10):1.

[0024] In one embodiment of the present invention, the culture medium is prepared by dissolving 10% skim milk, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract in water.

[0025] In one embodiment of the present invention, the pH of the culture medium is 6.8.

[0026] In one embodiment of the present invention, the drug contains Bifidobacterium longum subsp. infantis CCFM111, a drug carrier, and / or pharmaceutical excipients.

[0027] In one embodiment of the present invention, the drug carrier comprises microcapsules, microspheres, nanoparticles, and liposomes.

[0028] In one embodiment of the present invention, the pharmaceutical excipient comprises excipients and additives.

[0029] In one embodiment of the present invention, the pharmaceutical excipients include anti-adhesives, penetration enhancers, buffers, plasticizers, surfactants, defoamers, thickeners, encapsulating agents, absorbents, humectants, solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, pH adjusters, binders, disintegrants, fillers, lubricants, wetting agents, integrators, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, foaming agents, suspending agents, coating materials, fragrances, diluents, flocculants and anti-flocculation agents, filter aids, and release inhibitors.

[0030] In one embodiment of the present invention, the additives comprise microcrystalline cellulose, hydroxypropyl methylcellulose, and refined lecithin.

[0031] In one embodiment of the present invention, the dosage form of the medicine includes granules, capsules, tablets, pills, or oral liquid.

[0032] The present invention also provides the use of the above-mentioned Bifidobacterium longum subsp. infantis CCFM111, or the above-mentioned microbial preparation, in the preparation of medicines for the prevention and / or treatment of allergic rhinitis.

[0033] In one embodiment of the present invention, the main indicator for clinically diagnosing allergic rhinitis is serum specific allergen-sIgE.

[0034] In one embodiment of the present invention, the Bifidobacterium longum infant subspecies CCFM111 has achieved the effect of relieving allergic rhinitis in animal models and has achieved the effect of relieving and preventing allergic rhinitis in clinical treatment.

[0035] In one embodiment of the present invention, improving the pathological features of allergic rhinitis in an animal model includes: alleviating weight loss in mice, alleviating allergic rhinitis symptoms, alleviating pulmonary inflammatory infiltration in mice, downregulating serum OVA-sIgE and total IgE levels in mice, inhibiting the levels of pro-inflammatory cytokines (IL-4, IL-13, IL-6, IL-17) in the lungs, and increasing the levels of anti-inflammatory cytokines (IL-10) in the lungs.

[0036] In one embodiment of the present invention, improving the pathological characteristics of allergic rhinitis in clinical treatment includes: reducing the CSMS score of allergic rhinitis, increasing the proportion of symptom-free days, downregulating the serum levels of house dust mite and house dust mite-sIgE, downregulating the serum levels of pro-inflammatory cytokines (IL-13), upregulating the serum levels of anti-inflammatory cytokines (IL-10), and downregulating the levels of pro-inflammatory cytokines (IL-13) and eosinophil cationic protein (ECP) in the patient's nasal irrigation fluid.

[0037] In one embodiment of the present invention, the number of *Bifidobacterium longum* subsp. infantis CCFM111 cells in the drug is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g. Beneficial effects:

[0038] 1. The *Bifidobacterium longum* subsp. infantis CCFM111 provided by this invention can alleviate allergic rhinitis symptoms in animal models, specifically, compared with the model group:

[0039] (1) Alleviates weight loss in mice with allergic rhinitis;

[0040] (2) Relieves allergic symptoms in mice with allergic rhinitis;

[0041] (3) Reduced lung inflammation infiltration in mice with allergic rhinitis;

[0042] (4) The serum OVA-sIgE level in mice with allergic rhinitis decreased by 60.4% and the serum total IgE level decreased by 50.1%;

[0043] (5) In mice with allergic rhinitis, the expression levels of IL-4 in the lungs decreased by 46.3%, IL-13 by 69.2%, IL-6 by 62.3%, IL-17 by 57.0%, and IL-10 by 291.9%.

[0044] 2. The *Bifidobacterium longum* subsp. infantis CCFM111 provided by this invention can alleviate the symptoms of allergic rhinitis and reduce medication use in clinical treatment, playing an adjunctive therapeutic role. Specifically, compared with the control group (conventional medication group):

[0045] (1) After 8 weeks of intervention, the comprehensive symptoms and medication scores (CSMS) of patients with allergic rhinitis decreased significantly, and the reduction was 1.67 times that of the control group (normal medication group);

[0046] (2) After 2 weeks of intervention, the CSMS scores of patients with allergic rhinitis decreased significantly, and the reduction was 6.25 times that of the control group (normal medication group), indicating that Bifidobacterium longum subsp. infantis CCFM111 was effective after 2 weeks of intervention.

[0047] (3) After 20 weeks of intervention, the CSMS scores of patients with allergic rhinitis decreased significantly, and the reduction was 10 times that of the control group (normal medication group), indicating that the longer the treatment of AR with Bifidobacterium infantis CCFM111 is combined with conventional drugs, the better the effect.

[0048] (4) During the intervention period of weeks 1-8, the proportion of asymptomatic days in patients with allergic rhinitis increased by 52.9%; during the intervention period of weeks 9-20, the proportion of asymptomatic days in patients with allergic rhinitis increased by 75.0%, indicating that adjuvant treatment with Bifidobacterium longum subsp. infantis CCFM111 is significantly helpful in reducing AR recurrence.

[0049] (5) After 20 weeks of intervention, the serum levels of house dust mite and household dust mite s-IgE in patients with allergic rhinitis were significantly reduced;

[0050] (6) After 20 weeks of intervention, the expression level of IL-13 in the serum of patients with allergic rhinitis was significantly reduced, while the expression level of IL-10 was significantly increased.

[0051] (7) After 20 weeks of intervention, the expression levels of IL-13 and ECP in the nasal irrigation fluid of patients with allergic rhinitis were significantly reduced.

[0052] Therefore, Bifidobacterium longum subsp. infantis CCFM111 has great application potential in the preparation of drugs for the prevention and / or treatment of allergic rhinitis.

[0053] Preservation of biological materials

[0054] A strain of *Bifidobacterium longum* subsp. *infantis*, CCFM111, taxonomically named *Bifidobacterium longum* subsp. *infantis*, was deposited on October 31, 2024, at the Guangdong Provincial Institute of Microbiology, Guangdong Province, with accession number GDMCC No: 65375. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0055] Figure 1: Changes in the percentage of body weight of mice in different groups.

[0056] Figure 2: Allergy symptom scores of mice in different groups.

[0057] Figure 3: Pathological sections of lung tissue from mice in different experimental groups.

[0058] Figure 4: Serum OVA-specific IgE and total IgE levels in mice from different experimental groups.

[0059] Figure 5: Cytokine content in lung tissue of mice in different groups.

[0060] Figure 6: Flowchart of animal experiments.

[0061] Figure 7: Changes in mean CSMS scores from baseline on Sundays 7-8 for patients in different groups.

[0062] Figure 8: Changes in mean CSMS scores from baseline on days 2, 4, 6, and 8 for patients in different groups.

[0063] Figure 9: Changes in mean CSMS scores from baseline on days 8, 10, 12, 14, 16, 18, and 20 for patients in different groups.

[0064] Figure 10: Differences in the proportion of symptom-free days in weeks 1-8 and weeks 9-20 among different patient groups.

[0065] Figure 11: Serum-specific IgE levels of house dust mites and house dust mites at the end of week 8 and 20 in different groups of patients.

[0066] Figure 12: Serum IL-13 and IL-10 levels in patients from different groups at the end of week 8 and week 20.

[0067] Figure 13: IL-13 and ECP levels in nasal lavage fluid at the end of week 8 and week 20 in different groups of patients.

[0068] Figure 14: Clinical trial flowchart. Detailed Implementation

[0069] The BALB / c female mice used in the following examples were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0070] The preparation method of the OVA solution involved in the following examples is as follows:

[0071] Sensitization solution: Each mouse was injected intraperitoneally with 0.2 mL of sensitization solution, which was prepared by mixing 0.1 mL of physiological saline containing 30 μg OVA (product number A5503, purchased from Sigma-Aldrich) and 0.1 mL of alum adjuvant (product number 77161, purchased from Thermo).

[0072] Triggering solution: 50 μL of triggering solution was administered intranasally to each mouse. The 50 μL triggering solution was physiological saline containing 50 μg OVA.

[0073] The culture media involved in the following examples are as follows:

[0074] MRS liquid culture medium formula (1L): 10g peptone, 10g beef extract, 5g yeast extract, 20g glucose, 2g K2HPO4, 2g diammonium citrate, 2g sodium acetate, 1mL Tween 80, 0.5g MgSO4·7H2O, 0.5g cysteine ​​hydrochloride, 0.25g MnSO4·4H2O, pH 7.2–7.4.

[0075] MRS solid culture medium formula (1L): 10g peptone, 10g beef extract, 5g yeast powder, 20g grapes, 2g K2HPO4, 2g diammonium citrate, 2g sodium acetate, 1mL Tween 80, 0.5g MgSO4·7H2O, 0.5g cysteine ​​hydrochloride, 0.25g MnSO4·4H2O, 20g agar, pH 7.2–7.4.

[0076] The detection methods involved in the following embodiments are as follows:

[0077] The method for detecting viable bacteria count was based on the national standard GB 4789.35-2016, "National Food Safety Standard for Microbiological Testing of Food - Lactic Acid Bacteria Detection".

[0078] Acidity testing method: GB 431334-2010.

[0079] Bifidobacterium longum infantis subspecies CCFM1192 is another strain isolated from different infant fecal samples using the same isolation method, and has been disclosed in patent CN114276961A.

[0080] The probiotic powder involved in the following examples is obtained by compounding the Bifidobacterium longum subsp. infantis CCFM111 bacterial powder from Example 9 with other substances. The specific formula is (by mass fraction): 40-50% fructooligosaccharides, 30-40% maltodextrin, 5-10% xylitol, 5-10% strawberry fruit powder, and 2.5-5% (3.5 × 10⁻⁶) Bifidobacterium longum subsp. infantis CCFM111 bacterial powder. 9 ~7×10 9 CFU).

[0081] Example 1: Screening, morphological observation and identification of Bifidobacterium longum subsp. infantis CCFM111

[0082] Take 1g of fresh feces from a healthy infant, serially dilute it, spread it on MRS solid medium containing 1% mupirocin, and incubate it at 37℃ for 72h in an anaerobic environment. Observe and record the colony morphology, pick colonies, streak them twice for purification, and incubate them at 37℃ for 48h. Observe the bacteria under a microscope. It was found that the colonies were milky white, round, raised, and smooth. The bacteria were slightly irregular in shape, with round ends, and usually existed singly or in small clusters.

[0083] The strains obtained from the above screening were cultured in fresh MRS liquid culture at 37℃ for 24 h. 1 mL of bacterial suspension was centrifuged at 3500×g to obtain bacterial sludge. Then, 1 mL of sterile water was added to wash the sludge, and the supernatant was removed by centrifugation under the same conditions. 1 mL of sterile water was added again to reconstitute the sludge, preparing a bacterial suspension template. The bacterial 16S rDNA 25 μL reaction system consisted of: 0.5 μL each of 27F forward primer and 1492R reverse primer, 12.5 μL of Taq enzyme, 1 μL of bacterial suspension template, and 10.5 μL of sterile water. PCR reaction conditions: ① 95℃, 5 min; ② 95℃, 10 s; ③ 55℃, 30 s; ④ 72℃, 30 s; ⑤ Steps 2-4 were repeated 30 times; ⑥ 72℃, 5 min; ⑦ 2℃, 2 min. The PCR amplification products were sent to a professional sequencing company for sequencing using a next-generation sequencer. The nucleotide sequence of the 16S rDNA amplified from CCFM111 is shown in SEQ ID NO.1. The sequenced results were aligned using BLAST, showing a 99.86% and 100.00% similarity to the nucleic acid sequence of *Bifidobacterium longum* subsp. *infantis*, respectively. The strain was named *Bifidobacterium longum* subsp. *infantis* CCFM111. The strain was stored at -80°C for future use.

[0084] Example 2: Culture of Bifidobacterium longum subsp. infantis CCFM111

[0085] Bifidobacterium longum subsp. infantis CCFM111 and CCFM1192 were inoculated into MRS liquid medium and cultured in an anaerobic incubator at 37°C for 24 h. Then, the bacterial culture was transferred to fresh MRS liquid medium at an inoculation rate of 4% and cultured under the same conditions for 16-24 h. The bacterial cells were centrifuged at 8000×g for 15 min, washed with 0.9% physiological saline, and centrifuged again at 8000×g for 10 min. The bacterial cells were collected, resuspended in 30% (m / v) sucrose solution, and frozen at -80°C for later use.

[0086] Preparation of bacterial suspension for gavage: When using Bifidobacterium longum subsp. infantis strain for gavage in mice, the strain was taken out from a -80℃ freezer, centrifuged at 4℃ and 8000×g for 10 min, the supernatant was discarded, and the suspension was resuspended in 0.9% physiological saline to obtain the probiotic suspension for gavage.

[0087] Example 3: The alleviating effect of Bifidobacterium longum subsp. infantis CCFM111 on weight loss in mice with allergic rhinitis

[0088] Six-week-old SPF-grade female BALB / c mice were divided into five groups: a blank control group, a model group, a positive control group (dexamethasone acetate tablets DXMS), and experimental groups (CCFM111 group and CCFM1192 group); eight mice were in each group. They were housed in the Experimental Animal Center of Jiangsu Institute of Schistosomiasis Control, fed with ordinary feed, kept at a constant temperature of 21-26℃, humidity of 40-70%, noise level of ≤60dB, and animal illumination of 15-20LX (all animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangsu Institute of Schistosomiasis Control).

[0089] The experiment lasted 34 days, and the experimental method is shown in Figure 6. The acclimatization period for mice was 7 days; from day 8 to day 33, the control group and model group were administered 0.2 mL of physiological saline solution by gavage daily, while the CCFM111 group and CCFM1192 group were administered 0.2 mL of live bacteria solution with a count of 1×10⁻⁶ cells by gavage daily. 9 CFU / mL corresponding bacterial suspensions were prepared using the same method as in Example 2. The positive control group was administered 0.2 mL of physiological saline solution by gavage daily from day 8 to day 28, and 0.2 mL of 0.45 mg / mL DXMS solution by gavage daily from day 29 to day 33. Mice in the model group, positive control group, and experimental group were sensitized by intraperitoneal injection of 200 μL of sensitization solution (containing 30 μg OVA and 1 mg aluminum hydroxide adjuvant) on days 8, 15, and 22. From day 29 to day 33, mice were treated with 50 μL of OVA stimulation solution (concentration 1 mg / mL) via nasal drops. Mice were sacrificed on day 34. All groups had free access to water and food during this period.

[0090] Mouse body weight was recorded before each sensitization and before the last challenge. The results are shown in Table 1 and Figure 1.

[0091] Table 1: Percentage change in mouse body weight (body weight on day 8 is 100%)

[0092] The experimental results are shown in Table 1 and Figure 1. The establishment of the AR model and the OVA-induced allergic reaction caused a decrease in mouse weight. As shown in Table 1 and Figure 1, compared with the model group, the CCFM111 group effectively alleviated the weight loss in mice caused by allergic rhinitis, and its allergic effect was better than that of the positive control group and the CCFM1192 group.

[0093] The above experimental results indicate that, compared with the positive control group and Bifidobacterium longum subsp. infantis CCFM1192, Bifidobacterium longum subsp. infantis CCFM111 is more effective in alleviating the degree of weight loss in mice with allergic rhinitis.

[0094] Example 4: The allergic effect of Bifidobacterium longum subsp. infantis CCFM111 on allergic symptoms in mice with allergic rhinitis.

[0095] For specific implementation details, please refer to Example 3. Within 30 minutes after the final stimulation, the pathological scores of the mice's allergic rhinitis symptoms were assessed, with the following scoring criteria:

[0096] Table 2: Behavioral Scoring Table for Allergic Rhinitis in Mice

[0097] The experimental results are shown in Figure 2. As shown in Figure 2, the control group mice had smooth and shiny fur and no allergic symptoms, while the model group mice had messy, dull fur with piloerection and exhibited significant sluggishness. Compared with the model group mice, the positive drug administered via gavage, Bifidobacterium longum infantis CCFM111, and Bifidobacterium longum infantis CCFM1192 all significantly alleviated the pathological characteristics of mice with allergic rhinitis; compared with the CCFM1192 group, the CCFM111 group showed more significant symptom relief.

[0098] The above experimental results indicate that, compared with Bifidobacterium longum subsp. infantis CCFM1192, Bifidobacterium longum subsp. infantis CCFM111 is more effective in alleviating allergic symptoms in mice with allergic rhinitis.

[0099] Example 5: The alleviating effect of Bifidobacterium longum subsp. infantis CCFM111 on the severity of lung lesions in mice with allergic rhinitis.

[0100] For specific implementation details, refer to Example 3. Mice were sacrificed on day 34, and tissue samples from the left lung were collected and fixed with 4% paraformaldehyde solution. The samples then underwent washing, dehydration, clearing, paraffin embedding, sectioning, spreading, mounting, baking, hematoxylin-eosin (HE) staining, differentiation, rinsing, counterstaining, dehydration, clearing, and mounting to prepare HE-stained lung tissue sections. The HE pathological section results of the lung tissue are shown in Figure 3.

[0101] As shown in Figure 3, compared with the control group, the model group mice showed extensive infiltration of eosinophils and lymphocytes in the trachea, bronchi, alveoli, and perivascular areas, significant mucosal epithelial hyperplasia, severe airway epithelial damage, and thickening of alveolar septa. The positive drug group, CCFM111 group, and CCFM1192 group mice showed significantly reduced lung pathological damage, significantly reduced inflammatory cell infiltration in the lung tissue and surrounding areas, mild mucosal epithelial hyperplasia, reduced airway epithelial damage, and less severe alveolar septal thickening; and the CCFM111 group showed better remission than the CCFM1192 group.

[0102] The above experimental results indicate that, compared with the positive control group and Bifidobacterium longum subsp. infantis CCFM1192, Bifidobacterium longum subsp. infantis CCFM111 is more effective in improving lung pathology and inhibiting inflammatory infiltration in mice with allergic rhinitis.

[0103] Example 6: The alleviating effect of Bifidobacterium longum subsp. infantis CCFM111 on serum OVA-specific IgE and total IgE levels in mice with allergic rhinitis.

[0104] For specific implementation details, refer to Example 3. On day 34, after enucleating the eyeballs and collecting blood, and euthanizing the mice, the mouse blood was allowed to stand for 2 hours and then centrifuged at 3000 r / min for 15 min. The mouse serum was collected, and the OVA-specific IgE content (catalog number 439807, purchased from BioLegend) and total IgE content (catalog number NBP3-18786, purchased from Novus) in the mouse serum were measured using an ELISA kit. The results are shown in Figure 4.

[0105] As shown in Figure 4A, after oral administration of Bifidobacterium longum subsp. infantis CCFM111 to mice, the serum OVA-specific IgE content was significantly reduced to 9.42±2.61 ng / mL, which was significantly lower than that in the model group (OVA-sIgE content was 23.78±1.32 ng / mL) (p<0.001), a reduction of 60.4%. This indicates that the strain Bifidobacterium longum subsp. infantis CCFM111 in this invention can significantly inhibit the production of OVA-specific IgE. After oral administration of Bifidobacterium longum infantis subsp. CCFM1192 to mice, the serum OVA-specific IgE level was significantly reduced to 16.85±4.14 ng / mL, which was significantly lower than that in the model group (p<0.05), a decrease of 29.1%. After oral administration of the positive drug to mice (OVA-sIgE level was 18.53±8.07 ng / mL), there was also a reduction compared with the model group, but the difference was not statistically significant.

[0106] As shown in Figure 4B, after oral administration of Bifidobacterium longum subsp. infantis CCFM111 to mice, the serum total IgE content was significantly reduced to 43.79±10.98 μg / mL, which was significantly lower than that of the model group (total IgE content was 87.69±28.89 μg / mL) (p<0.05), a reduction of 50.1%. There was no significant difference between the model group and the normal group (total IgE content was 27.95±4.06 μg / mL), indicating that the strain Bifidobacterium longum subsp. infantis CCFM111 in this invention can significantly inhibit the production of total IgE. After oral administration of Bifidobacterium longum subsp. infantis CCFM1192 to mice, the total IgE content in the serum was significantly reduced to 50.04±5.96 μg / mL, which was significantly lower than that in the model group (p<0.05), a decrease of 42.9%. After oral administration of the positive drug to mice (total IgE content was 59.76±48.25 μg / mL), there was also a reduction compared with the model group, but the difference was not statistically significant.

[0107] The above experimental results indicate that, compared with the positive control group and Bifidobacterium longum subsp. infantis CCFM1192, Bifidobacterium longum subsp. infantis CCFM111 can significantly reduce the levels of OVA-specific IgE and total IgE in the serum of mice with allergic rhinitis.

[0108] Example 7: Effects of Bifidobacterium longum subsp. infantis CCFM111 on cytokine expression levels in lung tissue of mice with allergic rhinitis

[0109] For specific implementation details, refer to Example 3. After euthanizing mice on day 34, tissue samples from the right lung were collected. The right lung tissue was rinsed with pre-cooled PBS to remove residual blood and surrounding adipose tissue. After weighing, the tissue was minced. The minced tissue was mixed with RIPA lysis buffer at a ratio of 1:9 (g:mL) and lysed using a tissue homogenizer at 65 Hz for 30 seconds (6-8 cycles). Finally, the tissue was centrifuged at 5000×g for 10 min. The supernatant was used to detect the expression level of pro-inflammatory factor IL-4 in the lung tissue using an ELISA kit (catalog number VAL603, purchased from R&D). The results are shown in Figure 5A. The supernatant was also used to detect the expression level of pro-inflammatory factor IL-13 in the lung tissue using an ELISA kit (catalog number M13000CB, purchased from R&D). The expression levels of pro-inflammatory factor IL-6 in lung tissue were measured using an ELISA kit (catalog number M6000B, purchased from R&D), and the results are shown in Figure 5B. Similarly, the expression levels of pro-inflammatory factor IL-17 in lung tissue were measured using an ELISA kit (catalog number VAL610, purchased from R&D), and the results are shown in Figure 5D. Finally, the expression levels of anti-inflammatory factor IL-10 in lung tissue were measured using an ELISA kit (catalog number M1000B-1, purchased from R&D), and the results are shown in Figure 5E.

[0110] As shown in Figure 5A, after oral administration of Bifidobacterium longum subsp. infantis CCFM111 to mice, the IL-4 content in lung tissue (3.23±0.43 pg / mg protein) was significantly reduced, significantly lower than that in the model group (IL-4 content 6.01±0.85 pg / mg protein) (p<0.01), a reduction of 46.3%; and lower than that in the blank group (IL-4 content 3.74±0.91 pg / mg protein) by 13.6%. This indicates that the strain Bifidobacterium longum subsp. infantis CCFM111 in this invention can inhibit the Th2 response. After oral administration of the positive control drug (IL-4 content 4.49±1.39 ng / mL) and CCFM1192 (IL-4 content 5.07±1.91 ng / mL) to mice, the IL-4 content was also reduced compared with the model group, but the difference was not statistically significant.

[0111] As shown in Figure 5B, after oral administration of Bifidobacterium longum subsp. infantis CCFM111 to mice, the IL-13 content in lung tissue (4.58±0.61 pg / mg protein) was significantly lower than that in the model group (14.89±4.98 pg / mg protein) (p=0.001), a decrease of 69.2%; and lower than that in the blank group (5.19±2.28 pg / mg protein) by 11.8%. This indicates that the strain Bifidobacterium longum subsp. infantis CCFM111 in this invention can inhibit the Th2 response. After oral administration of the positive control drug (IL-13 content 5.30±1.92 ng / mL) and CCFM1192 (IL-13 content 5.40±3.33 ng / mL) to mice, the IL-13 content was also significantly lower than that in the model group (p=0.002).

[0112] As shown in Figure 5C, after oral administration of Bifidobacterium longum subsp. infantis CCFM111 to mice, the IL-6 content in lung tissue (13.34±5.01 pg / mg protein) was significantly lower than that in the model group (35.36±9.12 pg / mg protein) (p<0.01), a decrease of 62.3%; and lower than that in the blank group (17.84±2.91 pg / mg protein) by 25.2%. This indicates that the strain Bifidobacterium longum subsp. infantis CCFM111 in this invention can inhibit the expression of pro-inflammatory factors. After oral administration of the positive drug (IL-6 content 14.96±1.24 ng / mL) and CCFM1192 (IL-6 content 19.73±2.29 ng / mL) to mice, the levels were also significantly lower than those in the model group, but the significance was not as great as that of CCFM111.

[0113] As shown in the 5D results, after oral administration of Bifidobacterium longum subsp. infantis CCFM111 to mice, the IL-17 content in lung tissue (0.80±0.09 pg / mg protein) was significantly lower than that in the model group (1.86±0.37 pg / mg protein) (p<0.01), a decrease of 57.0%; however, there was no significant decrease compared to the control group (0.31±0.26 pg / mg protein). This indicates that the strain Bifidobacterium longum subsp. infantis CCFM111 in this invention can inhibit the expression of pro-inflammatory factors. After oral administration of the positive control drug (IL-17 content 0.33±0.10 ng / mL) and CCFM1192 (IL-17 content 1.19±0.28 ng / mL) to mice, the levels were also significantly lower than those in the model group.

[0114] As shown in Figure 5E, after oral administration of Bifidobacterium longum subsp. infantis CCFM111 to mice, the IL-10 content in lung tissue (40.56±20.08 pg / mg protein) was significantly higher than that in the model group (10.35±0.76 pg / mg protein) (p<0.05), an increase of 291.9%; however, there was no significant increase compared to the blank group (16.57±0.80 pg / mg protein). This indicates that the strain Bifidobacterium longum subsp. infantis CCFM111 in this invention can promote the expression of anti-inflammatory factors. After oral administration of the positive drug (IL-10 content 12.68±6.01 ng / mL) and CCFM1192 (IL-10 content 18.27±13.85 ng / mL) to mice, there was also an increase compared to the model group, but the difference was not statistically significant.

[0115] The above experimental results indicate that, compared with Bifidobacterium longum subsp. infantis CCFM1192, Bifidobacterium longum subsp. infantis CCFM111 can significantly downregulate the levels of pro-inflammatory cytokines IL-4, IL-6, and IL-17 in the lung tissue of rhinitis mice, and upregulate the level of anti-inflammatory cytokine IL-10.

[0116] In summary, the *Bifidobacterium infantis* subspecies CCFM111 provided by this invention can effectively alleviate rhinitis symptoms caused by OVA. In regulating mouse body weight, OVA-IgE, total IgE, and IL-10, it achieves significantly better levels than the drug group; in regulating pro-inflammatory factors, it also achieves levels comparable to the drug group. Its overall regulatory effect is superior to the previously reported *Bifidobacterium infantis* subspecies CCFM1192, achieving unexpected technical results. Compared with strain B10 reported in the literature "Bifidobacterium infantis Relieves Allergic Asthma in Mice by Regulating Th1 / Th2", strain B10's regulatory effect on serum OVA-IgE and total IgE is comparable to the drug group, failing to achieve a superior effect. Therefore, the *Bifidobacterium infantis* subspecies CCFM111 provided by this invention is superior to strain B10 in alleviating allergy symptoms.

[0117] Example 8: Multicenter, prospective, randomized, double-blind, placebo-controlled clinical trial

[0118] I. The specific sample standards in this embodiment are as follows:

[0119] 1. Selection Criteria

[0120] Individuals must meet all of the following criteria to be eligible to participate in the trial:

[0121] (1) The child has a positive skin prick test (SPT) or abnormal specific IgE level for at least one of the four common perennial allergens (dust mites, German cockroaches, animal dander, and mold); (2) The child has two or more symptoms of allergic rhinitis, such as nasal congestion, runny nose, itching, and paroxysmal sneezing, and the daily symptoms last for or accumulate for more than 1 hour; (3) The child has moderate or severe perennial allergic rhinitis, and the classification and judgment criteria are based on the "Chinese Guidelines for the Diagnosis and Treatment of Allergic Rhinitis 2022 Edition"; (4) The child is 3-12 years old, male or female; (5) The child has good compliance and can follow the requirements of this clinical trial without violating the regulations.

[0122] 2. Exclusion criteria

[0123] Individuals who meet any of the following criteria will be excluded from this trial:

[0124] (1) Allergy or intolerance to probiotic products or placebo; (2) Immunosuppressed patients; patients with a history of malignant diseases; patients with respiratory diseases, especially asthma (excluding mild intermittent asthma); patients with mental illness; and patients with any other diseases that may interfere with the assessment of trial results; (3) Patients receiving AIT during treatment; (4) Patients who have repeatedly taken probiotic products or consumed yogurt in the previous month; (5) Patients (legal guardians) who refuse to sign informed consent forms, or patients whose compliance is estimated to be poor and whose follow-up is unlikely; (6) Other circumstances that do not meet the inclusion criteria.

[0125] 3. Criteria for quitting midway

[0126] (1) Cases that do not meet the inclusion criteria but are mistakenly included; (2) Cases that have never taken probiotic products or have interrupted taking probiotics for more than 10 days or have consumed other probiotic powder-related products; (3) Cases that use drugs other than the concomitant treatments permitted by the study to treat AR; (4) Cases with poor compliance and no trial records during the trial; (5) Cases that use T-cell inhibitors or T-cell modulation ointments during the trial; (6) Cases with acute bacterial or viral infection complicated by sinusitis.

[0127] 4. Shedding Standard

[0128] (1) The patient needs to withdraw from the trial after a professional evaluation by the doctor; (2) The patient voluntarily requests to withdraw from the trial; (3) Administrative factors: such as moving, economic factors, inability to follow up regularly, etc.

[0129] II. Patient Grouping and Intervention

[0130] In this embodiment, all patients recruited had their informed consent forms signed by their guardians, and the process was approved by the Ethics Committees of Wuxi Children's Hospital and Wuxi Maternal and Child Health Hospital. The recruitment process and grouping for this embodiment are shown in Figure 14. A total of 182 AR patients were recruited and randomly divided into three groups using a simple randomization method.

[0131] (1) 8-week probiotic group: First take probiotic powder for 8 weeks (1 sachet per day), then take placebo for 12 weeks (1 sachet per day);

[0132] (2) 20-week probiotic group: taking probiotic powder (1 sachet per day) for 20 weeks;

[0133] (3) Control group (normal medication group): took placebo for 20 weeks (1 sachet per day).

[0134] Among them: Probiotic powder (1.5g / bag), the formula is (by mass fraction): fructooligosaccharides 48%, maltodextrin 33.5%, xylitol 7%, strawberry fruit powder 7%, Bifidobacterium longum subsp. infantis CCFM11 13.5% (5×10 9 CFU); Placebo (1.5g / sachet), formula (by mass fraction): 48% fructooligosaccharides, 38% maltodextrin, 7% xylitol, 7% strawberry fruit powder. The probiotic powder and placebo are identical in packaging and appearance, both produced by Microcare Probiotics (Suzhou) Co., Ltd.

[0135] During the intervention period, both the probiotic group and the control group (normal medication group) were only permitted to use oral / intranasal antihistamines (H1A), intranasal corticosteroids (INCS), and oral corticosteroids (OCS).

[0136] III. Research Steps and Related Inspections

[0137] (1) The patient’s parents should evaluate the patient’s daily AR symptoms and daily medication use according to the combined symptom and medication score (CSMS) criteria (see Table 3) and record them in the online form.

[0138] (2) Blood samples, nasal secretion samples and stool samples were collected from patients before intervention (baseline), at the end of week 8 and at the end of week 20.

[0139] (3) Patients need to be followed up at weeks 2, 4, 8, 12, 16 and 20 and report symptoms and adverse events to the doctor.

[0140] Table 3: Comprehensive Symptom and Medication Rating Scale (CSMS)

[0141] IV. Endpoint Indicators

[0142] 1. Primary endpoint: The efficacy of probiotics versus placebo as adjunctive therapy for AR was evaluated by comparing the change in mean daily CSMS scores at week 7–8 from baseline scores (CSMS scores before intervention) in different groups of patients (ΔCSMS = mean daily CSMS at week 7–8 - mean daily CSMS at baseline).

[0143] 2. Secondary endpoint:

[0144] (1) The onset time of probiotics was evaluated by comparing the changes in the average daily CSMS score of different groups of patients from baseline every 2 weeks (ΔCSMS = average daily CSMS at each time point - average daily CSMS at baseline); the effect of continuous intervention for 20 weeks; and the duration of the effect of probiotics after intervention was stopped for 8 weeks.

[0145] (2) The effect of probiotics on AR relapse was evaluated by comparing the proportion of symptom-free days in different groups of patients during weeks 1-8 and weeks 9-20 (proportion of symptom-free days = number of symptom-free days / total number of recorded days);

[0146] (3) Compare the changes in serum levels of house dust mite, house dust mite-specific IgE, and cytokines interleukin-13 (IL-13) and IL-10 in different groups of patients at the end of week 8 and week 20 compared with baseline.

[0147] (4) Compare the changes in the levels of the inflammatory factor IL-13 and eosinophil cationic protein (ECP) in the nasal irrigation fluid of different groups of patients at the end of week 8 and week 20 compared with baseline;

[0148] (5) Safety analysis of probiotics.

[0149] V. Statistical Analysis

[0150] Efficacy evaluation analyses employed a Full Analysis Set (FAS), which included eligible and dropout cases but excluded excluded cases. For dropout cases with baseline efficacy data but missing primary efficacy endpoints, their final efficacy endpoint results were considered final outcomes for statistical analysis based on Intention To Treat (ITT) data. Safety metrics were analyzed using a Security Data Set (SS), defined as all participants who used the investigational product at least once.

[0151] SPSS 26 statistical software was used to analyze baseline data, symptom improvement data, etc. The Mann-Whitney test was used to statistically analyze differences between two independent samples, the Wilcoxon matched-pairs signed rank test was used to statistically analyze differences between two paired samples, and the non-parametric Kruskal-Wallis test was used to analyze the significance of differences among multiple groups. P < 0.05 was considered statistically significant. * indicates within-group difference P < 0.05, # indicates between-group difference P < 0.05, ** and ## indicate P < 0.01, and *** and ### indicate P < 0.001.

[0152] VI. Experimental Results

[0153] 1. Patient Basic Information

[0154] The probiotic group, comprising 115 participants (the two probiotic intervention groups were combined and referred to as the probiotic group for data analysis within the first 8 weeks), and the control group, comprising 54 participants, were ultimately included in the data analysis. Basic information of the included patients is shown in Table 4. There were no significant differences in baseline characteristics between the probiotic group and the control group.

[0155] Table 4 Baseline data of patients in each group Note: TNSS stands for Total Nasal Symptom Score.

[0156] 2. The adjunctive therapeutic effect of probiotics on AR

[0157] The changes in the daily average ΔCSMS of the probiotic group and the control group (normal medication group) during weeks 7-8 (ΔCSMS = daily average CSMS during weeks 7-8 - daily average CSMS during the baseline period) are shown in Figure 7.

[0158] The results showed that, under the same routine treatment regimen, the ΔCSMS of patients in the probiotic group (-0.87±0.94) was significantly lower than that in the control group (normal medication group, -0.15±0.66) (p<0.001). The decrease in CSMS in the probiotic group was 1.67 times that in the control group, indicating that after 8 weeks of adjuvant treatment with Bifidobacterium longum subsp. infantis CCFM111, it can effectively alleviate patients' allergy symptoms and medication use.

[0159] The above experimental results indicate that Bifidobacterium longum subsp. infantis CCFM111, as an adjunct to conventional drug treatment, can significantly alleviate allergic symptoms in patients with acute rheumatoid arthritis (AR).

[0160] 3. Effects of probiotic intervention on patients' CSMS scores at 2, 4, 6, and 8 weeks.

[0161] The changes in the daily average ΔCSMS of the probiotic group and the control group at weeks 2, 4, 6 and 8 (ΔCSMS = daily average CSMS at each time point - daily average CSMS at baseline) are shown in Figure 8.

[0162] As shown in Figure 8, in the second week, the ΔCSMS of the probiotic group (-0.75±0.82) was significantly lower than that of the control group (-0.12±0.60) (P<0.001). At this time, the decrease in CSMS in the probiotic group was 6.25 times that in the control group. This indicates that probiotics are very helpful in assisting conventional drug treatment compared with drug treatment alone in the second week.

[0163] The above experimental results indicate that Bifidobacterium longum subsp. infantis CCFM111 is effective after 2 weeks of intervention.

[0164] 4. Effects of probiotic intervention on patients' CSMS scores at 8, 10, 12, 14, 16, 18, and 20 weeks.

[0165] The daily average ΔCSMS changes for the three groups in weeks 8, 10, 12, 14, 16, 18, and 20 (ΔCSMS = daily average CSMS at each time point - daily average CSMS at the baseline period) are shown in Figure 9 and Table 5.

[0166] Table 5. Changes in ΔCSMS in each group of patients at weeks 8, 10, 12, 14, 16, 18, and 20.

[0167] As shown in Figure 9 and Table 5, the ΔCSMS of patients in the probiotic group (Figure 9 right) was significantly different from that in the control group (Figure 9 left) at weeks 8, 10, 12, 14, 16, 18, and 20 (P < 0.001). At week 20, the decrease in CSMS in the probiotic group (-1.40 ± 1.00) was 10 times that of the control group (-0.14 ± 1.09). Table 5 shows that the difference between the 20-week group and the control group increased with the duration of probiotic intervention, indicating that the longer the probiotic intervention combined with conventional drug treatment, the better the effect. The ΔCSMS of patients in the 8-week probiotic group (Figure 9 middle) was significantly different from that in the control group (Figure 9 left) at week 12 (P < 0.01). At this time, the decrease in CSMS in the 8-week group was 6.29 times that in the control group. This indicates that the adjuvant therapeutic effect of probiotics is continuous and can be maintained for 4 weeks after the probiotic intervention is stopped.

[0168] The above experimental results indicate that the adjunctive therapeutic effect of Bifidobacterium longum subsp. infantis CCFM111 increases with the duration of intervention; its effect can be maintained for 4 weeks after the intervention is stopped at 8 weeks.

[0169] 5. The effect of probiotic intervention on the proportion of symptom-free days in patients

[0170] The differences in the proportion of asymptomatic days among the three groups during weeks 1-8 and 9-20 (proportion of asymptomatic days = number of asymptomatic days / total number of recorded days) are shown in Figure 10.

[0171] As shown in Figure 10 (left), during the intervention period of weeks 1-8, the proportion of symptom-free days in the probiotic group (0.26±0.25) was significantly higher than that in the control group (0.17±0.22) (P<0.05), an increase of 52.9%. This indicates that probiotic intervention for 8 weeks has a positive effect on reducing AR symptom onset. As shown in Figure 10 (right), during the intervention period of weeks 9-20, the proportion of symptom-free days in the probiotic group (0.42±0.37) at week 20 was also significantly higher than that in the control group (0.24±0.27) (P<0.05), an increase of 75%. Furthermore, the intra-group difference was significantly increased compared to the probiotic group in weeks 1-8 (P<0.001), an increase of 61.5%. This indicates that probiotic intervention for 20 weeks is helpful in reducing AR symptom onset, and its effect is better than that of 8 weeks of intervention.

[0172] The above experimental results indicate that intervention with Bifidobacterium longum subsp. infantis CCFM111 is very helpful in reducing the occurrence of AR symptoms, and the effect is better with the increase of intervention time.

[0173] 6. Effects of probiotic intervention on changes in serum house dust mite and household dust mite-specific IgE levels from baseline in patients.

[0174] The changes in serum Δ house dust mite-sIgE levels in the three groups at the end of week 8 and week 20 (Δ house dust mite-sIgE = house dust mite-sIgE levels at the end of week 8 and week 20 - house dust mite-sIgE levels at baseline) are shown in Figure 11A, and the changes in serum Δ house dust mite-sIgE levels (Δ house dust mite-sIgE = house dust mite-sIgE levels at the end of week 8 and week 20 - house dust mite-sIgE levels at baseline) are shown in Figure 11B.

[0175] As shown in Figure 11A, after 8 weeks of probiotic intervention, the serum level of Δ house dust mite-sIgE was 1.44±15.76 kU. A The concentration of 5.52 kDa / L was lower than that of the control group (5.52 ± 15.54 kU). AThe result ( / L) indicates that increasing probiotic intervention is more effective than drug treatment alone in inhibiting the production of house dust mite-sIgE in the body. After 20 weeks of probiotic intervention, the serum level of Δ house dust mite-sIgE was (-4.65±24.38kU). A The concentration of 11.41 kDa ( / L) was lower than that of the control group (11.41 ± 23.54 kU). A The level of ΔD / L was significantly reduced (P < 0.05), indicating that probiotic intervention for 20 weeks could significantly downregulate the expression of house dust mite-sIgE in the serum of atopic individuals; at this point, the serum ΔD / L level of house dust mite-sIgE in the probiotic group after 8 weeks was 1.14 ± 24.76 kU. A The concentration of probiotics in the urine was 99.9% (L), which was still lower than that in the control group, indicating that the effect of probiotics is sustained.

[0176] As shown in Figure 11B, after 8 weeks of probiotic intervention, the serum level of ΔHouse dust mite-sIgE was 1.69±11.23 kU. A The concentration of 4.24 kU / L was lower than that of the control group (4.24 ± 13.18 kU). A The result ( / L) indicates that probiotic intervention is more effective than drug treatment alone in inhibiting the production of house dust mite-sIgE in the body. After 20 weeks of probiotic intervention, the serum level of Δ house dust mite-sIgE was (-7.41±12.37kU). A The concentration of 22.14 kU / L was lower than that of the control group (22.14 ± 25.35 kU). A The level of ΔD. sIgE in the serum of the probiotic group was significantly reduced (P < 0.05), indicating that 20 weeks of probiotic intervention could significantly downregulate the expression of ΔD. sIgE in the serum of atopic individuals; at this point, the level of ΔD. sIgE in the serum of the probiotic group after 8 weeks was -3.08 ± 14.43 kU. A The concentration of probiotics in the urine was 99.9% (L), which was still lower than that in the control group, indicating that the effect of probiotics is sustained.

[0177] The above experimental results indicate that intervention with Bifidobacterium longum subsp. infantis CCFM111 can significantly downregulate the levels of dust mite-specific IgE in the serum of patients.

[0178] 7. Effects of probiotic intervention on changes in serum IL-13 and IL-10 levels from baseline in patients.

[0179] The changes in serum ΔIL-13 levels in the three groups at the end of week 8 and week 20 (ΔIL-13 = IL-13 level at the end of week 8 and week 20 - IL-13 level at baseline) are shown in Figure 12A, and the changes in ΔIL-10 levels (ΔIL-10 = IL-10 level at the end of week 8 and week 20 - IL-10 level at baseline) are shown in Figure 12B.

[0180] As shown in Figure 12A, after 8 weeks of probiotic intervention, the serum ΔIL-13 level (-190.51±700.07 pg / mL) was lower than that of the control group (-22.91±741.96 pg / mL), indicating that probiotic intervention is more effective than drug treatment in inhibiting the production of IL-13 in the body. After 20 weeks of probiotic intervention, the serum ΔIL-13 level (-319.22±1016.54 pg / mL) was significantly lower than that of the control group (275.60±1273.66 pg / mL) (P<0.05), indicating that 20 weeks of probiotic intervention can significantly downregulate the expression of the pro-inflammatory factor IL-13 in the serum of atopic individuals. At this time, the serum ΔIL-13 level in the probiotic group after 8 weeks was -248.64±1258.03 pg / mL, which was still lower than that in the control group, indicating that the effect of probiotics is sustained.

[0181] As shown in Figure 12B, after 8 weeks of probiotic intervention, the serum ΔIL-10 level (0.73±3.90 pg / mL) was significantly higher than that of the control group (-1.96±4.76 pg / mL) (P<0.01), indicating that probiotic intervention is more effective than drug treatment in promoting the production of IL-10 in the body. After 20 weeks of probiotic intervention, the serum ΔIL-10 level (2.97±5.94 pg / mL) was significantly higher than that of the control group (-1.52±6.87 pg / mL) (P<0.01), indicating that 20 weeks of probiotic intervention can significantly upregulate the expression of the anti-inflammatory factor IL-10 in the serum of atopic individuals; at this time, the serum ΔIL-10 level in the probiotic group after 8 weeks was 1.58±9.31 pg / mL, which was still higher than that in the control group, indicating that the effect of probiotics is sustained.

[0182] The above experimental results indicate that intervention with Bifidobacterium longum subsp. infantis CCFM111 can significantly downregulate the level of pro-inflammatory factor IL-13 in the serum of patients and significantly upregulate the level of anti-inflammatory factor IL-10 in the serum of patients.

[0183] 8. Effects of probiotic intervention on baseline changes in IL-13 and ECP levels in nasal lavage fluid of patients.

[0184] The changes in ΔIL-13 levels in nasal lavage fluid at the end of weeks 8 and 20 for the three groups are shown in Figure 13A, and the changes in ΔECP levels (ΔECP = ECP levels at the end of weeks 8 and 20 - ECP levels at baseline) are shown in Figure 13B.

[0185] As shown in Figure 13A, after 8 weeks of probiotic intervention, the level of ΔIL-13 in nasal irrigation fluid (-306.26±738.31 pg / mL) was significantly lower than that in the control group (161.02±757.56 pg / mL) (P<0.05). After 20 weeks of probiotic intervention, the level of ΔIL-13 in nasal irrigation fluid (-427.21±964.29 pg / mL) was significantly lower than that in the control group (-3.63±191.20 pg / mL) (P<0.05). This indicates that 8 weeks of probiotic intervention can significantly reduce the expression of the pro-inflammatory factor IL-13 in the nose of AR patients, and 20 weeks of intervention can further inhibit the production of IL-13 in the nose of patients. At this time, the level of ΔIL-13 in nasal irrigation fluid of the probiotic group after 8 weeks was -340.70±967.56 pg / mL, which was still lower than that in the control group, indicating that the effect of probiotics is sustained.

[0186] Eosinophils are active substances released after activation through various pathways in allergic inflammatory responses. ECP is a specific marker of eosinophils and can be used as an indicator for monitoring tracheal inflammation and guiding anti-inflammatory treatment. As shown in Figure 13B, after 8 weeks of probiotic intervention, the level of ΔECP in nasal lavage fluid (-139.63±288.40 pg / mL) was lower than that in the control group (-57.39±378.87 pg / mL). This indicates that increasing probiotic intervention is more effective than drug treatment alone in reducing eosinophil accumulation in the nasal cavity. After 20 weeks of probiotic intervention, the level of ΔECP in nasal irrigation fluid (-212.55±327.26 pg / mL) was significantly lower than that in the control group (11.54±243.72 pg / mL) (P<0.05), indicating that 20 weeks of probiotic intervention can significantly reduce the accumulation of eosinophils in the nose of atopic individuals. At this time, the level of ΔECP in nasal irrigation fluid of the probiotic group after 8 weeks was -84.89±209.17 pg / mL, which was still lower than that in the control group, indicating that the effect of probiotics is sustained.

[0187] The above experimental results indicate that intervention with Bifidobacterium longum subsp. infantis CCFM111 can significantly downregulate the levels of the pro-inflammatory factor IL-13 and ECP in the nasal cavity of patients.

[0188] 9. Safety analysis of probiotics

[0189] Safety and tolerability were monitored at each visit during the study period, with participants reporting symptoms and undergoing regular blood tests for laboratory examinations. The probiotic group, comprising 120 participants, was included in the safety analysis (for data analysis within the first 8 weeks, the two probiotic intervention groups were combined and referred to as the probiotic group), while the control group consisted of 59 participants.

[0190] Table 6. Blood component indicators of patients in the control group at different time points.

[0191] Table 7. Blood component indicators of patients in the probiotic group at different time points.

[0192] Blood samples were collected from patients in each group before intervention and at 8 and 20 weeks of intervention for laboratory testing. Results (see Tables 6-7) showed no differences in any indicators among the groups, indicating that the intervention with *Bifidobacterium longum* subsp. infantis CCFM111 had no significant effect on blood components. No side effects or adverse events were recorded during the study, suggesting that patients tolerated the 5 × 10⁵ samples daily. 9 CFU-based intervention with Bifidobacterium longum infantis CCFM111 was well tolerated.

[0193] The above results indicate that Bifidobacterium longum subsp. infantis CCFM111 has an acceptable safety profile and is well-tolerated in patients with allergic rhinitis.

[0194] In summary, the Bifidobacterium longum infantis CCFM111 provided by this invention can effectively reduce the overall symptoms and drug scores of patients with allergic rhinitis, effectively reduce the levels of house dust mite-specific IgE and pro-inflammatory factor IL-13 in the serum of patients, effectively increase the level of anti-inflammatory factor IL-10 in the serum of patients, and effectively reduce the levels of pro-inflammatory factor IL-13 and ECP in the nasal irrigation fluid of patients, and has good safety and tolerability.

[0195] Example 9: Preparation of powder containing Bifidobacterium longum subsp. infantis CCFM111

[0196] (1) Preparation of seed culture of Bifidobacterium longum subsp. infantis CCFM111

[0197] Bifidobacterium longum subsp. infant CCFM111 was inoculated into MRS liquid medium and cultured at 37℃ for 16 h to prepare seed culture of Bifidobacterium longum subsp. infant CCFM111.

[0198] (2) The seed culture of Bifidobacterium longum subsp. infant CCFM111 was inoculated into MRS medium at an inoculation amount of 3% of the total mass of the medium and cultured at 37°C for 30 h to obtain the culture medium.

[0199] Centrifuge the culture medium and collect the bacterial cells; wash the bacterial cells three times with phosphate buffer at pH 7.2 and then resuspend them in 100 g / L trehalose lyophilization protectant, controlling the mass ratio of lyophilization protectant to bacterial cells to be 2:1 to obtain the resuspended solution.

[0200] The resuspended solution was pre-cooled at -80℃ for 1.5 hours and then immediately transferred to a freeze dryer for 24 hours to obtain Bifidobacterium longum infantis subsp. CCFM111 bacterial powder.

[0201] Example 10: Fermented food containing Bifidobacterium longum subsp. infantis CCFM111

[0202] Fresh vegetables are selected, washed, and juiced. The juice is then subjected to high-temperature instantaneous sterilization at 140°C for 2 seconds, followed by immediate cooling to 37°C. The resulting bacterial powder of *Bifidobacterium longum* subsp. infantis (CCFM111) prepared according to this invention is then inoculated and fermented until its concentration reaches 10. 6 Fruit and vegetable beverages containing live bacteria of Bifidobacterium longum subsp. infantis CCFM111 were obtained by storing the beverage at 4°C with a concentration of CFU / mL or higher.

[0203] Other fermented foods, such as solid foods, liquid foods, and semi-solid foods, can be produced using the *Bifidobacterium longum* subsp. infantis CCFM111 described in this invention. These fermented foods include dairy products, soy products, and fruit and vegetable products. Dairy products include milk, sour cream, and cheese; fruit and vegetable products include cucumber, carrot, beet, celery, and cabbage products.

[0204] Example 11: Preparation of a probiotic product containing Bifidobacterium longum subsp. infantis CCFM111 and functional excipients

[0205] To ensure that *Bifidobacterium longum subsp. infantis* CCFM111 maintains a sufficient number of viable bacteria and effectively colonizes after being transmitted through the digestive tract, this invention uses 48% fructooligosaccharides, 34.5% maltodextrin, 7% xylitol, and 7% strawberry fruit powder as functional excipients, along with 3.5% *Bifidobacterium longum* subsp. infantis CCFM111 bacterial powder (with a viable count of 5 × 10⁻⁶). 9 Fructooligosaccharides (FOS) are compounded to prepare probiotic products. FOS serves as a specific carbon source for the bacterial strains, while maltodextrin acts as a highly efficient protective carrier. Together, they ensure that the strains maintain a sufficient number of live bacteria upon reaching the intestines. Xylitol and strawberry fruit powder are used only to optimize flavor and do not affect the core protective efficacy.

[0206] Other probiotic products with different compositions were prepared according to the formulations in Table 8. The effects of the components in the probiotic products on the gastrointestinal fluid tolerance and intestinal cell adhesion rate of *Bifidobacillus salivarius* subsp. *infantica* CCFM111 were investigated. The viable cell counts in the different probiotic products were kept the same. The effectiveness of the above formulations was verified using an in vitro gastrointestinal tract simulation experiment (viable cell counts were detected after treatment with artificial gastric and intestinal fluids) and a preliminary colonization ability experiment after reaching the intestine (using Caco-2 cell adhesion rate as the evaluation index). The in vitro gastrointestinal tract simulation experiment method was based on "Development and application of an in vitro methodology to determine the transit tolerance of potentially probiotic *Lactobacillus* and *Bifidobacterium* species in the upper human gastrointestinal tract," and the preliminary colonization ability experiment method was based on "In vitro evaluation of the probiotic potential of *Lactobacillus salivarius* SMXD51." The specific results are shown in Table 8 below. Compared with single-component groups and other excipient groups, the functional excipient combination used in this invention significantly improves the protective effect and colonization ability of Bifidobacterium longum subsp. infantis CCFM111, ensuring a survival rate of 39.1±2.7% and an adhesion rate of 2.95±0.35%, which helps to promote the function of the strain in relieving allergic rhinitis.

[0207] Table 8. Effects of different functional excipients (combinations) on improving the survival rate and colonization ability of Bifidobacterium longum subsp. infantis CCFM111.

[0208] Verification has shown that the following percentage concentrations of each component in the probiotic powder achieve the aforementioned effects: fructooligosaccharides 40-50%, maltodextrin 30-40%, xylitol 5-10%, strawberry fruit powder 5-10%, and Bifidobacterium longum subsp. infantis CCFM111 probiotic powder 2.5-5% (3.5 × 10⁻⁶). 9 ~7×10 9 CFU).

[0209] 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. A strain of Bifidobacterium longum subsp. infantis CCFM111, characterized in that, The described Bifidobacterium longum infant subspecies CCFM111 has been deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 65375.

2. A microbial preparation containing the Bifidobacterium longum subsp. infantis CCFM111 as described in claim 1.

3. The microbial preparation of claim 2, wherein The number of bacteria of Bifidobacterium longum subsp. infantis CCFM111 in the microbial preparation is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

4. The microbial preparation of claim 3, wherein The microbial preparation contains, by weight percentage: 40-50% fructooligosaccharides, 30-40% maltodextrin, 5-10% xylitol, 5-10% strawberry fruit powder, and 2.5-5% Bifidobacterium longum subsp. infantis CCFM111 bacterial powder; the number of Bifidobacterium longum subsp. infantis CCFM111 bacteria in the microbial preparation is 3.5 × 10⁻⁶. 9 ~7×10 9 CFU.

5. The microbial preparation of claim 4, wherein The Bifidobacterium longum infant subspecies CCFM111 bacterial powder also contains trehalose.

6. A product characterized by, The product contains live cells, inactivated cells, supernatant, lysate and / or metabolites of Bifidobacterium longum subsp. infantis CCFM111 as described in claim 1.

7. The product of claim 6, wherein, The products include food, medicine, or health products.

8. The product of claim 7, wherein, The food products include dairy products, soy products, meat products, or fruit and vegetable products.

9. The product of claim 8, wherein, The drug also contains a drug carrier and / or pharmaceutical excipients.

10. The product of claim 9, wherein, The drug carrier comprises microcapsules, microspheres, nanoparticles, or liposomes; the pharmaceutical excipients comprise anti-adhesion agents, penetration enhancers, buffers, plasticizers, surfactants, defoamers, thickeners, inclusion agents, absorbents, humectants, solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, pH adjusters, binders, disintegrants, fillers, lubricants, wetting agents, integrators, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, foaming agents, suspending agents, coating materials, fragrances, diluents, flocculants and anti-flocculation agents, filter aids, or release inhibitors.

11. The use of the Bifidobacterium longum subsp. infantis CCFM111 as described in claim 1 or any of the microbial preparations described in claims 2 to 5 in the preparation of a medicament for the prevention and / or treatment of allergic rhinitis.

12. The use according to claim 11, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The number of bacteria of Bifidobacterium longum subsp. infantis CCFM111 in the medicine is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

Images