Application of Streptococcus thermophilus CT-02 in the preparation of products to relieve pollen-induced allergic rhinitis

Abstract

This invention relates to the use of Streptococcus thermophilus CT-02 in the preparation of products for relieving pollen-induced allergic rhinitis; the CT-02 strain is Streptococcus thermophilus with accession number GDMCC No:64474. Streptococcus thermophilus CT-02 strain. This invention develops a new use for Streptococcus thermophilus CT-02, providing a research basis for its widespread application. It also develops a novel strategy for alleviating pollen-induced allergic rhinitis. This invention finds that Streptococcus thermophilus CT-02 has a very significant effect in alleviating symptoms related to pollen-induced allergic rhinitis, specifically by significantly improving symptoms such as nasal mucosal lesions, nasal itching, sneezing, runny nose, asthma, and intestinal flora imbalance caused by pollen-induced allergic rhinitis.

Description

Technical Field

[0001] This invention belongs to the field of probiotic technology and relates to a new use of Streptococcus thermophilus CT-02, specifically the application of Streptococcus thermophilus CT-02 in the preparation of products that relieve pollen-induced allergic rhinitis. Background Technology

[0002] Allergic diseases, such as allergic rhinitis (AR) caused by pollen allergy, are seeing a continuous rise in prevalence worldwide, significantly impacting patients' quality of life. Statistics show that there are currently 200-300 million people with pollen allergies in my country. Over the past few decades, large-scale urban greening and afforestation projects in my country have resulted in the widespread presence of highly allergenic plants, especially wormwood. These plants produce large quantities of lightweight pollen that can be dispersed by wind over long distances, making them major allergens.

[0003] In recent years, the prevalence of pollen-induced allergic rhinitis has been rising steadily, becoming a serious public health challenge. The disease's progression is generally divided into two key stages: the immune sensitization induction stage and the symptom onset response stage. Current mainstream clinical interventions (such as antihistamines, leukotriene receptor antagonists, and nasal corticosteroids) primarily target the response stage, alleviating symptoms by antagonizing or inhibiting released allergic mediators. However, effective intervention strategies for the immune dysregulation in the induction stage are lacking. Therefore, exploring novel, safe, and long-lasting interventions that can regulate immune balance from an early stage is of great significance.

[0004] thermophilic streptococci ( Streptococcus thermophilus Streptococcus thermophilus, as a recognized safe food-grade microorganism (GRAS ingredient), has long been widely used as a core starter culture in fermented dairy products such as yogurt, boasting a long history of safe consumption and excellent gastrointestinal tolerance. Previous studies have revealed that specific strains of Streptococcus thermophilus can modulate immune responses, for example, downregulating the pro-inflammatory factor IL-6 in human intestinal epithelial cells in in vitro experiments. However, as a classic food fermentation bacterium, its research depth and breadth in the field of probiotic immunomodulation are far less than that of "model bacteria" such as Lactobacillus or Bifidobacterium. Although existing technologies indicate that supplementing with probiotics to regulate the gut microbiota shows acceptable benefits in improving allergic rhinitis, to date, no studies have systematically explored and clarified whether Streptococcus thermophilus has a relieving effect on allergic rhinitis induced by pollen, a widely distributed seasonal airborne allergen. Therefore, providing a product that can effectively relieve allergic rhinitis induced by major allergenic pollens native to China (such as wormwood pollen) has broad application prospects. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a new use of Streptococcus thermophilus CT-02, specifically the use of Streptococcus thermophilus CT-02 in the preparation of products that relieve pollen-induced allergic rhinitis.

[0006] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides the use of Streptococcus thermophilus CT-02 in the preparation of products that alleviate pollen-induced allergic rhinitis; The CT-02 strain is a thermophilic streptococcus with accession number GDMCC No: 64474. Streptococcus thermophilus CT-02 strain.

[0007] This invention explores a new use for Streptococcus thermophilus CT-02, providing a research foundation for its widespread application. It also develops a novel strategy for alleviating pollen-induced allergic rhinitis. This invention reveals that Streptococcus thermophilus CT-02 has a significant effect on relieving symptoms associated with pollen-induced allergic rhinitis, specifically by significantly improving symptoms such as nasal mucosal lesions, nasal itching, sneezing, runny nose, asthma, and intestinal flora imbalance caused by pollen-induced allergic rhinitis. Furthermore, Streptococcus thermophilus CT-02 is a probiotic, ensuring high product safety and minimizing the likelihood of resistance development.

[0008] Preferably, the pollen includes Artemisia pollen.

[0009] Preferably, the dosage form of the product includes solution, powder, tablet, granule or capsule.

[0010] Preferably, the product also contains auxiliary materials.

[0011] Preferably, the excipients include any one or a combination of at least two of the following: protective agents, fillers, binders, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, colorants, pH regulators, antioxidants, antibacterial agents, or buffers.

[0012] Preferably, the product is in the form of a solution, which is prepared by the following method: The strain was inoculated into a culture medium and activated and fermented sequentially to obtain a fermentation broth; the fermentation broth was centrifuged and resuspended in a solvent to obtain the product.

[0013] Preferably, the product is in powder form, which is prepared by the following method: The strain was inoculated into a culture medium and activated and fermented sequentially to obtain a fermentation broth. The fermentation broth was centrifuged, mixed with a protective agent, and then dried to obtain the product.

[0014] Secondly, the present invention also provides the application of Streptococcus thermophilus CT-02 in the preparation of products that regulate eosinophil levels; The CT-02 strain is a thermophilic streptococcus with accession number GDMCC No: 64474. Streptococcus thermophilus CT-02 strain.

[0015] According to the research results of the present invention, products containing Streptococcus thermophilus CT-02 can effectively regulate the level of eosinophils in the body to achieve immunomodulation. That is, the above-mentioned products containing Streptococcus thermophilus CT-02 can be made into an experimental preparation that acts on eosinophils to investigate the immune response process involving eosinophils. The application claimed above in the preparation of eosinophil level regulators is not for relieving symptoms of pollen-induced allergic rhinitis, eliminating the cause or lesion, but a separate application.

[0016] Thirdly, the present invention also provides the application of Streptococcus thermophilus CT-02 in the preparation of products that regulate cytokine levels; The CT-02 strain is a thermophilic streptococcus with accession number GDMCC No: 64474. Streptococcus thermophilus CT-02 strain; The cytokines include any one or a combination of at least two of interferon-γ, interleukin-4, or interleukin-10.

[0017] According to the research results of the present invention, products containing Streptococcus thermophilus CT-02 can effectively regulate the secretion level of interferon-γ / interleukin-4 / interleukin-10 in the body to achieve immunomodulation. That is, the above-mentioned products containing Streptococcus thermophilus CT-02 can be made into an experimental preparation that acts specifically on interferon-γ / interleukin-4 / interleukin-10 to explore the immune response process involving interferon-γ / interleukin-4 / interleukin-10. The application claimed above in the preparation of secretion regulators of interferon-γ / interleukin-4 / interleukin-10 is not for relieving symptoms of pollen-induced allergic rhinitis, eliminating the cause or lesion, but is a separate application.

[0018] Fourthly, the present invention also provides the application of Streptococcus thermophilus CT-02 in the preparation of products that enhance the level of intestinal flora; The CT-02 strain is a thermophilic streptococcus with accession number GDMCC No: 64474. Streptococcus thermophilus CT-02 strain.

[0019] Preferably, the gut microbiota includes Pediococcus , Adlercreutzia , Butyribacter or Candidatus arthromitus Any one or at least two of them.

[0020] According to the research results of the present invention, products containing Streptococcus thermophilus CT-02 can effectively regulate the metabolic activities of intestinal flora to achieve the regulation of immune or allergic responses. That is, the above-mentioned products containing Streptococcus thermophilus CT-02 can be made into an experimental preparation that acts on the structure of intestinal flora to explore the immune response process involving the activity of intestinal flora. The application claimed above in the preparation of intestinal flora level regulators is not for relieving symptoms of pollen-induced allergic rhinitis, eliminating causes or lesions, but a separate application.

[0021] Compared with the prior art, the present invention has the following beneficial effects: This invention develops a novel strategy for alleviating pollen-induced allergic rhinitis. It discovers that *Streptococcus thermophilus* CT-02 has a significant effect on relieving symptoms associated with pollen-induced allergic rhinitis, specifically improving symptoms such as nasal mucosal lesions, nasal itching, sneezing, runny nose, asthma, and gut microbiota imbalance caused by pollen-induced allergic rhinitis. Furthermore, *Streptococcus thermophilus* CT-02 is a probiotic, ensuring high product safety and minimizing the likelihood of resistance development. Additionally, it has been found that the product can also be used to prepare eosinophil level regulators, cytokine secretion regulators, or gut microbiota structure regulators.

[0022] The CT-02 strain involved in this invention is classified and named as follows: Streptococcus thermophilus The depositary institution is Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 64474, deposit date March 29, 2024, and deposit address 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0023] Figure 1 This is a flowchart of mouse modeling and drug administration for the prevention and treatment groups; Figure 2 This is a graph showing the behavioral scores of mice in each group; Figure 3 This is a graph showing the pathological scores of nasal mucosa HE sections in the prevention group and the statistical results of the proportion of eosinophils under 400x magnification. Eosinophils are marked by yellow arrows in the graph. Figure 4 This is a graph showing the pathological scores of HE sections of nasal mucosa in the treatment group and the statistical results of the proportion of eosinophils under 400x magnification. Eosinophils are marked with yellow arrows in the graph. Figure 5This is a graph showing the statistical results of the serum IgE and IgG antibody levels and the IgE transcriptional expression level in the nasal mucosa tissue of the prevention group. Figure 6 This is a graph showing the statistical results of the serum IgE and IgG antibody levels and the IgE transcriptional expression level in the nasal mucosa tissue of the treatment group. Figure 7 This is a statistical graph showing the results of the endpoint IL-4 protein expression level (top), transcriptional expression level (bottom left), and serum IL-4 cytokine content (bottom right) in the prevention group. Figure 8 This is a statistical graph showing the expression levels of IL-4 protein in nasal mucosa tissue (top), transcriptional expression levels (bottom left), and serum IL-4 cytokine levels (bottom right) at the midpoint of the prevention group. Figure 9 This is a statistical graph showing the results of the endpoint IFN-γ protein expression level (top), transcriptional expression level (bottom left), and serum IFN-γ cytokine content (bottom right) in the prevention group. Figure 10 This is a statistical graph showing the expression levels of IFN-γ protein in nasal mucosa tissue (top), transcriptional expression levels (bottom left), and serum IFN-γ cytokine levels (bottom right) at the midpoint of the prevention group. Figure 11 This is a statistical chart showing the expression levels of IL-4 protein in nasal mucosa tissue (top), transcriptional expression levels (bottom left), and serum IL-4 cytokine levels (bottom right) in the treatment group. Figure 12 This is a statistical chart showing the expression levels of IFN-γ protein in nasal mucosa tissue (top), transcriptional expression levels (bottom left), and serum IFN-γ cytokine levels (bottom right) in the treatment group. Figure 13 The graph shows the statistical results of the serum IFN-γ / IL-4 (left 1) and IL-13 cytokine levels (left 3) at the endpoint of the prevention group and the serum IFN-γ / IL-4 (left 2) and IL-13 cytokine levels (left 4) at the midpoint of the prevention group. Figure 14 This is a graph showing the statistical results of the serum IFN-γ / IL-4 (left) and IL-13 cytokine levels (right) in the treatment group; Figure 15 The results of the analysis are as follows: community composition analysis of the prevention group - genus level (top), community structure similarity analysis - PCoA (middle), and community structure diversity analysis - β diversity (bottom). Figure 16 The results of the analysis of the microbial community composition of the treatment group - genus level (top), microbial community structure similarity analysis - NMDS (middle), and microbial community structure diversity analysis - β diversity (bottom) are shown in the figure. Figure 17 This is a statistical graph showing the results of linear discriminant analysis (LDA) of gut microbiota scores at the midpoint (top) and endpoint (bottom) of the prevention group. Figure 18 This is a statistical chart of the linear discriminant analysis (LDA) scores of the gut microbiota in the treatment group. Detailed Implementation

[0024] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0025] The following classification and naming of CT-02 strains Streptococcus thermophilus The accession number is GDMCC No: 64474.

[0026] The following methods for preparing bacterial suspensions and powders are as follows: After activating the bacterial strain, it is inoculated into a culture medium for cultivation to obtain a culture solution; the culture solution is centrifuged, and the bacterial suspension is resuspended to obtain a bacterial suspension, or a protectant is added for freeze drying to obtain a freeze-dried bacterial powder product.

[0027] The mice used in the following test cases were SPF-grade female BALB / c mice, 6 weeks old, weighing 15±2g, provided by Beijing SPF Biotechnology Co., Ltd. Five mice were randomly assigned to each cage, and the experimental animals were housed at the SPF-grade Laboratory Animal Center of China Agricultural University. The animals were housed in a barrier environment with a temperature of 23±1℃ and humidity of 50±3%, with a light-dark cycle of 12 h.

[0028] In the following test results data, This means p < 0.0001. This means p < 0.001. This means p < 0.01. (#) represents p<0.05, and ns represents no significant difference.

[0029] Example 1 (1) Laboratory animals Six-week-old SPF-grade female BALB / c mice, weighing 15±2g, were randomly assigned to cages of five and housed at the SPF-grade Laboratory Animal Center of China Agricultural University. The animals were housed in a barrier environment with a temperature of 23±1℃ and humidity of 50±3%, with a 12-hour light-dark cycle.

[0030] (2) Animal grouping and administration method Grouping: Blank control group (Control), model group (AR), positive drug group (LOR), prevention group - Streptococcus thermophilus CT-02, treatment group - Streptococcus thermophilus CT-02.

[0031] Administration method: ① Blank control group: A total of 20 mice were administered PBS (0.2 mL / mouse) by gavage daily from day 0 to day 30; and 40 μL PBS was administered intranasally once daily from day 21 to day 30 for 10 consecutive days. ② Model group: A total of 20 mice were administered PBS (0.2 mL / mouse) by gavage daily from day 0 to day 30. Sensitization was achieved on days 0, 3, 6, 9, 12, and 15 by intraperitoneal injection of Artemisia pollen and aluminum hydroxide immunoadjuvant (0.1 mL of a mixture of 2 mg / mL Artemisia pollen and 5% aluminum hydroxide immunoadjuvant was injected intraperitoneally every 3 days); allergic rhinitis was induced in mice by nasal instillation of a suspension of Artemisia pollen and aluminum hydroxide immunoadjuvant on days 21-30 (40 μL of a mixture of 10 mg / mL Artemisia pollen and 5% immunoadjuvant was instilled intranasally once daily for 10 consecutive days). ③ Positive drug group: A total of 20 mice were administered loratadine by gavage daily from day 0 to day 30 (0.2 mL / mouse, loratadine ground into powder and dissolved in DMSO, concentration of 0.1625 mg / mL). Sensitization was achieved on days 0, 3, 6, 9, 12, and 15 by intraperitoneal injection of Artemisia pollen and aluminum hydroxide immunoadjuvant (0.1 mL of a mixture of 2 mg / mL Artemisia pollen and 5% aluminum hydroxide immunoadjuvant was injected intraperitoneally every 3 days); allergic rhinitis was induced in mice by nasal instillation of a suspension of Artemisia pollen and aluminum hydroxide immunoadjuvant on days 21-30 (40 μL of a mixture of 10 mg / mL Artemisia pollen and 5% immunoadjuvant was instilled intranasally once daily for 10 consecutive days). ④ Prevention group: A total of 16 mice were administered Streptococcus thermophilus CT-02 bacterial suspension (0.2 mL / mouse) via gavage once daily from day 1 to day 20. The gavage dose was 10... 9 CFU / day; Sensitization was achieved on days 0, 3, 6, 9, 12, and 15 by intraperitoneal injection of Artemisia pollen and aluminum hydroxide immunoadjuvant (0.1 mL of a mixture of 2 mg / mL Artemisia pollen and 5% aluminum hydroxide immunoadjuvant was injected intraperitoneally every 3 days); allergic rhinitis was induced in mice by nasal instillation of a suspension of Artemisia pollen and aluminum hydroxide immunoadjuvant on days 21-30 (40 μL of a mixture of 10 mg / mL Artemisia pollen and 5% immunoadjuvant was instilled intranasally once daily for 10 consecutive days). ⑤ Treatment group: A total of 10 mice were administered Streptococcus thermophilus CT-02 bacterial suspension (0.2 mL / mouse) via gavage once daily from day 21 to 30. The gavage dose was 10 mL / mouse. 9 CFU / day; Sensitization was achieved on days 0, 3, 6, 9, 12, and 15 by intraperitoneal injection of Artemisia pollen and aluminum hydroxide immunoadjuvant (0.1 mL of a mixture of 2 mg / mL Artemisia pollen and 5% aluminum hydroxide immunoadjuvant was injected intraperitoneally every 3 days); allergic rhinitis was induced in mice by nasal instillation of a suspension of Artemisia pollen and aluminum hydroxide immunoadjuvant on days 21-30 (40 μL of a mixture of 10 mg / mL Artemisia pollen and 5% immunoadjuvant was instilled intranasally once daily for 10 consecutive days). On day 21, eight mice from each group except the treatment group were sacrificed; on day 31, eight mice from each group were sacrificed for subsequent testing, and the mice's condition was continuously monitored during the period.

[0032] (3) Efficacy test ① Animal behavioral analysis On day 30, after the last nasal stimulation, each mouse was placed in an individual cage for 10 minutes for observation. The occurrence and severity of nasal symptoms such as nose scratching, sneezing, and runny nose were observed. The mice were scored based on their nasal symptoms, as shown in Table 1. A total score was calculated using an additive method; a total score greater than 5 indicated successful model establishment. The scoring results are as follows: Figure 2 As shown.

[0033] Table 1 The results in the figure show that the model group (AR) had a strong allergic reaction with a score greater than 6, indicating successful modeling. Compared with the model group mice, the blank control group mice showed mild nose scratching and a small number of sneezes. p <0.01); In the treatment group, both the positive drug group (LOR) and CT-02 significantly reduced allergy symptoms ( p <0.01); In the prevention group, both loratadine and CT-02 significantly reduced allergy symptoms ( p <0.01). Compared with the model group mice, the effect of CT-02 in reducing allergy symptoms in the treatment group was lower than that in the LOR group and not significant; the effect of CT-02 in reducing allergy symptoms in the prevention group was higher than that in the LOR group but not significant. These results indicate that both preventive and therapeutic interventions with the CT-02 strain can significantly reduce the occurrence and severity of symptoms such as nose scratching and sneezing, and help alleviate allergy symptoms.

[0034] ② Pathological analysis of paraffin sections of nasal mucosa Observe the pathological sections of mouse nasal mucosa, including the proliferation of epithelial cells, ciliary arrangement, tissue structure, presence of edema in the mucosal epithelium and intercellular spaces, goblet cell proliferation, infiltration of eosinophils and plasma cells, and glandular hyperplasia. Increased eosinophil and lymphocyte infiltration, and goblet cell proliferation are representative pathological symptoms of allergic rhinitis. Scoring was performed based on the pathological symptoms of the mouse nasal mucosa, and the specific scoring criteria are shown in Table 2. The test results of the prevention group (samples from mice sacrificed on day 31) are as follows: Figure 3 As shown, the treatment group's test results (mice sacrificed on day 31) are as follows: Figure 4 As shown.

[0035] Table 2 like Figure 3 It can be seen that in the pathological scores of nasal mucosa HE sections: the model group (AR) had the highest pathological score, indicating a significant allergic reaction; compared with AR: the intervention of the positive drug group (LOR) and CT-02 resulted in a decrease in pathological scores, but not significantly. Compared with LOR: the trend of decreasing pathological scores after CT-02 intervention was higher than that of LOR, but not significantly.

[0036] In the eosinophil percentage under 400x magnification: the model group (AR) had the highest eosinophil percentage, indicating a significant allergic reaction; compared to AR: LOR intervention suppressed the eosinophil percentage in allergic mice. p <0.01); CT-02 intervention suppressed the proportion of eosinophils in allergic mice, but not significantly. Compared with LOR: the trend of CT-02 inhibiting the proportion of eosinophils in allergic mice was lower than that of LOR and not significant. The above results indicate that preventive intervention with CT-02 strain can appropriately reduce nasal mucosal pathological symptoms and the proportion of eosinophils under 400x magnification, and has a certain effect in alleviating allergy symptoms.

[0037] like Figure 4 It can be seen that in the pathological scores of nasal mucosa HE sections: the model group (AR) had the highest pathological score, indicating a significant allergic reaction; compared with AR: intervention in the positive drug group (LOR) resulted in a decrease in the pathological score, but not significantly; intervention in CT-02 resulted in a significant decrease in the pathological score. p <0.01). Compared with LOR: the trend of pathological score reduction after CT-02 intervention was higher than that after LOR, but not significantly.

[0038] In the eosinophil percentage under 400x magnification: the model group (AR) had the highest eosinophil percentage, indicating a significant allergic reaction; compared to AR: both LOR and CT-02 inhibited the eosinophil percentage in allergic mice (LOR compared to AR, p <0.05; CT-02 compared to AR, p<0.01). Compared with LOR: CT-02 showed a greater trend in inhibiting the proportion of eosinophils in allergic mice than LOR. These results indicate that therapeutic intervention with CT-02 can significantly reduce nasal mucosal pathological symptoms and the proportion of eosinophils at 400x magnification, thus helping to alleviate allergy symptoms.

[0039] ③ Analysis of IgE and IgG expression levels in serum and nasal mucosa The levels of IgE and IgG in mouse serum were detected using enzyme-linked immunosorbent assay (ELISA), and the expression level of IgE transcription in mouse nasal mucosa was detected using the Q-PCR method. Results of the prevention group (samples from mice sacrificed on day 31) are as follows. Figure 5 As shown, the treatment group's test results (mice sacrificed on day 31) are as follows: Figure 6 As shown.

[0040] like Figure 5 As shown, the serum antibody levels of IgE and IgG were significantly higher in the allergy model group (AR) than in the control group (PA). p <0.01); Compared with AR: the antibody levels of IgE and IgG in the positive drug group (LOR) and CT-02 were significantly decreased ( p <0.01). Compared with LOR: CT-02 showed a higher trend in reducing the allergic response to IgE and IgG in mice than LOR, but the effect was not significant.

[0041] The expression level of IgE transcription in nasal mucosa tissue was significantly higher in the allergy model group (AR) than in the control group; compared with AR: the expression level of IgE transcription in LOR showed a decreasing trend but was not significant; the expression level of IgE transcription in CT-02 was significantly decreased. p <0.05). Compared with LOR: CT-02 showed a greater trend in reducing the transcriptional expression level of allergic antibody IgE than LOR. These results indicate that preventive intervention with CT-02 can significantly reduce the levels of serum IgE and IgG antibodies and the transcriptional expression level of IgE in nasal mucosa tissue, which helps to reduce allergic reactions and alleviate allergy symptoms.

[0042] like Figure 6 As shown, the serum levels of IgE and IgG antibodies were significantly higher in the allergy model group (AR) than in the control group (B). p＜ 0.01 ); Compared with AR: the levels of IgE and IgG antibodies in the positive drug group (LOR) and CT-02 were significantly decreased ( p＜0.01 Compared with LOR, CT-02 showed a higher but not significant trend in reducing the allergic response to IgE and IgG in mice.

[0043] The level of IgE transcription in nasal mucosa tissue was significantly higher in the allergy model group (AR) than in the control group (p＜0.01 ); Compared with AR: IgE transcriptional expression levels in LOR showed a decreasing trend but were not significant; IgE transcriptional expression levels in CT-02 were significantly decreased ( p＜0.05 Compared to LOR, CT-02 showed a greater trend in reducing the transcriptional expression of allergic antibody IgE than LOR. These results indicate that therapeutic intervention with CT-02 can significantly reduce serum IgE and IgG antibody levels and IgE transcriptional expression in nasal mucosa tissue, helping to reduce allergic reactions and alleviate allergy symptoms.

[0044] ④ Immunofluorescence staining of nasal mucosa, analysis of transcriptional expression levels and serum cytokine levels The expression levels of IFN-γ and IL-4 secreted proteins in nasal mucosa tissue were measured. Simultaneously, the levels of IFN-γ, IL-4, and IL-13 cytokines in mouse serum were detected using enzyme-linked immunosorbent assay (ELISA), and the expression levels of IFN-γ and IL-4 cytokine transcriptional levels in mouse nasal mucosa were detected using Q-PCR. The test samples used for the intermediate point in the prevention group were mice sacrificed on day 21; the test samples used for the endpoint in the prevention group were mice sacrificed on day 31.

[0045] The results of the endpoint tests for IL-4 protein expression, transcriptional expression, and serum IL-4 cytokine levels in the prevention group are as follows: Figure 7 As shown; the expression levels of IL-4 protein in nasal mucosa tissue, transcriptional expression levels, and serum IL-4 cytokine levels at the midpoint of the prevention group are as follows: Figure 8 As shown; the endpoints for the prevention group included the expression levels of IFN-γ protein in nasal mucosa tissue, the transcriptional expression level, and the serum IFN-γ cytokine content, as shown in the figure. Figure 9 As shown; the results of the tests on IFN-γ protein expression level, transcriptional expression level, and serum IFN-γ cytokine content at the midpoint of the prevention group are as follows. Figure 10 As shown; the results of the tests on IL-4 protein expression level, transcriptional expression level, and serum IL-4 cytokine content in the treatment group are as follows. Figure 11 As shown; the results of the tests on IFN-γ protein expression level, transcriptional expression level, and serum IFN-γ cytokine content in the treatment group are as follows. Figure 12 As shown.

[0046] like Figure 7 As shown, IL-4 levels in the model group (AR) tissues were significantly higher than those in the control group (AR). p <0.01); Compared with AR: the positive drug group (LOR) significantly inhibited the protein expression of IL-4 in tissues ( p<0.05), inhibited transcriptional expression but not significantly, and inhibited serum IL-4 cytokine levels but not significantly; CT-02 significantly inhibited IL-4 protein expression in tissues ( p <0.01), it inhibited transcriptional expression but not significantly, and it inhibited the cytokine content of IL-4 in serum but not significantly. Compared with LOR: In tissues, CT-02 had a higher but not significant inhibitory effect on IL-4 protein expression and transcriptional expression, while in serum, CT-02 had a lower and not significant inhibitory effect on IL-4 cytokine content. These results indicate that the endpoint of CT-02 intervention in the prevention group can significantly downregulate the expression of IL-4 protein in tissues, thereby helping to downregulate B cell activation and IgE production, and alleviate allergy symptoms.

[0047] like Figure 8 As shown, IL-4 levels in the model group (AR) tissues were significantly higher than those in the control group (AR). p <0.01); Compared with AR: In tissues, the positive drug group (LOR) significantly inhibited the protein expression of IL-4 ( p <0.05), which inhibited the transcriptional expression of IL-4 but not significantly, and the cytokine content of IL-4 in serum but not significantly; in tissues, CT-02 significantly inhibited the protein expression of IL-4 ( p The concentration of CT-02 (<0.05) inhibited the transcriptional expression of IL-4, but not significantly, and also inhibited the cytokine content of IL-4 in serum, but not significantly. Compared with LOR, CT-02 had a lower inhibitory effect on the expression of IL-4 protein in tissues, the transcriptional expression level, and the IL-4 cytokine content in serum. These results indicate that intervention with CT-02 at the midpoint of the prevention group can significantly downregulate the expression of IL-4 protein in tissues, thereby helping to downregulate B cell activation and IgE production, and alleviate allergy symptoms.

[0048] like Figure 9 As shown, IFN-γ in tissues and serum was significantly decreased in the model group (AR); compared with AR, the positive drug group (LOR) significantly upregulated the protein expression of IFN-γ in tissues. p <0.05), upregulating but not significantly increasing the transcriptional expression of IFN-γ, and significantly upregulating the cytokine content of IFN-γ in serum. p <0.01); In tissues, CT-02 upregulated IFN-γ protein expression but not significantly, and significantly upregulated transcriptional expression. p<0.05), serum IFN-γ cytokine levels were upregulated but not significantly. Compared with LOR: CT-02 had a lower and insignificant upregulation effect on IFN-γ protein expression in tissues than LOR, a higher but insignificant upregulation effect on IFN-γ transcriptional expression in tissues, and a significantly lower upregulation effect on serum IFN-γ cytokine levels than LOR. p <0.05). The above results indicate that intervention in the prevention endpoint group CT-02 can significantly upregulate the transcriptional expression level of IFN-γ in tissues, inhibit Th2 cell function, and alleviate allergic reactions.

[0049] like Figure 10 As shown, IFN-γ was significantly decreased in tissues and serum in the model group (AR); compared with AR, the protein expression of IFN-γ was significantly upregulated in tissues in the positive drug group (LOR). p <0.05), upregulated transcriptional expression but not significantly, and significantly upregulated serum IFN-γ cytokine levels ( p <0.05); CT-02 upregulated IFN-γ protein expression, transcriptional expression, and serum IFN-γ cytokine levels in both tissues and serum, but the effect was not significant. Compared with LOR: the upregulation effect of CT-02 on IFN-γ protein expression, transcriptional expression, and serum IFN-γ cytokine levels was lower than that of LOR and not significant. These results indicate that intervention with CT-02 in the intermediate point group can appropriately upregulate IFN-γ protein expression, transcriptional expression, and serum IFN-γ cytokine levels, and has a certain inhibitory effect on Th2 cell function.

[0050] like Figure 11 As shown, tissue and serum IL-4 levels in the model group (AR) were significantly higher than those in the control group (AR). p <0.01); Compared with AR: In tissues, the positive drug group (LOR) significantly inhibited the protein expression of IL-4 ( p <0.01), which inhibited the transcriptional expression of IL-4 but not significantly, and downregulated the cytokine content of IL-4 in serum but not significantly; in tissues, CT-02 significantly inhibited the protein expression of IL-4. p <0.01), which inhibited transcriptional expression but not significantly, and significantly downregulated the cytokine content of IL-4 in serum. p<0.05). Compared with LOR: In tissues and serum, CT-02 had a greater inhibitory effect on IL-4 protein expression, transcriptional expression, and serum IL-4 cytokine levels than LOR. These results indicate that CT-02 intervention in the treatment group can significantly downregulate IL-4 protein expression in tissues and cytokine levels in serum, thereby helping to downregulate B cell activation and IgE production, and alleviate allergy symptoms.

[0051] like Figure 12 As shown, IFN-γ levels in the model group (AR) tissues and serum were significantly decreased. Compared to AR, the positive control group (LOR) upregulated IFN-γ protein expression, transcriptional expression, and serum IFN-γ cytokine levels in both tissues and serum, but the effect was not significant. Similarly, CT-02 upregulated IFN-γ protein expression, transcriptional expression, and serum IFN-γ cytokine levels in both tissues and serum, but the effect was not significant. Compared to LOR, CT-02 upregulated IFN-γ protein expression, transcriptional expression, and serum IFN-γ cytokine levels in tissues, but the effect was not significant. These results indicate that CT-02 intervention in the treatment group can appropriately upregulate IFN-γ protein expression, transcriptional expression, and serum cytokine levels, and has a certain inhibitory effect on Th2 cell function.

[0052] ⑤ Analysis of immune balance and IL-13 cytokine levels The results of serum Th1 / Th2 (IFN-γ / IL-4) and IL-13 cytokine levels at the endpoint and intermediate points in the prevention group are as follows: Figure 13 As shown; the results of serum Th1 / Th2 and IL-13 cytokine levels in the treatment group are as follows. Figure 14 As shown, the test sample used at the midpoint of the prevention group was the mouse sacrificed on day 21; the test sample used at the endpoint of the prevention group was the mouse sacrificed on day 31.

[0053] like Figure 13 As shown, the serum Th1 / Th2 ratio in the model group (AR) was significantly decreased; compared to AR, the intervention in the endpoint-positive drug group (LOR) significantly increased the serum Th1 / Th2 ratio. p<0.01), intervention at the midpoint LOR in the prevention group increased the serum Th1 / Th2 ratio, but not significantly; intervention at the endpoint and midpoint CT-02 in the prevention group increased the serum Th1 / Th2 ratio, but not significantly; compared with LOR: the effect of intervention at the endpoint and midpoint CT-02 in the prevention group on increasing the serum Th1 / Th2 ratio was lower than that of LOR and not significant. The cytokine level of IL-13 in the model group (AR) was significantly increased. Compared with AR: intervention at the endpoint and midpoint LOR in the prevention group significantly downregulated IL-13 levels ( p <0.01); In the prevention group, the intervention of CT-02 at both the endpoint and midpoint downregulated serum IL-13 levels, but the effect was not significant. Compared with LOR: The effect of CT-02 in downregulating IL-13 at both the endpoint and midpoint in the prevention group was lower than that in LOR and was not significant. The above results indicate that the intervention of CT-02 in the endpoint and midpoint groups of the prevention group can appropriately upregulate the serum Th1 / Th2 ratio and reduce the level of IL-13 cytokine, and has a certain effect on regulating immune imbalance caused by allergies and reducing Th2 cytokines.

[0054] like Figure 14 As shown, the serum Th1 / Th2 ratio in the model group (AR) was significantly decreased. Compared to AR, intervention in the positive drug group (LOR) increased the serum Th1 / Th2 ratio, but not significantly; intervention in CT-02 significantly increased the serum Th1 / Th2 ratio, but not significantly. Compared to LOR, the intervention effect of CT-02 in increasing the serum Th1 / Th2 ratio was higher than that of LOR, but not significantly. The IL-13 cytokine level in the model group (AR) was significantly increased. Compared to AR, interventions in LOR and CT-02 significantly downregulated the IL-13 cytokine level (p < 0.01). Compared to LOR, the intervention effect of CT-02 in downregulating the IL-13 cytokine level was higher than that of LOR, but not significantly. These results indicate that intervention in the treatment group with CT-02 can significantly upregulate the serum Th1 / Th2 ratio and reduce the IL-13 cytokine level, playing a significant role in regulating immune imbalance caused by allergies and reducing Th2-type cytokines, thus helping to alleviate allergic reactions.

[0055] ⑥ Intestinal flora analysis Microbial community structure similarity analysis was performed using Bray-Curtis distance-based principal coordinate analysis (PCoA) to detect differences in the structure of different gut microbiota. Microbial community structure diversity analysis—β-diversity—presented the significant differences in community structure among different groups. The results of the prevention group test are shown below. Figure 15 As shown, the test analysis results of the treatment group are as follows: Figure 16 As shown.

[0056] like Figure 15As shown, after CT-02 intervention: Genus-level bacterial community composition results showed significant changes in the dominant species composition of the CT-02 group compared to the model group (AR) and control group (Control), with significant changes in Bacteroides and Lactobacillus genera; PCoA results showed that the CT-02 group was not completely separated from Control and AR, and the changes in bacterial community structural similarity were relatively small; β-diversity index results showed that the CT-02 group was significantly higher than other groups, and its community structure differed significantly from that of the AR and Control groups. These results indicate that CT-02 intervention in the prevention group can alter the genus-level bacterial community composition and structural similarity, significantly increase bacterial community structural diversity, and help alleviate related symptoms in allergic mice.

[0057] like Figure 16 As shown, after CT-02 intervention: Genus-level bacterial community composition results showed significant changes in the dominant species composition of the CT-02 group compared to the model group (AR) and control group (Control), with significant changes in *Lactobacillus* and *Actinomyces* genera; PCoA results showed complete separation of the CT-02 group from Control and AR, with a significant alteration in bacterial community structural similarity; β-diversity index results showed that the CT-02 group was significantly higher than all other groups, with smaller differences in community structure compared to the Control and AR groups. These results indicate that CT-02 intervention in the treatment group can alter the genus-level bacterial community composition and structural similarity, appropriately increase bacterial community structural diversity, and help alleviate related symptoms in allergic mice.

[0058] Linear discriminant analysis (LDA) effect size analysis (LEfSe) is a type of LDA that tests for differences at multiple levels, analyzes differential species at multiple levels, and uses the LDA value to measure the magnitude of the species' influence on the differential effect. It visually demonstrates the influence of the identified marker species between different groups on the differential effect. The results of the prevention group analysis are as follows: Figure 17 As shown, the analysis results of the treatment group are as follows: Figure 18 As shown.

[0059] like Figure 17 As shown, the linear discriminant analysis (LDA) scores of gut microbiota in mice at both the midpoint and endpoint of the prevention group were ≥3. After CT-02 intervention: the gut microbiota at the midpoint of the prevention group... Pediococcus , Litchfieldia Upward adjustment, among which, Pediococcus Related to anti-allergy effects, upregulation of this bacterium helps alleviate allergic reactions; prevention endpoints include gut microbiota. Harryflintia , Angelakisella It is upregulated and not related to anti-allergy effects.

[0060] like Figure 18As shown, the linear discriminant analysis (LDA) score of the gut microbiota in the treatment group was ≥2.5. After CT-02 intervention: within the gut microbiota of the treated group, Pediococcus, Adlercreutzia, Butyribacter, Candidatus arthromitus, Turicibacter Upward adjustment, among which, Pediococcus Related to anti-allergy Pediococcus The upregulation helps alleviate allergic reactions; Adlercreutzia, Candidatus arthromitus Related to anti-inflammatory and immune-modulating effects. Adlercreutzia、 Candidatus arthromitus Upregulation may help alleviate inflammatory responses caused by allergies or regulate allergies caused by immune imbalances; Butyribacter They are beneficial bacteria. Butyribacter An increase in these substances may play a role in regulating the balance of gut microbiota, which can help alleviate allergy symptoms.

[0061] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0062] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0063] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. Application of Streptococcus thermophilus CT-02 in the preparation of products to relieve pollen-induced allergic rhinitis; The CT-02 strain is a thermophilic streptococcus with accession number GDMCC No: 64474. Streptococcus thermophilus CT-02 strain.

2. The application as described in claim 1, characterized in that, The dosage forms of the product include solutions, powders, tablets, granules, or capsules.

3. The application as described in claim 1, characterized in that, The product also contains auxiliary materials.

4. The application as described in claim 3, characterized in that, The excipients include any one or a combination of at least two of the following: protective agents, fillers, binders, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, colorants, pH regulators, antioxidants, antibacterial agents, or buffers.

5. The application as described in claim 2, characterized in that, The product is in the form of a solution, which is prepared by the following method: The strain was inoculated into a culture medium and activated and fermented sequentially to obtain a fermentation broth; the fermentation broth was centrifuged and resuspended in a solvent to obtain the product.

6. The application as described in claim 2, characterized in that, The product is in powder form and is prepared by the following method: The strain was inoculated into a culture medium and activated and fermented sequentially to obtain a fermentation broth. The fermentation broth was centrifuged, mixed with a protective agent, and then dried to obtain the product.

7. Application of Streptococcus thermophilus CT-02 in the preparation of products that regulate eosinophil levels; The CT-02 strain is a thermophilic streptococcus with accession number GDMCC No: 64474. Streptococcus thermophilus CT-02 strain.

8. Application of Streptococcus thermophilus CT-02 in the preparation of products that regulate cytokine levels; The CT-02 strain is a thermophilic streptococcus with accession number GDMCC No: 64474. Streptococcus thermophilus CT-02 strain; The cytokines include any one or a combination of at least two of interferon-γ, interleukin-4, or interleukin-10.

9. Application of Streptococcus thermophilus CT-02 in the preparation of products that enhance intestinal flora levels; The CT-02 strain is a thermophilic streptococcus with accession number GDMCC No: 64474. Streptococcus thermophilus CT-02 strain.

10. The application as described in claim 9, characterized in that, The gut microbiota includes Pediococcus , Adlercreutzia , Butyribacter or Candidatusarthromitus Any one or at least two of them.

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