Bifidobacterium longum subsp.infantis capable of relieving and preventing allergic rhinitis and application of bifidobacterium longum subsp.infantis

Abstract

The invention discloses bifidobacterium longum subsp.infantis capable of relieving and preventing allergic rhinitis and application of the bifidobacterium longum subsp.infantis, and belongs to the technical field of microorganisms and the technical field of medicines. The bifidobacterium longum subsp. Infantis CCFM111 can relieve allergic reaction of allergic mice and relieve and prevent allergic diseases of perennial allergic rhinitis patients, and is specifically shown as follows: (1) weight loss and symptoms caused by allergy of the mice are relieved; lung inflammation infiltration is reduced; the OVA-sIgE level and the total IgE level of serum are reduced; the expression of a proinflammatory factor and an anti-inflammatory factor is regulated. (2) allergy symptoms and medication scores of patients are reduced; the asymptomatic day number proportion of the patient is increased; the serum dermatophagoides farinae and dermatophagoides farinae-sIgE level of patients is reduced; and the expression of cytokines in serum of a patient and nasal irrigation solution is regulated. Therefore, the bifidobacterium longum subsp. Infantis CCFM111 has a huge application prospect in preparation of products for preventing and / or treating allergic rhinitis.

Description

Technical Field

[0001] The invention relates to a strain of Bifidobacterium longum infantis subspecies capable of relieving and preventing allergic rhinitis and application thereof, belonging to the technical field of microorganisms and the technical field of medicine. Background Art

[0002] Allergic rhinitis (AR) is a non-infectious chronic inflammatory disease of the nasal mucosa mediated mainly by immunoglobulin E (IgE) after atopic individuals are exposed to allergens (allergens). Typical symptoms include paroxysmal sneezing, clear watery nasal discharge, nasal itching and nasal congestion; it may be accompanied by eye symptoms, including itchy eyes, tearing, red eyes and burning sensation. According to research statistics, AR affects 10%-40% of adults and 2%-25% of children worldwide. In recent years, the increase in the types of allergens has, to a certain extent, led to a significant increase in the number of AR patients. Today, AR has become one of the most common chronic inflammatory diseases of the respiratory tract. The World Health Organization has listed AR as a global health problem, which has attracted great attention from the public and society. At present, there is no cure for 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 in use include: (1) avoidance of contact with allergens, (2) conventional drug treatment, and (3) allergen-specific immunotherapy (AIT). However, first of all, it is difficult to completely avoid contact with some allergens, especially inhaled allergens (such as dust mites, pollen, mold, etc.). Secondly, due to the recurring nature of AR, its treatment drugs usually need to be taken for life, and these drugs can only relieve the patient's immediate allergic symptoms, and will not have any positive effect on the occurrence and development of AR diseases. In addition, the side effects of long-term use of chemical drugs are also worthy of attention. In addition, AIT has problems such as long treatment cycle (usually 2-3 years), unstable compliance and high treatment costs. These factors have led to the current treatment status of AR being quite difficult.

[0003] From an immunological perspective, AR is an inflammatory reaction of the nasal mucosa caused by an imbalance of Th1 / Th2 and Treg / Th17 immune responses, with Th2 and Th17 immune responses as the main immune responses. Atopic individuals in a sensitized state produce more specific allergen-specific IgE (sIgE) and total IgE than healthy individuals. When they are exposed to allergens again, they recruit and activate immune cells such as mast cells, eosinophils and basophils, which in turn promote the release of pro-inflammatory factors by Th2 and Th17 cells, while inhibiting the secretion of anti-inflammatory factors by Th1 and Treg. In the pathogenesis of allergic diseases, the relationship between intestinal flora structure and immune function has attracted widespread attention. A large number of epidemiological surveys have shown that intestinal flora disorders affect the course of AR to a certain extent. Probiotics are a type of active microorganisms that are beneficial to the host by colonizing in the human body and changing the composition of the flora in a certain part of the host. Probiotics can enhance the intestinal barrier function by affecting the composition and metabolism of intestinal flora, regulate the host immune system to downregulate the levels of pro-inflammatory factors (such as interleukin-4 (IL-4), IL-13, IL-6 and IL-17), and upregulate the levels of anti-inflammatory factors (such as IL-10) to restore the Th1 / Th2 and Th17 / Treg immune balance, thereby alleviating AR symptoms.

[0004] Clinically, the level of specific allergens-sIgE in the body's serum is the main objective indicator for judging AR. For AR patients themselves, they are more concerned about subjective feelings such as relieving AR symptoms, reducing the number of times therapeutic drugs are used, and preventing repeated attacks of AR. The combined symptom and medication score (CSMS) scale is used to subjectively score the patient's nasal symptoms, eye symptoms and conventional medications, and the scoring results are used to evaluate the effect of probiotics on the relief of patients' AR conditions. Due to the persistence of perennial AR patients' disease attacks (symptom attacks ≥ 4 days / week, and ≥ 4 consecutive weeks), the preventive effect of probiotics on AR conditions is evaluated by comparing the proportion of symptom-free days in patients. In the prior art, no edible probiotics that can efficiently regulate allergic body serum allergens-sIgE, and whose regulatory effect is better than the effect of drugs, have been found, nor have probiotics that can achieve significant inhibitory effects on AR conditions in clinical applications. Summary of the invention

[0005] The present invention screened out a food safety strain that can efficiently downregulate animal and human serum specific allergens - sIgE and "effectively relieve patients' AR symptoms, reduce drug use, and reduce AR recurrence". It provides new ideas and solutions for the current situation that allergic rhinitis is difficult to cure, prone to recurrence, and has great side effects of drug treatment, and provides a powerful tool for clinical application.

[0006] The present invention provides a strain of Bifidobacterium longum subsp. infantis CCFM111, which is classified and named Bifidobacterium longum subsp. infantis. It has been deposited in Guangdong Provincial Microbiological Culture Collection Center on October 31, 2024, with a deposit number of GDMCC No: 65375, and the deposit address is Guangdong Provincial Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] In one embodiment of the present invention, the Bifidobacterium longum subsp. infantis CCFM111 is isolated from feces of healthy infants fed exclusively with breast milk in Changsha, Hunan. The strain is sequenced and analyzed, and its 16S rDNA sequence is shown in SEQ ID NO.1. The sequence obtained by sequencing is compared with the nucleic acid sequence of NCBI's BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and the result shows that the nucleic acid sequence similarity with that of Bifidobacterium longum subsp. infantis is as high as 99.86%, and it is named Bifidobacterium longum subsp. infantis CCFM111.

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

[0009] In one embodiment of the present invention, the number 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.

[0010] The present invention also provides a product, which contains the living cells, inactivated cells, supernatant, lysate and / or metabolites of the Bifidobacterium longum subsp. infantis CCFM111.

[0011] In one embodiment of the present invention, the amount of Bifidobacterium longum subsp. infantis CCFM111 in the product is not less than 1×106 CFU / mL or 1×10 6 CFU / g.

[0012] In one embodiment of the present invention, the product comprises a food or a medicine.

[0013] In one embodiment of the present invention, the products include but are not limited to ordinary foods, special foods and medicines.

[0014] In one embodiment of the present invention, the food is a food containing Bifidobacterium longum subsp. infantis CCFM111 or its fermentation metabolite.

[0015] In one embodiment of the present invention, the food is a dairy product, bean product, meat product or fruit and vegetable product produced using Bifidobacterium longum subsp. infantis CCFM111 or a starter containing Bifidobacterium longum subsp. infantis CCFM111.

[0016] In one embodiment of the present invention, the food is a solid beverage containing Bifidobacterium longum subsp. infantis CCFM111.

[0017] In one embodiment of the present invention, the preparation method of the starter is as follows: inoculating Bifidobacterium longum infantis subspecies CCFM111 into a culture medium at an inoculation amount of 2 to 4% of the total mass of the culture medium, and culturing at 37°C for 30 hours to obtain a culture solution; centrifuging the culture solution to collect the bacteria; washing the bacteria three times with a phosphate buffer having a pH of 7.2 and then resuspending with a freeze-drying protective agent to obtain a resuspension; freeze-drying the resuspension by a vacuum freezing method to obtain a starter of Bifidobacterium longum infantis subspecies CCFM111.

[0018] In one embodiment of the present invention, the mass ratio of the lyoprotectant to the bacteria is 2:1.

[0019] In one embodiment of the present invention, the lyophilization protective agent contains skimmed milk powder, maltodextrin and sodium L-glutamate; wherein the ratio of skimmed milk powder: maltodextrin: sodium L-glutamate is (8-10): (8-10): 1.

[0020] In one embodiment of the present invention, the culture medium is prepared by dissolving 10% of skim milk, 0.5% of glucose, 1.5% of tryptone and 0.3% of yeast extract accounting for the total mass of the culture medium in water.

[0021] In one embodiment of the present invention, the pH of the culture medium is 6.8.

[0022] In one embodiment of the present invention, the medicine contains Bifidobacterium longum subsp. infantis CCFM111, a drug carrier and / or a pharmaceutical excipient.

[0023] In one embodiment of the present invention, the drug carrier comprises microcapsules, microspheres, nanoparticles and liposomes.

[0024] In one embodiment of the present invention, the pharmaceutical excipient comprises an excipient and an additive.

[0025] In one embodiment of the present invention, the pharmaceutical excipients include anti-adhesive agents, penetration enhancers, buffers, plasticizers, surfactants, defoamers, thickeners, inclusion agents, absorbents, humectants, solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, pH regulators, adhesives, disintegrants, fillers, lubricants, wetting agents, integrators, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, foaming agents, suspending agents, coating materials, fragrances, diluents, flocculants and deflocculating agents, filter aids and release retardants.

[0026] In one embodiment of the present invention, the additive comprises microcrystalline cellulose, hydroxypropyl methylcellulose and refined lecithin.

[0027] In one embodiment of the present invention, the dosage form of the drug comprises granules, capsules, tablets, pills or oral liquids.

[0028] The present invention also provides the use of the Bifidobacterium longum subsp. infantis CCFM111 or the microbial preparation in the preparation of medicines for preventing and / or treating allergic rhinitis.

[0029] In one embodiment of the present invention, clinically, the index for judging allergic rhinitis is mainly: serum specific allergen-sIgE.

[0030] In one embodiment of the present invention, the Bifidobacterium longum subspecies infantis CCFM111 has the effect of relieving allergic rhinitis in an animal model and has the effect of relieving and preventing allergic rhinitis in clinical treatment.

[0031] In one embodiment of the present invention, improving the pathological characteristics of allergic rhinitis in an animal model includes: alleviating weight loss in mice, alleviating symptoms of allergic rhinitis, alleviating pulmonary inflammatory infiltration in mice, downregulating serum OVA-sIgE levels and total IgE levels in mice, inhibiting lung proinflammatory cytokine (IL-4, IL-13, IL-6, IL-17) levels, and increasing lung anti-inflammatory cytokine (IL-10) levels.

[0032] In one embodiment of the present invention, improving the pathological characteristics of allergic rhinitis in patients during clinical treatment includes: lowering the CSMS score of allergic rhinitis, increasing the proportion of symptom-free days in patients, downregulating the levels of dust mites and house dust mite-sIgE in the patient's serum, downregulating the level of proinflammatory cytokines (IL-13) in the patient's serum, upregulating the level of anti-inflammatory cytokines (IL-10) in the patient's serum, and downregulating the levels of proinflammatory cytokines (IL-13) and eosinophil cationic protein (ECP) in the patient's nasal lavage fluid.

[0033] In one embodiment of the present invention, the number of cells of Bifidobacterium longum subspecies infantis CCFM111 in the drug is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0034] Beneficial effects:

[0035] 1. The Bifidobacterium longum infantis subspecies CCFM111 provided by the present invention can alleviate the symptoms of allergic rhinitis in an animal model, as shown in the following aspects:

[0036] (1) Alleviate weight loss in mice with allergic rhinitis;

[0037] (2) Alleviate allergic symptoms in mice with allergic rhinitis;

[0038] (3) Reduced lung inflammatory infiltration in mice with allergic rhinitis;

[0039] (4) The serum OVA-sIgE level of mice with allergic rhinitis decreased by 60.4% and the serum total IgE level decreased by 50.1%;

[0040] (5) In the lungs of mice with allergic rhinitis, the expression levels of IL-4, IL-13, IL-6, IL-17, IL-17, and IL-10 were decreased by 46.3%, 69.2%, 62.3%, 57.0%, and 291.9%, respectively.

[0041] 2. The Bifidobacterium longum infantis subspecies CCFM111 provided by the present invention can relieve the symptoms of allergic rhinitis and reduce medication in clinical treatment, and play an auxiliary therapeutic effect, which is specifically reflected in that compared with the control group (conventional medication group):

[0042] (1) After 8 weeks of intervention, the comprehensive symptom and medication score (CSMS) of patients with allergic rhinitis decreased significantly, and the reduction was 1.67 times that of the control group (normal medication group);

[0043] (2) After 2 weeks of intervention, the CSMS scores of patients with allergic rhinitis decreased significantly, and the decrease was 6.25 times that of the control group (normal medication group), indicating that Bifidobacterium longum subspecies infantis CCFM111 was effective after 2 weeks of intervention;

[0044] (3) After 20 weeks of intervention, the CSMS scores of patients with allergic rhinitis decreased significantly, which was 10 times that of the control group (normal medication group), indicating that the longer the Bifidobacterium longum subsp. infantis CCFM111 was used as an adjunct to conventional medication to treat AR, the better the effect.

[0045] (4) During the intervention period of weeks 1 to 8, the proportion of symptom-free days in patients with allergic rhinitis increased by 52.9%; during the intervention period of weeks 9 to 20, the proportion of symptom-free days in patients with allergic rhinitis increased by 75.0%, indicating that adjuvant therapy with Bifidobacterium longum subspecies infantis CCFM111 is significantly helpful in reducing the recurrence of AR.

[0046] (5) After 20 weeks of intervention, the serum s-IgE levels of dust mites and house dust mites in patients with allergic rhinitis were significantly reduced;

[0047] (6) After 20 weeks of intervention, the expression level of IL-13 in the serum of patients with allergic rhinitis was significantly decreased, while the expression level of IL-10 was significantly increased;

[0048] (7) After 20 weeks of intervention, the expression levels of IL-13 and ECP in the nasal lavage fluid of patients with allergic rhinitis were significantly reduced.

[0049] Therefore, Bifidobacterium longum subsp. infantis CCFM111 has great application prospects in the preparation of medicines for preventing and / or treating allergic rhinitis.

[0050] Biomaterial Deposit

[0051] A strain of Bifidobacterium longum subsp. infantis CCFM111, taxonomically named Bifidobacterium longum subsp. infantis, was deposited in the Guangdong Provincial Microbiological Culture Collection on October 31, 2024, with the deposit number GDMCC No: 65375, and the deposit address is Guangdong Provincial Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 : Changes in body weight percentage of experimental mice in different groups.

[0053] Figure 2 : Allergy symptom scores of experimental mice in different groups.

[0054] Figure 3 :Pathological sections of lung tissues of experimental mice in different groups.

[0055] Figure 4 : Levels of serum OVA-specific IgE and total IgE in different groups of experimental mice.

[0056] Figure 5 : Cytokine contents in lung tissues of experimental mice in different groups.

[0057] Figure 6 :Illustration of the animal experiment process.

[0058] Figure 7 : The changes of average CSMS scores of patients in different groups on the 7th and 8th day compared with the baseline.

[0059] Figure 8 :The changes of the average CSMS scores of patients in different groups on the 2nd, 4th, 6th and 8th days compared with the baseline.

[0060] Fig. 9 :The changes of the average CSMS scores of patients in different groups on the 8th, 10th, 12th, 14th, 16th, 18th and 20th days compared with the baseline.

[0061] Fig.10 : Differences in the proportion of symptom-free days in different groups of patients during weeks 1-8 and weeks 9-20.

[0062] Fig.11 : The levels of Dermatophagoides farinae and Dermatophagoides pteronyssinus-specific IgE in serum of patients in different groups at the end of the 8th and 20th week.

[0063] Fig.12 : The levels of IL-13 and IL-10 in serum of patients in different groups at the end of the 8th and 20th week.

[0064] Fig.13 : The contents of IL-13 and ECP in nasal lavage fluid of patients in different groups at the end of the 8th and 20th week.

[0065] Fig.14 :Diagram of clinical trial process. DETAILED DESCRIPTION

[0066] The BALB / c female mice involved in the following examples were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

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

[0068] Sensitization solution: Each mouse was intraperitoneally injected with 0.2 mL of sensitization solution, which was mixed with 0.1 mL of normal saline containing 30 μg OVA (Product No. A5503, purchased from Sigma-Aldrich) and 0.1 mL of alum adjuvant (Product No. 77161, purchased from Thermo).

[0069] Provocation solution: 50 μL of provocation solution was dripped into the nose of each mouse. The 50 μL provocation solution was physiological saline containing 50 μg OVA.

[0070] The culture medium involved in the following examples is as follows:

[0071] MRS liquid culture medium formula (1L): peptone 10g, beef extract 10g, yeast powder 5g, glucose 20g, K2HPO42g, diammonium citrate 2g, sodium acetate 2g, Tween 80 1mL, MgSO4·7H2O 0.5g, cysteine ​​hydrochloride 0.5g, MnSO4·4H2O 0.25g, pH 7.2-7.4.

[0072] MRS solid culture medium formula (1L): peptone 10g, beef extract 10g, yeast powder 5g, grape 20g, K2HPO42g, diammonium citrate 2g, sodium acetate 2g, Tween 80 1mL, MgSO4·7H2O 0.5g, cysteine ​​hydrochloride 0.5g, MnSO4·4H2O0.25g, agar 20g, pH 7.2-7.4.

[0073] The detection methods involved in the following embodiments are as follows:

[0074] Detection method of viable bacteria count: adopt the national standard "GB 4789.35-2016 National Food Safety Standard Food Microbiology Detection Lactic Acid Bacteria Detection".

[0075] Acidity detection method: adopt national standard GB 431334-2010.

[0076] Bifidobacterium longum subspecies infantis CCFM1192 is another strain isolated from different infant feces samples using the same isolation method and has been disclosed in patent CN114276961A.

[0077] Example 1: Screening, morphological observation and identification of Bifidobacterium longum subsp. infantis CCFM111

[0078] Take 1g of fresh feces from a healthy infant, spread it on MRS solid culture medium containing 1% mupirocin after gradient dilution, and culture it at 37℃ for 72h in an anaerobic environment; observe and record the colony morphology, pick colonies and streak purification twice, and after culturing at 37℃ for 48h, observe the bacteria under a microscope and find that the colonies are milky white, round and smooth, and the shape of the bacteria is slightly irregular, with rounded ends, usually single and in small clusters.

[0079] The strains screened above were cultured with fresh MRS liquid medium at 37°C for 24h, and then 1mL of bacterial solution was centrifuged at 3500×g to obtain bacterial mud, and then 1mL of sterile water was added to wash the bacterial mud, and the supernatant was removed by centrifugation under the same conditions, and then 1mL of sterile water was added to re-dissolve to prepare a bacterial suspension template. Bacterial 16S rDNA 25μL reaction system: 0.5μL each of 27F forward primer and 1492R reverse primer, 12.5μL Taq enzyme, 1μL bacterial suspension template, and 10.5μL sterile water. PCR reaction conditions: ①95℃, 5min; ②95℃, 10s; ③55℃, 30s; ④72℃, 30s; ⑤Steps 2-4 were repeated for 30 cycles; ⑥72℃, 5min; ⑦2℃, 2min. The PCR amplification product was sent to a professional sequencing company and sequenced using a second-generation sequencer. The nucleotide sequence of 16S rDNA amplified from CCFM111 is shown in SEQ ID NO.1. The sequence results obtained by sequencing were compared with BLAST for nucleic acid sequence. The results showed that the nucleic acid sequence similarity with Bifidobacterium longum subsp. infantis was as high as 99.86% and 100.00%, respectively. The strain was named Bifidobacterium longum subsp. infantis CCFM111. The strain was stored in a -80°C refrigerator for future use.

[0080] Example 2: Cultivation of Bifidobacterium longum subsp. infantis CCFM111

[0081] Bifidobacterium longum subsp. infantis CCFM111 and Bifidobacterium longum subsp. infantis CCFM1192 were inoculated into MRS liquid culture medium and cultured in an anaerobic chamber at 37°C for 24 hours, then the bacterial solution was transferred into fresh MRS liquid culture medium according to an inoculum volume of 4%, and cultured under the same conditions for 16-24 hours, the bacteria were centrifuged at 8000×g for 15 minutes, the bacteria were washed with 0.9% saline and centrifuged again at 8000×g for 10 minutes, the bacteria were collected, resuspended with 30% (m / v) sucrose solution, and frozen in a -80°C refrigerator for use.

[0082] Preparation of bacterial suspension for oral administration: When Bifidobacterium longum subsp. infantis was used for oral administration to mice, it was taken out from a -80°C refrigerator, centrifuged at 4°C and 8000×g for 10 min, the supernatant was discarded, and the suspension was resuspended in 0.9% saline to obtain a probiotic suspension for oral administration.

[0083] Example 3: Effect of Bifidobacterium longum subsp. infantis CCFM111 on the alleviation of weight loss in mice with allergic rhinitis

[0084] Six-week-old SPF-grade BALB / c female mice were divided into five groups, namely, a blank group, a model group, a positive control group (dexamethasone acetate tablets DXMS) and an experimental group (CCFM111 group and CCFM1192 group); 8 mice in each group were housed in the Experimental Animal Center of Jiangsu Schistosomiasis Control and Prevention Institute, fed with ordinary feed, and maintained at a constant temperature of 21-26°C, a humidity of 40-70%, a noise level of less than or equal to 60 dB, and an animal illumination of 15-20 LX (all animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangsu Schistosomiasis Control and Prevention Institute).

[0085] The experimental period was 34 days in total, and the experimental methods were as follows: Figure 6 The mice were adapted for 7 days. From the 8th day to the 33rd day, the blank group and the model group were gavaged with 0.2 mL of saline solution every day, and the CCFM111 group and CCFM1192 group were gavaged with 0.2 mL of viable bacteria every day. 9 CFU / mL of the corresponding bacterial suspension, the preparation method of the bacterial suspension is the same as that in Example 2; the positive control group was gavaged with 0.2 mL of normal saline solution every day from the 8th day to the 28th day, and 0.2 mL of 0.45 mg / ml DXMS solution every day from the 29th day to the 33rd day. The 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 the 8th, 15th and 22nd days; from the 29th day to the 33rd day, 50 μL of OVA provocation solution (concentration of 1 mg / mL) was used for nasal drops; the mice were sacrificed on the 34th day. All groups were free to drink water and eat during this period.

[0086] The weight of mice was recorded before each sensitization and before the last challenge. The results are shown in Table 1 and Figure 1 As shown:

[0087] Table 1: Changes in body weight of mice (body weight on day 8 was 100%)

[0088]

[0089] The experimental results are shown in Table 1 and Figure 1As shown in Table 1. The establishment of AR model and OVA-induced allergic reaction will cause the mice to lose weight. Figure 1 It can be seen that compared with the blank group and the model group, the CCFM111 group effectively alleviated the weight loss of mice caused by allergic rhinitis, and its alleviating effect was better than that of the positive control group and the CCFM1192 group.

[0090] The above experimental results show that compared with the positive control group and Bifidobacterium longum subsp. infantis CCFM1192, Bifidobacterium longum subsp. infantis CCFM111 can more effectively alleviate the degree of weight loss in mice with allergic rhinitis.

[0091] Example 4: Effect of Bifidobacterium longum subsp. infantis CCFM111 on allergic symptoms in mice with allergic rhinitis

[0092] Specific implementation method: Refer to Example 3. The pathological score of allergic rhinitis symptoms of mice was performed within 30 minutes after the last challenge. The score requirements are as follows:

[0093] Table 2: Behavioral scoring table for allergic rhinitis in mice

[0094]

[0095] The experimental results are as follows Figure 2 As shown. Figure 2 It can be seen that the blank group mice had smooth and shiny hair and no allergic symptoms, while the model group mice had messy and dull hair, and erect hair, with obvious slow reaction. Compared with the model group mice, oral administration of positive drugs, Bifidobacterium longum subsp. infantis CCFM111 and Bifidobacterium longum subsp. infantis CCFM1192 significantly alleviated the pathological characteristics of mice with allergic rhinitis; compared with the CCFM1192 group, the symptoms of the CCFM111 group mice were more significantly relieved.

[0096] The above experimental results show that compared with Bifidobacterium longum subsp. infantis CCFM1192, Bifidobacterium longum subsp. infantis CCFM111 is more capable of alleviating the allergic symptoms of mice with allergic rhinitis.

[0097] Example 5: Effect of Bifidobacterium longum subsp. infantis CCFM111 on the severity of lung tissue lesions in mice with allergic rhinitis

[0098] Specific implementation method Refer to Example 3. The mice were killed on the 34th day, and the left lung tissue samples of the mice were collected and fixed with 4% paraformaldehyde solution, followed by washing, dehydration, transparency, waxing, embedding, slicing, spreading, sticking, baking, staining with hematoxylin-eosin (HE), differentiation, rinsing, counterstaining, dehydration, transparency and sealing to produce lung tissue HE staining sections. The results of lung tissue HE pathological sections are shown in Figure 3 .

[0099] Depend on Figure 3 It can be seen that compared with the blank group, the model group mice had pathological manifestations such as a large number of eosinophils and lymphocytes infiltration around the trachea, bronchi, alveoli and blood vessels, obvious mucosal epithelial hyperplasia, severe airway epithelial damage and thickening of alveolar septa. The pathological damage of the lungs of mice in the positive drug group, CCFM111 group and CCFM1192 group was significantly reduced, the infiltration of inflammatory cells in the lung tissue and the surrounding area was significantly reduced, the mucosal epithelium was mildly hyperplastic, the airway epithelial damage was reduced and the thickening of the alveolar septa was reduced; and the relief effect of CCFM111 group was better than that of CCFM1192.

[0100] The above experimental results show that compared with the positive control group and Bifidobacterium longum subsp. infantis CCFM1192, Bifidobacterium longum subsp. infantis CCFM111 can better improve the lung pathology of mice with allergic rhinitis and inhibit inflammatory infiltration.

[0101] Example 6: Alleviating effect of Bifidobacterium longum subsp. infantis CCFM111 on serum OVA-specific IgE level and total IgE level in mice with allergic rhinitis

[0102] Specific implementation method Refer to Example 3. On the 34th day, the eyeballs were removed and blood was collected and the mice were killed. The mouse blood was allowed to stand for 2 hours and then centrifuged at 3000r / min for 15 minutes. The mouse serum was collected and the OVA-specific IgE content (Cat. No. 439807, purchased from BioLegend) and total IgE content (Cat. No. NBP3-18786, purchased from Novus) in the mouse serum were determined by ELISA kits. The results are shown in Figure 4 shown.

[0103] Depend on Figure 4A shows that after mice were gavaged with Bifidobacterium longum infant subspecies CCFM111, the content of OVA-specific IgE in serum was significantly reduced, the content was: 9.42±2.61ng / mL, which was significantly lower than the model group (the content of OVA-sIgE was 23.78±1.32ng / mL) (p<0.001), and decreased by 60.4%, indicating that the strain Bifidobacterium longum infant subspecies CCFM111 in the present invention can significantly inhibit the production of OVA-specific IgE. After mice were gavaged with Bifidobacterium longum infant subspecies CCFM1192, the content of OVA-specific IgE in serum was also significantly reduced, the content was: 16.85±4.14ng / mL, which was significantly lower than the model group (p<0.05), and decreased by 29.1%; and after mice were gavaged with positive drugs (the content of OVA-sIgE was 18.53±8.07ng / mL), there was also a reduction effect compared with the model group, but there was no significant difference.

[0104] Depend on Figure 4 B shows that after mice were gavaged with Bifidobacterium longum infantis subspecies CCFM111, the total IgE content in the serum was significantly reduced, and the content was: 43.79±10.98μg / mL, which was significantly lower than that of the model group (the total IgE content was 87.69±28.89μg / mL) (p<0.05), a decrease of 50.1%; there was no significant difference between the normal group (the total IgE content was 27.95±4.06μg / mL), indicating that the strain Bifidobacterium longum infantis subspecies CCFM111 in the present invention can significantly inhibit the production of total IgE. After mice were gavaged with Bifidobacterium longum infantis subspecies CCFM1192, the total IgE content in the serum was also significantly reduced, to 50.04±5.96μg / mL, which was significantly lower than that in the model group (p<0.05), a decrease of 42.9%; and after mice were gavaged with positive drugs (the total IgE content was 59.76±48.25μg / mL), there was also a decrease compared with the model group, but there was no significant difference.

[0105] The above experimental results show 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.

[0106] Example 7: Effect of Bifidobacterium longum subsp. infantis CCFM111 on cytokine expression levels in lung tissue of mice with allergic rhinitis

[0107] For a specific implementation, refer to Example 3. After the mice were killed on the 34th day, the right lung tissue samples of the mice were collected, the right lung tissue was rinsed with pre-cooled PBS, residual blood was removed, the surrounding fat tissue was removed, and it was cut into pieces after weighing. The cut tissue was mixed with RIPA lysis buffer at a ratio of 1:9 (g:mL), and crushed using a tissue crusher at 65HZ, 30s (cycled 6-8 times), and finally centrifuged at 5000×g for 10min. The supernatant was taken and the expression level of the pro-inflammatory factor IL-4 in the lung tissue was detected by ELISA kit (Cat. No. VAL603, purchased from R&D). The results are shown in Figure 5 A; The supernatant was taken and the expression level of pro-inflammatory factor IL-13 in lung tissue was detected by ELISA kit (Cat. No. M13000CB, purchased from R&D). The results are shown in Figure 5 B; The supernatant was taken and the expression level of pro-inflammatory factor IL-6 in lung tissue was detected by ELISA kit (Cat. No. M6000B, purchased from R&D). The results are shown in Figure 5 C; The supernatant was taken and the expression level of pro-inflammatory factor IL-17 in lung tissue was detected by ELISA kit (Cat. No. VAL610, purchased from R&D). The results are shown in Figure 5 D; The supernatant was taken and the expression level of anti-inflammatory factor IL-10 in lung tissue was detected by ELISA kit (Cat. No. M1000B-1, purchased from R&D). The results are shown in Figure 5 E.

[0108] Depend on Figure 5 A shows that after mice were gavaged with Bifidobacterium longum infantis subspecies CCFM111, the IL-4 content in the lung tissue (content was 3.23±0.43pg / mg protein) was significantly reduced, which was significantly lower than that in the model group (IL-4 content was 6.01±0.85pg / mg protein) (p<0.01), a decrease of 46.3%; it was reduced by 13.6% compared with the blank group (IL-4 content was 3.74±0.91pg / mg protein); this indicates that the strain Bifidobacterium longum infantis subspecies CCFM111 in the present invention can inhibit Th2 response; and after mice were gavaged with positive drugs (IL-4 content was 4.49±1.39ng / mL) and CCFM1192 (IL-4 content was 5.07±1.91ng / mL), there was also a decrease compared with the model group, but there was no significant difference.

[0109] Depend on Figure 5B shows that after mice were gavaged with Bifidobacterium longum infantis subspecies CCFM111, the IL-13 content in the lung tissue (content was 4.58±0.61pg / mg protein) was significantly lower than that in the model group (IL-13 content was 14.89±4.98pg / mg protein) (p=0.001), a decrease of 69.2%; compared with the blank group (IL-13 content was 5.19±2.28pg / mg protein), it was reduced by 11.8%; this indicates that the strain Bifidobacterium longum infantis subspecies CCFM111 in the present invention can inhibit Th2 response; after mice were gavaged with positive drugs (IL-13 content was 5.30±1.92ng / mL) and CCFM1192 (IL-13 content was 5.40±3.33ng / mL), it was also significantly lower than that in the model group (p=0.002).

[0110] Depend on Figure 5 C It can be seen that after mice were gavaged with Bifidobacterium longum infantis subspecies CCFM111, the IL-6 content in the lung tissue (content was 13.34±5.01pg / mg protein) was significantly lower than that in the model group (IL-6 content was 35.36±9.12pg / mg protein) (p<0.01), a decrease of 62.3%; compared with the blank group (IL-6 content was 17.84±2.91pg / mg protein), it was reduced by 25.2%; indicating that the strain Bifidobacterium longum infantis subspecies CCFM111 in the present invention can inhibit the expression of proinflammatory factors; after mice were gavaged with positive drugs (IL-6 content was 14.96±1.24ng / mL) and CCFM1192 (IL-6 content was 19.73±2.29ng / mL), there was also a significant decrease compared with the model group, but its significance was not as high as that of CCFM111.

[0111] As can be seen from 5D, after mice were gavaged with Bifidobacterium longum infantis subspecies CCFM111, the IL-17 content in the lung tissue (content was 0.80±0.09pg / mg protein) was significantly lower than that in the model group (IL-17 content was 1.86±0.37pg / mg protein) (p<0.01), a decrease of 57.0%; there was no significant decrease compared with the blank group (IL-17 content was 0.31±0.26pg / mg protein); this indicates that the strain Bifidobacterium longum infantis subspecies CCFM111 in the present invention can inhibit the expression of proinflammatory factors; after mice were gavaged with positive drugs (IL-17 content was 0.33±0.10ng / mL) and CCFM1192 (IL-17 content was 1.19±0.28ng / mL), there was also a significant decrease compared with the model group.

[0112] Depend on Figure 5E It can be seen that after mice were gavaged with Bifidobacterium longum infantis subspecies CCFM111, the IL-10 content in the lung tissue (content was 40.56±20.08pg / mg protein) was significantly increased (p<0.05) compared with the model group (IL-10 content was 10.35±0.76pg / mg protein), an increase of 291.9%; there was no significant increase compared with the blank group (IL-10 content was 16.57±0.80pg / mg protein); this shows that the strain Bifidobacterium longum infantis subspecies CCFM111 in the present invention can promote the expression of anti-inflammatory factors; after mice were gavaged with positive drugs (IL-10 content was 12.68±6.01ng / mL) and CCFM1192 (IL-10 content was 18.27±13.85ng / mL), it was also increased compared with the model group, but there was no significant difference.

[0113] The above experimental results show that compared with Bifidobacterium longum subsp. infantis CCFM1192, Bifidobacterium longum subsp. infantis CCFM111 can significantly downregulate the levels of proinflammatory 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.

[0114] In summary, the Bifidobacterium longum infantis subspecies CCFM111 provided by the present invention can effectively relieve the symptoms of rhinitis caused by OVA, and in the regulation of mouse weight, OVA-IgE, total IgE, and IL-10, it reaches a level significantly better than that of the drug group; in the regulation of proinflammatory factors, it also reaches a level equivalent to that of the drug group; the overall regulatory effect is better than the reported same strain Bifidobacterium longum infantis subspecies CCFM1192, and an unexpected technical effect is achieved. Compared with the strain B10 reported in the document "Bifidobacterium infantis Relieves Allergic Asthma in Mice by Regulating Th1 / Th2", the regulatory effect of strain B10 on serum OVA-IgE and total IgE is equivalent to that of the drug group, and it cannot achieve an effect better than that of the drug. Therefore, the Bifidobacterium longum infantis subspecies CCFM111 provided by the present invention is better than strain B10 in relieving allergic symptoms.

[0115] Example 8: Multicenter, prospective, randomized, double-blind, placebo-controlled clinical trial

[0116] 1. The sample standards in this embodiment are as follows:

[0117] 1. Selection criteria

[0118] Individuals must meet all of the following criteria to be eligible for participation in the trial:

[0119] (1) Children have 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) Two or more symptoms of allergic rhinitis, such as nasal congestion, runny nose, itching, and paroxysmal sneezing, occur, and the daily symptoms last or accumulate for more than 1 hour; (3) Children with moderate or above perennial allergic rhinitis, refer to the classification and judgment criteria of the "Guidelines for the Diagnosis and Treatment of Allergic Rhinitis in China 2022 Edition"; (4) Children aged 3-12 years, male or female; (5) Good compliance, able to comply with the requirements of this clinical trial and not violate the regulations.

[0120] 2. Exclusion criteria

[0121] Individuals who meet any of the following criteria will be excluded from this trial:

[0122] (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 (except mild intermittent asthma); patients with mental illness; and patients with any other diseases that may interfere with the evaluation of trial results; (3) patients receiving AIT during treatment; (4) patients who repeatedly took probiotic products or consumed yogurt in the previous month; (5) patients who refused to sign the informed consent (legal guardians), or patients with estimated poor compliance and poor follow-up possibility; (6) other situations that do not meet the inclusion criteria.

[0123] 3. Exit criteria

[0124] (1) Patients who did not meet the inclusion criteria but were mistakenly included; (2) Patients who had never taken a probiotic product or had discontinued taking probiotics for more than 10 days or consumed other probiotic powder-related products; (3) Patients who used drugs other than concomitant treatments permitted by the study to treat AR; (4) Patients with poor compliance and no trial records during the trial; (5) Patients who used T cell inhibitors or T cell regulating ointments during the trial; (6) Patients with acute bacterial or viral infection complicated by sinusitis.

[0125] 4. Shedding Standard

[0126] (1) After professional evaluation by a physician, the patient needs to withdraw from the trial; (2) The patient actively requests to withdraw from the trial; (3) Administrative factors: such as relocation, economic factors, and inability to follow up regularly.

[0127] II. Patient Grouping and Intervention

[0128] The patients recruited in this example have all signed informed consent forms by their guardians and have been approved by the Ethics Committee of Wuxi Children's Hospital and the Ethics Committee of Wuxi Maternal and Child Health Hospital. Fig.14 As shown in the figure, a total of 182 children with AR were screened and recruited, and they were divided into three groups by simple random method:

[0129] (1) 8-week probiotic group: first take probiotic powder for 8 weeks (1 bag per day), then take placebo for 12 weeks (1 bag per day); (2) 20-week probiotic group: take probiotic powder (1 bag per day) for 20 weeks;

[0130] (3) Control group (normal medication group): took placebo for 20 weeks (1 bag per day).

[0131] Among them: probiotic powder (1.5g / bag), the formula is (by mass fraction): oligofructose 48%, maltodextrin 33.5%, xylitol 7%, strawberry fruit powder 7%, Bifidobacterium longum infantis subspecies CCFM1113.5% (5×10 9 CFU); placebo (1.5g / bag), the formula is (by mass fraction): oligofructose 48%, maltodextrin 38%, xylitol 7%, strawberry fruit powder 7%. The packaging and appearance of probiotic powder and placebo are the same, both are produced by Weikang Probiotics (Suzhou) Co., Ltd.

[0132] During the intervention period, both the probiotic group and the control group (normal medication group) were only allowed to use oral / intranasal antihistamines (H1A), intranasal corticosteroids (INCS), and oral corticosteroids (OCS).

[0133] 3. Research steps and related inspections

[0134] (1) Parents were required to 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 an online form.

[0135] (2) Patients underwent blood, nasal secretion, and stool sample collection before intervention (baseline), at the end of the 8th week, and at the end of the 20th week.

[0136] (3) Patients are required to undergo follow-up visits at weeks 2, 4, 8, 12, 16, and 20 and report symptoms and adverse events to their physicians.

[0137] Table 3: Comprehensive Symptom and Medication Rating Scale (CSMS)

[0138]

[0139] IV. Endpoint indicators

[0140] 1. Primary endpoint: To evaluate the adjuvant therapeutic effect of probiotics versus placebo on AR by comparing the changes in the daily average CSMS scores of patients in different groups at week 7-8 compared with the baseline score (CSMS score before the patient received the intervention) (ΔCSMS = daily average CSMS at week 7-8 - daily average CSMS at baseline).

[0141] 2. Secondary endpoints:

[0142] (1) The onset time of probiotics was evaluated by comparing the changes in the daily average CSMS scores of patients in different groups every 2 weeks compared with the baseline (ΔCSMS = daily average CSMS at each time point - daily average CSMS during the baseline period); the effect of continuous intervention for 20 weeks; and the duration of the probiotics effect after 8 weeks of intervention.

[0143] (2) To evaluate the preventive effect of probiotics on AR recurrence 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);

[0144] (3) To compare the changes in serum levels of dust mite-specific IgE and cytokines interleukin-13 (IL-13) and IL-10 compared with the baseline in patients in different groups at the end of the 8th and 20th week;

[0145] (4) To compare the changes of inflammatory factor IL-13 levels and eosinophil cationic protein (ECP) in nasal lavage fluid at the end of the 8th and 20th week compared with the baseline in different groups of patients;

[0146] (5) Safety analysis of probiotics.

[0147] V. Statistical Analysis

[0148] The effectiveness evaluation analysis adopts the Full Analysis Set (FAS) analysis, which includes qualified cases and dropped cases, but does not include excluded cases. Dropped cases, for example, have baseline efficacy data, but the main efficacy index is missing. According to the intention to treat (ITT) analysis, the final efficacy index result can be regarded as the final result for statistical analysis. Safety indicators are analyzed using the Security Data Set (SS). The SS set is defined as all participants who have used the trial product at least once.

[0149] SPSS26 statistical software was used to analyze the baseline data and symptom improvement data. The Mann-Whitney test was used to analyze the differences between the two independent samples, the Wilcoxon matched-pairs signed rank test was used to analyze the differences between the two paired samples, and the non-parametric Kruskal-Wallis test was used to analyze the significance of the differences between multiple groups. P<0.05 was considered statistically significant, * indicated that the difference within the group was P<0.05, # indicated that the difference between the groups was P<0.05, ** and ## indicated that P<0.01, and *** and ### indicated that P<0.001.

[0150] 6. Experimental Results

[0151] 1. Basic information of the patient

[0152] The final number of patients included in the probiotic group was 115 (when analyzing the data within the first 8 weeks, the two probiotic intervention groups were collectively referred to as the probiotic group), and the number of patients in the control group was 54. The basic information of the included patients is shown in Table 4. There was no significant difference in the baseline data between the probiotic group and the control group.

[0153] Table 4 Baseline data of patients in each group

[0154]

[0155] Note: TNSS is Total Nasal Symptom Score

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

[0157] The results of the daily average ΔCSMS changes in the probiotic group and the control group (normal medication group) at week 7-8 (ΔCSMS = daily average CSMS at week 7-8 - daily average CSMS at baseline) are as follows: Figure 7 shown.

[0158] The results showed that under the same conventional treatment regimen, the ΔCSMS of the probiotic group (-0.87±0.94) was significantly lower than that of 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 of the control group, indicating that after 8 weeks of auxiliary treatment with Bifidobacterium longum infantis subspecies CCFM111, the patients' allergic symptoms and medication conditions can be effectively relieved.

[0159] The above experimental results show that Bifidobacterium longum subsp. infantis CCFM111 can significantly alleviate the allergic symptoms of AR patients in addition to conventional drug treatment.

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

[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 the figure. Figure 8 shown.

[0162] Depend on Figure 8 It can be seen that at 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 of the control group, which shows that probiotics-assisted conventional drug treatment is very helpful compared with simple drug treatment at the second week.

[0163] The above experimental results show that Bifidobacterium longum subsp. infantis CCFM111 takes effect after 2 weeks of intervention.

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

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

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

[0167]

[0168] Depend on Fig. 9 As shown in Table 5, the probiotic group ( Fig. 9 Right) ΔCSMS of patients and control group ( Fig. 9 Compared with the control group (left), there were significant differences between the two groups 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). As can be seen from Table 5, as the probiotic intervention time increased, the difference between the 20-week group and the control group became greater, indicating that the longer the probiotic-assisted conventional drug treatment intervention time, the better the effect. Fig. 9 ΔCSMS of patients (middle) and control group ( Fig. 9Compared with the control group (left), there was still a significant difference at the 12th week (P < 0.01). At this time, the decrease in CSMS in the 8-week group was 6.29 times that of the control group, which shows that the auxiliary treatment effect of probiotics is continuous and its effect can be maintained for 4 weeks after stopping the probiotic intervention.

[0169] The above experimental results show that the auxiliary therapeutic effect of Bifidobacterium longum subsp. infantis CCFM111 increases with the increase of intervention time; its effect can be maintained for 4 weeks after the intervention is stopped at 8 weeks.

[0170] 5. Effect of probiotic intervention on the proportion of symptom-free days in patients

[0171] The differences in the proportion of symptom-free days between the three groups in weeks 1-8 and 9-20 (proportion of symptom-free days = number of symptom-free days / total number of recorded days) are shown in Table 1. Fig.10 shown.

[0172] Depend on Fig.10 As can be seen from the left, during the intervention period of weeks 1 to 8, the proportion of symptom-free days in the probiotic group (0.26±0.25) increased significantly (P<0.05) compared with the control group (0.17±0.22), an increase of 52.9%, indicating that 8 weeks of probiotic intervention is positively helpful in reducing the onset of AR symptoms. Fig.10 As can be seen from the right, during the intervention period of weeks 9-20, the proportion of symptom-free days in the 20-week probiotic group (0.42±0.37) increased significantly (P<0.05) compared with the control group (0.24±0.27), an increase of 75%, and the intra-group difference also increased significantly compared with the probiotic group in weeks 1-8 (P<0.001), an increase of 61.5%. This shows that 20 weeks of probiotic intervention is helpful in reducing the onset of AR symptoms, and is better than 8 weeks of intervention.

[0173] The above experimental results show that Bifidobacterium longum subsp. infantis CCFM111 intervention is very helpful in reducing the onset of AR symptoms, and the effect is better as the intervention time increases.

[0174] 6. Effect of probiotic intervention on the changes of serum dust mite and house dust mite specific IgE levels compared with baseline

[0175] The results of the changes in the serum Δ dust mite-sIgE levels of the three groups at the end of the 8th and 20th week (Δ dust mite-sIgE = dust mite-sIgE levels at the end of the 8th and 20th week - dust mite-sIgE levels at the baseline period) are as follows: Fig.11As shown in A, the results of the change in Δ house dust mite-sIgE level (Δ house dust mite-sIgE = house dust mite-sIgE level at the end of the 8th and 20th weeks - house dust mite-sIgE level at the baseline period) are as follows Fig.11 As shown in B.

[0176] Depend on Fig.11 A shows that after 8 weeks of probiotic intervention, the level of Δ dust mite-sIgE in serum (1.44±15.76kU A / L) was lower than that of the control group (5.52±15.54 kU A / L), which indicates that the addition of probiotics intervention can better inhibit the production of dust mite-sIgE in the body than drug treatment alone. After 20 weeks of probiotic intervention, the level of Δ dust mite-sIgE in serum (-4.65±24.38kU A / L) compared with the control group (11.41±23.54 kU A / L) was significantly reduced (P<0.05), which indicates that probiotic intervention for 20 weeks can significantly downregulate the expression of Δ dust mite-sIgE in the serum of atopic individuals; at this time, the level of Δ dust mite-sIgE in the serum of the 8-week probiotic group was 1.14±24.76kU A / L, which was still lower than that of the control group, indicating that the effect of probiotics was sustained.

[0177] Depend on Fig.11 B shows that after 8 weeks of probiotic intervention, the level of Δ house dust mite-sIgE in serum (1.69±11.23 kU A / L) was lower than that of the control group (4.24±13.18 kU A / L), which indicates that the addition of probiotics intervention can better inhibit the production of house dust mite-sIgE in the body than drug treatment alone. After 20 weeks of probiotic intervention, the level of Δ dust mite-sIgE in serum (-7.41±12.37kU A / L) compared with the control group (22.14±25.35 kU A / L) was significantly reduced (P<0.05), which indicates that probiotic intervention for 20 weeks can significantly downregulate the expression of house dust mite-sIgE in the serum of atopic individuals; at this time, the level of Δ dust mite-sIgE in the serum of the 8-week probiotic group was -3.08±14.43kU A / L, which was still lower than that of the control group, indicating that the effect of probiotics was sustained.

[0178] The above experimental results show that Bifidobacterium longum subsp. infantis CCFM111 intervention can significantly downregulate the levels of dust mite and house dust mite-specific IgE in patient serum.

[0179] 7. Effect of probiotic intervention on the changes of serum IL-13 and IL-10 levels compared with baseline

[0180] The results of the changes in serum ΔIL-13 levels of the three groups at the end of the 8th and 20th week (ΔIL-13 = IL-13 levels at the end of the 8th and 20th week - IL-13 levels at the baseline period) are as follows: Fig.12 As shown in A, the results of the change in ΔIL-10 levels (ΔIL-10 = IL-10 levels at the end of the 8th and 20th weeks - IL-10 levels at the baseline period) are shown in Fig.12 As shown in B.

[0181] Depend on Fig.12 A shows that after 8 weeks of probiotic intervention, the level of ΔIL-13 in serum (-190.51±700.07pg / mL) was lower than that in the control group (-22.91±741.96pg / mL), which indicates that increasing probiotic intervention can better inhibit the production of IL-13 in the body than simple drug treatment. After 20 weeks of probiotic intervention, the level of ΔIL-13 in serum (-319.22±1016.54pg / mL) was significantly lower than that in the control group (275.60±1273.66pg / mL) (P<0.05), which indicates that 20 weeks of probiotic intervention can significantly downregulate the expression of proinflammatory factor IL-13 in the serum of atopic individuals; at this time, the level of ΔIL-13 in the serum of the 8-week probiotic group was -248.64±1258.03pg / mL, which was still lower than that of the control group, indicating that the effect of probiotics is sustained.

[0182] Depend on Fig.12 B shows that after 8 weeks of probiotic intervention, the level of ΔIL-10 in serum (0.73±3.90pg / mL) was significantly higher than that in the control group (-1.96±4.76pg / mL) (P<0.01), which indicates that increasing probiotic intervention can better promote the production of IL-10 in the body than simple drug treatment. After 20 weeks of probiotic intervention, the level of ΔIL-10 in serum (2.97±5.94pg / mL) was significantly higher than that in the control group (-1.52±6.87pg / mL) (P<0.01), which indicates that 20 weeks of probiotic intervention can significantly upregulate the expression of anti-inflammatory factor IL-10 in the serum of atopic individuals; at this time, the level of ΔIL-10 in the serum of the 8-week probiotic group was 1.58±9.31pg / mL, which was still higher than that of the control group, indicating that the effect of probiotics is sustained.

[0183] The above experimental results show that Bifidobacterium longum subsp. infantis CCFM111 intervention can significantly downregulate the level of pro-inflammatory factor IL-13 in the patient's serum and significantly upregulate the level of anti-inflammatory factor IL-10 in the patient's serum.

[0184] 8. Effect of probiotic intervention on the changes of IL-13 and ECP levels in patients' nasal lavage fluid compared with baseline

[0185] The results of the changes in ΔIL-13 levels in the nasal lavage fluid of the three groups at the end of the 8th and 20th week were as follows: Fig.13 As shown in A, the results of the ΔECP level change (ΔECP = ECP level at the end of the 8th and 20th weeks - ECP level at the baseline period) are as follows Fig.13 As shown in B.

[0186] Depend on Fig.13 A shows that after 8 weeks of probiotic intervention, the level of ΔIL-13 in nasal lavage fluid (-306.26±738.31pg / mL) was significantly lower than that in the control group (161.02±757.56pg / mL) (P<0.05). After 20 weeks of probiotic intervention, the level of ΔIL-13 in nasal lavage fluid (-427.21±964.29pg / mL) was significantly lower than that in the control group (-3.63±191.20pg / mL) (P<0.05), which indicates that 8 weeks of probiotic intervention can significantly reduce the expression of IL-13, a proinflammatory factor 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 the nasal lavage fluid of the 8-week probiotic group was -340.70±967.56pg / mL, which was still lower than that of the control group, indicating that the effect of probiotics is sustained.

[0187] Eosinophils are active substances released after being activated by various pathways in allergic inflammatory reactions. ECP is a specific marker of eosinophils and can be used as an indicator to monitor tracheal inflammation and to guide anti-inflammatory treatment. Fig.13 B shows that after 8 weeks of probiotic intervention, the level of ΔECP in nasal lavage fluid (-139.63±288.40pg / mL) was lower than that in the control group (-57.39±378.87pg / mL), which indicates that increasing probiotic intervention can better reduce the enrichment of eosinophils in the patient's nose than simple drug treatment. After 20 weeks of probiotic intervention, the level of ΔECP in nasal lavage fluid (-212.55±327.26pg / mL) was significantly lower than that in the control group (11.54±243.72pg / mL) (P<0.05), which indicates that 20 weeks of probiotic intervention can significantly reduce the enrichment of nasal acidophils in atopic individuals; at this time, the level of ΔECP in the nasal lavage fluid of the 8-week probiotic group was -84.89±209.17pg / mL, which was still lower than that of the control group, indicating that the effect of probiotics is sustained.

[0188] The above experimental results show that Bifidobacterium longum subsp. infantis CCFM111 intervention can significantly downregulate the levels of nasal proinflammatory factors IL-13 and ECP in patients.

[0189] 9. Safety analysis of probiotics

[0190] Safety and tolerability were monitored at each visit during the study, with symptoms reported by the subjects and blood drawn regularly for laboratory tests. There were 120 people in the probiotic group included in the safety analysis (when analyzing the data within the first 8 weeks, the two probiotic intervention groups were collectively referred to as the probiotic group), and 59 people in the control group.

[0191] Table 6 Blood component indicators of patients in the control group at different time points

[0192]

[0193] Table 7 Blood component indicators of patients in the probiotic group at different time points

[0194]

[0195] Patients in each group provided blood samples for laboratory tests before intervention, at 8 weeks and 20 weeks of intervention. The results (as shown in Tables 6-7) showed that there were no differences in the indicators of the patients in each group, indicating that the intervention of Bifidobacterium longum infantis subspecies CCFM111 had no significant effect on the blood components of the patients. No side effects or adverse events were recorded during the study, which indicated that the patients were not sensitive to 5×10 9 The intervention with CFU of Bifidobacterium longum subsp. infantis CCFM111 was well tolerated.

[0196] The above results show that Bifidobacterium longum subsp. infantis CCFM111 has acceptable safety and is well tolerated in patients with allergic rhinitis.

[0197] In summary, the Bifidobacterium longum infantis subspecies CCFM111 provided by the present invention can effectively reduce the comprehensive symptoms and drug scores of patients with allergic rhinitis, effectively reduce the dust mite and house dust mite-specific IgE levels and the proinflammatory factor IL-13 levels in the patient's serum, effectively increase the anti-inflammatory factor IL-10 level in the patient's serum, effectively reduce the proinflammatory factor IL-13 level and ECP level in the patient's nasal lavage fluid, and has good safety and tolerability.

[0198] Example 9: Preparation of bacterial powder containing Bifidobacterium longum subsp. infantis CCFM111

[0199] (1) Preparation of Bifidobacterium longum subspecies infantis CCFM111 seed solution

[0200] The Bifidobacterium longum subsp. infantis CCFM111 was inoculated into an MRS liquid culture medium and cultured at 37° C. for 16 h to prepare a seed solution of the Bifidobacterium longum subsp. infantis CCFM111;

[0201] (2) inoculating the prepared seed solution of Bifidobacterium longum infantis subspecies CCFM111 into MRS medium at an inoculum amount of 3% of the total mass of the medium, and culturing at 37° C. for 30 h to obtain a culture solution;

[0202] The culture solution was centrifuged to collect the bacterial cells; the bacterial cells were washed three times with a phosphate buffer solution having a pH of 7.2, and then resuspended with a trehalose freeze-drying protective agent having a concentration of 100 g / L, and the mass ratio of the freeze-drying protective agent to the bacterial cells was controlled to be 2:1 to obtain a resuspension;

[0203] The resuspension was precooled at -80°C for 1.5 hours and then immediately transferred to a freeze dryer for drying for 24 hours to obtain Bifidobacterium longum subsp. infantis CCFM111 bacterial powder.

[0204] Example 10: Fermented food containing Bifidobacterium longum subsp. infantis CCFM111

[0205] Fresh vegetables were selected and washed and juiced, and then sterilized at high temperature for 2 seconds at 140°C, and then immediately cooled to 37°C, and then the powder of Bifidobacterium longum infantis subspecies CCFM111 prepared by the present invention was added and fermented to a concentration of 10 6 CFU / mL or above, and stored in a refrigerator at 4°C to obtain a fruit and vegetable beverage containing live bacteria of Bifidobacterium longum infantis subspecies CCFM111.

[0206] The Bifidobacterium longum infantis subspecies CCFM111 of the present invention is used to ferment and produce other fermented foods, such as solid foods, liquid foods, and semi-solid foods. The fermented foods include dairy products, bean products, and fruit and vegetable products, wherein dairy products include milk, sour cream, and cheese; and fruit and vegetable products include cucumber, carrot, beet, celery, and cabbage products.

[0207] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Bifidobacterium longum subsp. infantis CCFM111, characterized in that: The Bifidobacterium longum subspecies infantis CCFM111 has been deposited in Guangdong Provincial Microbiological Culture Collection Center with the deposit number of GDMCC No: 65375.

2. A microbial preparation containing the Bifidobacterium longum subspecies infantis CCFM111 according to claim 1.

3. The microbial preparation according to claim 2, characterized in that In the microbial preparation, the number of cells of Bifidobacterium longum infantis subspecies CCFM111 is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

4. A product, characterized in that The product contains living cells, inactivated cells, supernatant, lysate and / or metabolites of Bifidobacterium longum subspecies infantis CCFM111 according to claim 1.

5. The product according to claim 4, characterized in that The products include food, medicine or health products.

6. The product according to claim 5, characterized in that The food includes dairy products, soy products, meat products or fruit and vegetable products.

7. The product according to claim 6, characterized in that The medicine also contains a drug carrier and / or a pharmaceutical excipient.

8. The product according to claim 7, characterized in that The drug carrier comprises microcapsules, microspheres, nanoparticles or liposomes; the pharmaceutical excipients comprise anti-adhesive agents, penetration enhancers, buffers, plasticizers, surfactants, defoamers, thickeners, inclusion agents, absorbents, humectants, solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, pH regulators, adhesives, disintegrants, fillers, lubricants, wetting agents, integrators, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, foaming agents, suspending agents, coating materials, fragrances, diluents, flocculants and deflocculating agents, filter aids or release retardants.

9. Use of the Bifidobacterium longum subsp. infantis CCFM111 according to claim 1 or the microbial preparation according to claim 2 in the preparation of a medicament for preventing and / or treating allergic rhinitis.

10. The use according to claim 9, characterized in that: In the drug, the number of cells of Bifidobacterium longum infantis subspecies CCFM111 is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

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