Paradigm shift in the treatment of allergic rhinitis

Abstract

This invention belongs to the field of microbial technology, specifically relating to a strain of *Pseudomonas dignitaries* and its application in relieving allergic rhinitis. *Pseudomonas dignitaries* is deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC No. 38360, deposit date April 22, 2026, and classified and named accordingly. Parabacteroides distasonis In this invention, *Pseudomonas diffusa* HGI-04 can reduce the frequency of sneezing and nose scratching in mice with Artemisia argyi pollen-induced allergic rhinitis, decrease the number of eosinophils in the nasal mucosa, and regulate immune metabolites related to tryptophan metabolism and short-chain fatty acid metabolism. By regulating the "gut microbiota-metabolism-immunity" pathway, it alleviates allergic rhinitis, providing a new microbial resource for the microecological intervention of allergic rhinitis, and laying the application foundation for the development of precision probiotic products.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of *Pseudomonas diffusa* isolated from the intestines of healthy individuals and its application in relieving allergic rhinitis. Background Technology

[0002] Allergic rhinitis is a common immune-related disease induced by allergens and characterized by chronic inflammation of the nasal mucosa. Its core mechanism is type I hypersensitivity reaction mediated by immunoglobulin E.

[0003] Currently, common treatments for allergic rhinitis mainly include antihistamines, nasal corticosteroids, and allergen-specific immunotherapy. However, these treatments have drawbacks such as high recurrence rates and poor long-term adherence, necessitating the development of safe and long-lasting new intervention methods. Recent studies have confirmed that gut microbiota imbalance can damage the mucosal barrier, disrupt immune homeostasis, and induce allergic inflammation via the gut-nose / gut-lung axis. Allergic individuals generally exhibit gut microbiota diversity and low levels of short-chain fatty acids. Reshaping the gut microbiota can suppress nasal allergic inflammation, making microecological intervention a new direction for the prevention and treatment of allergic rhinitis.

[0004] Probiotics can regulate mucosal immunity, inhibit Th2 inflammation, and induce immune tolerance, but their efficacy exhibits significant strain specificity. Different isolates of the same species show vastly different colonization, metabolism, and anti-allergic effects. Existing related studies show stable results in animal experiments, but clinical efficacy varies. Human-derived functional strains suitable for Artemisia argyi pollen-induced allergic rhinitis and possessing both gut microbiota regulation and alleviation of allergy symptoms remain scarce. Intestinal isolates from healthy individuals show good host compatibility and high development potential. Therefore, screening for a clearly derived *Pseudomonas diffusa* HGI-04 that can improve Artemisia argyi pollen-induced allergic rhinitis and simultaneously regulate gut microbiota and metabolic levels could improve the microecological system of allergic rhinitis and provide a basis for the development of anti-allergic functional bacterial preparations. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a strain of *Pseudomonas diffusa* and its application in alleviating artemisia pollen-induced allergic rhinitis. This strain can effectively reduce the frequency of sneezing and nose scratching in mice, decrease the number of eosinophils in the nasal mucosa, regulate the intestinal flora structure, and improve the levels of immune metabolites associated with allergic inflammation.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a strain of *Pseudomonas dignitaria*, namely *Pseudomonas dignitaria* HGI-04, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38360, deposited on April 22, 2026, and classified as follows: Parabacteroides distasonis .

[0007] Preferably, the *Pseudomonas dilataniae* is isolated from the intestines of healthy individuals.

[0008] The *Pseudomonas diffusa* HGI-04 strain of this invention was isolated from fecal samples of healthy men. Phenotypic identification, molecular biological identification, and sequencing analysis confirmed the strain as *Pseudomonas diffusa*. Its hemolytic activity, antibiotic susceptibility, and bacterial pathogenicity were evaluated, confirming its safety. Furthermore, tolerance tests to artificial gastric and intestinal fluids demonstrated its gastrointestinal tolerance, indicating its suitability for gastrointestinal delivery.

[0009] Secondly, this invention provides the use of *Peribrobacter pylori* in the preparation of a medicament for the prevention and / or relief of allergic rhinitis, wherein the medicament is a probiotic powder or probiotic oral liquid. When used, the viable count of *Peribrobacter pylori* is not less than 1 × 10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

[0010] Preferably, *Pseudomonas dilataniae* alleviates allergic rhinitis by regulating immune metabolites associated with allergic rhinitis; the allergic rhinitis is induced by Artemisia argyi pollen, and the immune metabolites are one or more of indole-3-acetic acid, tryptophan, propionic acid, and butyric acid.

[0011] Thirdly, the present invention provides a medicament for the prevention and / or relief of allergic rhinitis, the medicament containing the aforementioned *Pseudomonas dilataniae*.

[0012] Preferably, the viable count of *Pseudomonas difficile* in the drug is not less than 1 × 10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

[0013] Fourthly, the present invention provides a microbial preparation for the prevention and / or relief of allergic rhinitis, said microbial preparation containing the aforementioned *Pseudomonas difficile* and excipients; the viable count of *Pseudomonas difficile* in the microbial preparation is 1.0 × 10⁻⁶. 5 -1.0×10 12 CFU / mL or 1.0×10 5 -1.0×10 12 CFU / g.

[0014] Preferably, the excipients include one or more of inulin, fructooligosaccharides, skim milk powder, lactoferrin, casein phosphopeptide, or hydrolyzed egg yolk powder.

[0015] Fourthly, the present invention provides the use of microbial preparations in the preparation of products for the prevention and / or relief of allergic rhinitis, said products including pharmaceuticals or animal feed.

[0016] Preferably, *Pseudomonas dilataniae* alleviates allergic rhinitis by regulating immune metabolites associated with allergic rhinitis; the allergic rhinitis is induced by Artemisia argyi pollen, and the immune metabolites are one or more of indole-3-acetic acid, tryptophan, propionic acid, and butyric acid.

[0017] This invention uses 6-week-old female BALB / c mice as an animal model to investigate the intervention effect of *Pseudomonas dignitaries* on allergic rhinitis induced by Artemisia argyi pollen. By recording the number of sneezing and nose scratching in mice, detecting the ratio of eosinophils and neutrophils in nasal mucosa tissue, observing eosinophil infiltration in nasal mucosa and pathological changes in nasal tissue, and combining gut microbiota metagenomic sequencing and targeted immune metabolite detection, the comprehensive evaluation of the ameliorative effect of *Pseudomonas dignitaries* HGI-04 on allergic rhinitis induced by Artemisia argyi pollen, related gut microbiota imbalance and immune metabolic disorders was conducted.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides *Pseudomonas dilataniae* HGI-04 ( Parabacteroides distasonis HGI-04 significantly reduced the frequency of sneezing and nose scratching in mice with Artemisia argyi pollen-induced allergic rhinitis, alleviated eosinophil infiltration in the nasal mucosa, maintained gut microbiota homeostasis in female mice with allergic rhinitis, and improved gut microbiota imbalance. It exerts its effect in alleviating allergic rhinitis through the "gut microbiota-metabolism-immunity" regulatory pathway, providing new bacterial resources and theoretical basis for the prevention and intervention of allergic rhinitis and related allergic diseases, and contributing to the development and application of precision probiotic products. Attached Figure Description

[0019] Figure 1 This is a colony morphology diagram of *Pseudomonas difficile* HGI-04 in an embodiment of the present invention; Figure 2 This is a microscopic morphological image of *Pseudomonas dilatatus* HGI-04 after staining in Example 1 of this invention; Figure 3 This is a circulated genome diagram of *Pseudomonas diffusa* HGI-04 in Example 1 of the present invention; Figure 4 This is a diagram showing the growth of *Pseudomonas diffusa* HGI-04 after treatment with artificial gastrointestinal fluid in Example 2 of this invention. Figure 5 This refers to the number of sneezes within 10 minutes after nasal stimulation with Artemisia argyi pollen at 24, 31, 38, and 45 days in Example 5 of this invention, compared with the control group, model group, and HGI-04 intervention group. This indicates that P < 0.05. This indicates that P < 0.01. ## indicates P < 0.001 compared to the model group; ## indicates P < 0.01. Figure 6 In Example 5 of this invention, after intranasal stimulation with Artemisia argyi pollen at days 24, 31, 38, and 45, the number of times mice in the control group, model group, and HGI-04 intervention group scratched their noses within 10 minutes after nasal instillation was compared with the control group. This indicates that P < 0.05. # indicates P < 0.01, compared with the model group; # indicates P < 0.05. Figure 7 This is a representative gating plot of the proportion of eosinophils in the nasal mucosa tissue of mice in each treatment group detected by flow cytometry in Example 5 of the present invention. A is the control group, B is the model group, and C is the HGI-04 treatment group. Figure 8 This is a representative gating plot of the proportion of neutrophils in mouse nasal mucosa tissue detected by flow cytometry in Example 5 of the present invention, where A is the control group, B is the model group, and C is the HGI-04 treatment group. Figure 9 This is the statistical result of the flow cytometry analysis of the proportions of eosinophils and neutrophils in the nasal mucosa tissue of mice in each treatment group in Example 5 of this invention. Where A represents the proportion of eosinophils and B represents the proportion of neutrophils. Compared with the control group, This indicates that P < 0.05. This indicates that P < 0.01; Figure 10 The results of eosinophil counts in the nasal mucosa tissue of mice in each treatment group in Example 5 of this invention are compared with those in the control group. # indicates P < 0.05, compared with the model group; # indicates P < 0.05. Figure 11 The images show sections of nasal mucosa tissue stained with hematoxylin and eosin (H&E) from control mice in Example 5 of this invention. A is a section of the nasal mucosa, and B is a magnified view of point ① in A. The red arrows indicate eosinophils. Figure 12 The images show sections of nasal mucosa tissue stained with hematoxylin and eosin (H&E) from mice in the model group of Example 5 of this invention. A is a section of the nasal mucosa, and B is a magnified view of point ① in A. The red arrows indicate eosinophils. Figure 13 This is a section of nasal mucosa tissue stained with hematoxylin and eosin (H&E) from mice in the HGI-04 group in Example 5 of the present invention. A is a section of the nasal mucosa, and B is a magnified view of ① in A. The red arrow indicates eosinophils. Figure 14 This refers to the α-diversity of the gut microbiota in mice of the control group, model group, and HGI-04 intervention group after 24 days in Example 6 of this invention, compared with the control group. ### indicates P<0.001, compared with the model group; ### indicates P<0.001. Figure 15 The β diversity of the gut microbiota in mice in the control group, model group, and HGI-04 intervention group after 24 days in Example 6 of this invention; Figure 16 This refers to the α-diversity of gut microbiota in mice of the control group, model group, and HGI-04 intervention group after 45 days in Example 6 of this invention, compared with the control group. # indicates P < 0.05, compared with the model group; # indicates P < 0.05. Figure 17 The β-diversity of the gut microbiota in mice in the control group, model group, and HGI-04 intervention group after 45 days in Example 6 of this invention; Figure 18 The figures for Example 7 of this invention show the changes in immune-related metabolites in mice after 45 days in the control group, model group, and HGI-04 intervention group. A represents the change in indole-3-acetic acid level, B represents the change in tryptophan level, C represents the change in propionic acid level, and D represents the change in butyric acid level. These changes are compared with the control group. This indicates that P < 0.05. The expression indicates that P < 0.01, compared with the model group. # indicates P < 0.05, ## indicates P < 0.01, and ### indicates P < 0.001.

[0020] Instructions for the Preservation of Biological Materials *Pseudomonas diffusa* HGI-04 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38360, on April 22, 2026. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. It is classified and named as follows: Parabacteroides distasonis . Detailed Implementation

[0021] The present invention will be further described in conjunction with the following embodiments to make the objectives, technical solutions, and advantages of the present invention clearer. However, the embodiments are merely illustrative and do not constitute any limitation on the content and scope of protection of the present invention. Experimental steps, conditions, raw materials, reagents, instruments, components, and dosages not specifically described in the following embodiments are all conventional methods known to those skilled in the art and can be performed according to the conventional experimental procedures or conditions described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0022] This application provides a strain of *Pseudomonas diffusa* HGI-04 that can alleviate allergic rhinitis induced by Artemisia argyi pollen. *Pseudomonas diffusa* HGI-04 is preferably derived from the intestines of healthy humans and exhibits excellent gastrointestinal fluid and acid / bile acid tolerance. In this embodiment, the mammalian BALB / c mouse is a conventional model organism in the prior art, purchased from Spiford (Beijing) Biotechnology Co., Ltd.

[0023] Example 1: Isolation and identification of *Pseudomonas dilataniae* HGI-04 (1) Separation and purification: *Pseudomonas dilatatus* HGI-04 was isolated from fecal samples from healthy men. Before inoculation, PBS buffer and Columbia blood agar plates (supplemented with heme chloride and vitamin K1) were placed in an anaerobic workstation for 24 h to deaerate. 1.0 g of fecal sample was picked from the anaerobic workstation, and 9 mL of PBS buffer was added, followed by thorough shaking to prepare an initial bacterial suspension. The initial bacterial suspension was then serially diluted with PBS buffer to obtain suitable dilutions (10⁻⁶). 6 ~10 7 (CFU / mL) 100 μL of the dilution was spread onto a Columbia blood agar plate and incubated anaerobicly at 37°C for 48 h. Single colonies meeting the initial screening criteria for the target bacteria were selected based on colony shape, size, edge, and transparency. Colonies were then picked using an inoculation loop and streaked onto Columbia blood agar plates for three-zone purification, and incubated anaerobicly at 37°C for 48 h. This process was repeated until pure colonies were obtained.

[0024] (2) Passage and cryopreservation: Colonies were scraped from the surface of Columbia blood agar plates using an inoculation loop and inoculated into 10 mL of Columbia blood culture medium. The culture was then anaerobic at 37°C for 48 h. After incubation, sterilized skim milk and the bacterial culture were mixed at a 1:1 volume ratio, thoroughly mixed, and stored at -80°C.

[0025] (3) Identification of strains: The *Pseudomonas dilatatus* HGI-04 strain was identified according to FMIC-QO01-001-2015, "Microbiological Detection and Identification Methods for Multiphasic Bacteria". The purified strain was inoculated onto Columbia blood agar plates and anaerobically cultured at 37°C for 48 h, after which colony morphology was observed. Results are as follows: Figure 1 As shown, *Pseudomonas dilataniae* HGI-04 colonies are grayish-white to off-white, round, smooth, opaque, with regular, slightly raised edges. Microscopic observation is as follows... Figure 2 As shown, the bacteria are rod-shaped, measuring 0.4–0.6 μm × 0.7–1.6 μm, and are arranged singly or in pairs. Gram staining results are negative.

[0026] (4) Phylogenetic analysis of the strains: Genomic DNA was extracted from *Pseudomonas diffusa* HGI-04 using a bacterial genome extraction kit, and the extracted genomic DNA underwent quality testing. After passing quality testing, a sequencing library was constructed and high-throughput sequencing was performed.

[0027] The obtained raw sequencing data underwent quality control, adapter removal, and low-quality sequence filtering to obtain high-quality sequencing data (clean reads). Subsequently, the high-quality sequencing data was assembled de novo to obtain the genome sequence of *Pseudomonas diffusa* HGI-04, and its coding genes, rRNA, tRNA, and other functional elements were predicted and annotated.

[0028] The genome sequence (whole genome) of *Pseudomonas digiri* HGI-04 was further compared with the genome sequences of closely related reference strains in the NCBI database. The average nucleotide identity (ANI) was calculated, and a phylogenetic tree was constructed based on the genome sequence to verify the strain's taxonomic position. According to the classification criteria for prokaryotes based on genome sequence, an ANI value of 95% or higher usually supports the classification of strains into the same species. Based on the results of the whole genome comparative analysis, this strain was identified as *Pseudomonas digiri* and named *Pseudomonas digiri* HGI-04.

[0029] Genome sequencing and analysis results showed that the genome of *Pseudomonas dilataniae* HGI-04 consisted of 80 contigs, with a total genome length of 5,120,961 bp and a GC content of 45.03%. Figure 3 As shown.

[0030] Example 2: Tolerance evaluation of *Pseudomonas dilatatus* HGI-04 Preparation of artificial gastric fluid: 40 ml of sterile PBS normal solution + 3.0 g / L pepsin (0.3%), then adjust the pH to 2.5 (adjusted with 0.1 mol / L HCl), filter through a 0.22 μm filter membrane into a sterile empty conical flask for later use; Preparation of artificial intestinal fluid: 40 ml of sterile PBS normal solution + 1.0 g / L trypsin (0.1%) + 1.8% bile salts, then adjust the pH to 8.0 (adjusted with 0.1 mol / L NaOH), filter through a 0.22 μm filter membrane into a sterile empty conical flask for later use.

[0031] After culturing *Pseudomonas diffusa* HGI-04 for 24 h, the bacterial cells were collected by centrifugation and washed twice with PBS buffer. The cells were then resuspended in 5 mL of PBS buffer to obtain the initial bacterial suspension.

[0032] 500 μL of the initial bacterial suspension was added to a test tube containing 4.5 mL of artificial gastric fluid and treated at 37 °C for 3 h. Subsequently, 500 μL of the bacterial suspension treated with artificial gastric fluid was added to a test tube containing 4.5 mL of artificial intestinal fluid and treated at 37 °C for another 8 h. The bacterial growth was measured at 0 h, 3 h after artificial gastric fluid treatment, and 8 h after artificial intestinal fluid treatment to evaluate the tolerance of *Pseudomonas diffusa* HGI-04 to artificial gastric and intestinal fluids.

[0033] The results are as follows Figure 4 As shown, after treatment with artificial gastrointestinal fluid, the OD values ​​of the supernatant samples at 3 h and 11 h were... 600 The values ​​were 0.0164 and 0.2935, respectively. Using the bacterial sludge group as a reference, the relative growth rates of the supernatant samples at 3 h and 11 h were 12.11% and 33.95%, respectively. The results indicate that *Pseudomonas dignitaria* HGI-04 still possesses a certain growth recovery ability after treatment with artificial gastric and intestinal fluids, suggesting that this strain has good tolerance to artificial gastric and intestinal fluids.

[0034] Example 3: Safety evaluation of *Pseudomonas dilatatus* HGI-04 (1) Drug susceptibility testing: This embodiment refers to the antimicrobial susceptibility testing method in the EFSA-related microbial characterization guidelines to test the antimicrobial susceptibility of *Pseudomonas difficile* HGI-04. The tested drugs included ampicillin, gentamicin, erythromycin, tetracycline, and chloramphenicol. The strain's antimicrobial susceptibility is expressed as the minimum inhibitory concentration (MIC).

[0035] The MIC test results are shown in Table 1. Parabacterium dysenteriae HGI-04 was sensitive to ampicillin, gentamicin, erythromycin, tetracycline and chloramphenicol.

[0036] Table 1. MIC values ​​and drug susceptibility of *Pseudomonas difficile* HGI-04

[0037] (2) Antibiotic resistance genes and virulence genes: The antibiotic resistance and virulence genes of *Pseudomonas dilatatus* HGI-04 were detected using the FMIC-QO01-024-2020 microbiological detection method.

[0038] Regarding antibiotic resistance genes, using the CARD database and RGI software, with Perfect and Strict algorithms as screening criteria, no antibiotic resistance-related genes were detected. Using sequence similarity ≥90% and sequence coverage ≥60% as screening criteria, no antibiotic resistance-related genes were detected.

[0039] Regarding virulence genes, the setA dataset, whose sequences in the database had been experimentally validated, was used as the analysis dataset. With sequence similarity ≥80% and sequence coverage ≥70% as screening criteria, no virulence-related genes were detected. Further screening with sequence similarity ≥90% and sequence coverage ≥60% as screening criteria also yielded no virulence-related genes.

[0040] The above results indicate that *Pseudomonas difficile* HGI-04 does not contain exogenous antibiotic resistance genes or virulence genes, and is safe for consumption.

[0041] (3) Animal pathogenicity testing: Animal pathogenicity was tested according to Appendix A of the Technical Guidelines for Safety Testing and Evaluation of Microbial Strains Used in Health Food Raw Materials (2020 Edition). The results of the intraperitoneal injection test are shown in Table 2. After 14 consecutive days of observation, there were no significant differences in the initial and final body weights of mice in the *P. dignitary* HGI-04 culture undiluted group compared to the saline control group (P>0.05), indicating that the *P. dignitary* HGI-04 culture had no effect on the body weight of female BALB / c mice during the experiment. The results of the acute toxicity observation are shown in Table 3. No obvious toxic reactions or deaths were observed in the test mice during the experiment, indicating that the *P. dignitary* HGI-04 culture did not show significant animal pathogenicity.

[0042] Table 2. Effects of *Pseudomonas difficile* HGI-04 culture on mouse body weight (intraperitoneal injection)

[0043] Table 3. Effects of *Pseudomonas difficile* HGI-04 culture on acute toxicity in mice (intraperitoneal injection).

[0044] Example 4: Preparation of *Pseudomonas dilatatus* HGI-04 bacterial powder A *Pseudomonas diffusa* HGI-04 bacterial powder, prepared using a conventional method, with the following steps: (1) Activation of the strain: The strain frozen at -80℃ was inoculated into the corresponding liquid culture medium sterilized at 121℃ for 15 min under anaerobic conditions, and then anaerobically cultured at 37℃ for 18-24 h to obtain the revived strain. Subsequently, a single colony was picked and inoculated into the liquid culture medium, and anaerobically cultured at 37℃ for 18-24 h. This process was repeated 1-2 times to obtain the activated strain.

[0045] (2) Preparation of seed solution: Centrifuge the activated bacterial solution from step (1) at 4℃ and 6000r / min for 15min to collect the bacterial sludge; wash with sterile water and centrifuge again, repeat the washing process, and finally resuspend in sterile water to obtain the seed solution.

[0046] (3) Inoculation and fermentation: The seed culture from step (2) was inoculated into the liquid culture medium at 1‰ and fermented for 36 h under controlled fermentation conditions. The fermentation conditions were controlled as follows: constant temperature culture at 30℃ in the early stage of fermentation, natural fermentation until pH 5.0; then the fermentation temperature was adjusted to constant temperature culture at 37℃, and the pH was controlled to maintain 6.0 to maintain anaerobic fermentation.

[0047] The pH is controlled by adding a neutralizing agent, NaOH, during the above process.

[0048] (4) Freeze-drying: a. Cell concentration: The high-density fermentation broth was concentrated into cells by centrifugation at 12000 g; b. Add protectant: Add 5 times the amount of protectant solution to the bacterial concentrate; the protectant can be skim milk, trehalose, sucrose, lactose, vitamin C, antioxidants, etc. c. Drying: The bacterial suspension with added preservatives was freeze-dried to obtain freeze-dried bacterial powder, with a total viable count of 1.0 × 10⁻⁶. 11 CFU / g or higher.

[0049] The total viable count of the *Pseudomonas difficile* HGI-04 bacterial powder was 1×10⁻⁶. 11 CFU / g.

[0050] The *Pseudomonas dilatatus* HGI-04 bacterial powder prepared in this embodiment can be used for pharmaceutical or animal feed purposes.

[0051] Example 5: Effect of oral administration of *Pseudomonas difficile* HGI-04 on AR in mice. The effects of oral administration of *Pseudomonas difficile* HGI-04 on acute respiratory syndrome (AR) in mice were investigated using the following methods: Establishment of the S1.AR mouse model: Mice were randomly divided into 3 groups of 10 mice each, and the treatment methods for each group were as follows: Control group: Mice were injected intraperitoneally with 200 μL of physiological saline at 0, 7 and 14 days; 20 μL of physiological saline was administered bilaterally via nasal drip at 15-24, 31, 38 and 45 days; 200 μL of physiological saline was administered by gavage at 25-45 days; mice were sacrificed on day 46.

[0052] Model group: Mice were sensitized by intraperitoneal injection of 200 μL of solution A at 0, 7, and 14 days; at 15-24, 31, 38, and 45 days, mice were given 20 μL of solution B via bilateral nasal drops; at 25-45 days, mice were given 200 μL of physiological saline by gavage; mice were sacrificed on day 46.

[0053] HGI-04 intervention group: Mice were sensitized by intraperitoneal injection of 200 μL of solution A on days 0, 7, and 14; on days 15-24, 31, 38, and 45, mice were given 20 μL of solution B via bilateral nasal drops; on days 25-45, mice were given 200 μL of HGI-04 solution by gavage; mice were sacrificed on day 46.

[0054] Solution A refers to the following: 1 mg of Artemisia pollen and 0.4 mg of aluminum hydroxide are weighed and dissolved in 200 μL of physiological saline to obtain solution A.

[0055] Solution B refers to the following: 200 μg of Artemisia argyi pollen and 20 μg of aluminum hydroxide are weighed and fully dissolved in 20 μL of physiological saline to obtain solution B.

[0056] The HGI-04 solution contains 2 × 10⁻⁶ viable bacteria. 9 CFU / mL of *Pseudomonas difficile* HGI-04 solution.

[0057] The number of times mice sneezed and scratched their noses was recorded at specified time points, and their mental state and fur condition were monitored daily throughout the experiment.

[0058] Assessment of nasal symptoms: Nasal symptoms were assessed by counting the number of times the nasal cavity was rubbed and the number of sneezes. After the intranasal stimulation with Artemisia argyi pollen ended at 24, 31, 38, and 45 days, the number of times the nasal cavity was rubbed and the number of sneezes were recorded within 10 minutes after the mice received the nasal drops, and the counts were performed by the observers.

[0059] The results are as follows Figures 5-6 As shown, on day 24, compared with the control group, the frequency of sneezing and nose scratching in the model group mice was significantly increased (P<0.05), indicating the successful establishment of the Artemisia argyi pollen-induced AR mouse model. On day 45, compared with the model group, the number of sneezing and nose scratching in the HGI-04 intervention group mice was significantly reduced (P<0.05), indicating that oral administration of Parabacterium dyseri HGI-04 can improve nasal symptoms in Artemisia argyi pollen-induced AR mice.

[0060] S2. Flow cytometry analysis of the ratio of eosinophils to neutrophils in mice: After the mice in S1 underwent modeling, they were euthanized, and nasal mucosa tissue samples were collected. These samples were washed in pre-cooled PBS to remove residual blood and impurities. The tissue samples were mechanically minced and digested appropriately according to their condition. After filtration through a cell strainer, the filtrate was collected, centrifuged, and the supernatant was discarded. Cells were washed with PBS to prepare a single-cell suspension. The resulting single-cell suspension was resuspended and adjusted to a suitable cell concentration for subsequent flow cytometry analysis.

[0061] The above-mentioned single-cell suspension was stained with fluorescent antibodies according to standard flow cytometry procedures. After staining, the cells were washed with PBS and resuspended, and the signals of FITC, PB450, and PC5.5 fluorescence channels were detected using flow cytometry. During the detection process, cell debris and obvious abnormalities were first excluded based on forward and side-scattered light, and diploid cells were further excluded to obtain an effective cell population; then, eosinophil and neutrophil populations were obtained separately according to a preset gating strategy.

[0062] The ratio of eosinophils to neutrophils is calculated using the following formula: Eosinophil or neutrophil percentage (%) = Number of cell events in the target gating region / Number of cell events in the parent gating region × 100%.

[0063] The obtained flow cytometry results, such as Figures 7-9 As shown in the figure, compared with the control group, the proportions of eosinophils and neutrophils in the model group mice were increased, suggesting that Artemisia argyi pollen stimulation can induce abnormal inflammatory cell proportions in AR mice. Compared with the model group, after oral administration of Parabacterium difficile HGI-04, the proportions of eosinophils and neutrophils in mice showed a decreasing trend, indicating that Parabacterium difficile HGI-04 can improve the abnormal inflammatory cell proportions induced by Artemisia argyi pollen in AR mice and has a regulatory effect on allergic inflammatory responses.

[0064] S3. Eosinophil count in mouse nasal mucosa: Mice that had completed the S1 model were sacrificed, and nasal mucosa tissue was collected and fixed in 4% paraformaldehyde at 4°C for 24 hours. After fixation, the tissue samples were dehydrated, cleared, and embedded in paraffin, and then paraffin sections were prepared. After dewaxing to water, the paraffin sections were stained with hematoxylin and chromogen solution. After staining, they were dehydrated, cleared, and mounted. The infiltration of eosinophils in the nasal mucosa tissue was observed under an optical microscope, and the eosinophils were counted.

[0065] The results are as follows Figure 10As shown, compared with the control group, the model group mice showed a significant increase in eosinophil infiltration in the nasal mucosa, suggesting that Artemisia argyi pollen stimulation can induce a local allergic inflammatory response in the nasal mucosa. Compared with the model group, after intervention with Parabacterium difficile HGI-04, the number of eosinophils in the nasal mucosa of mice decreased, indicating that HGI-04 can alleviate Artemisia argyi pollen-induced eosinophil infiltration in the nasal mucosa, thereby alleviating the local inflammatory response in AR mice.

[0066] S4. Histopathological examination of mouse nasal mucosa: After the mice in S1 underwent modeling, they were sacrificed, and nasal mucosa tissue was collected and fixed in 4% paraformaldehyde at 4°C for 24 h. After fixation, the tissue samples were dehydrated using a gradient of ethanol solutions, cleared, embedded in paraffin, and then prepared into 5 μm thick paraffin sections. Histopathological examination was performed using hematoxylin and eosin (H&E) staining.

[0067] The results are as follows Figures 11-13 As shown, the nasal mucosa of the model group mice showed structural abnormalities and increased inflammatory cell infiltration; after intervention with Parabacterium dysenteriae HGI-04, the above pathological changes were improved, suggesting that HGI-04 can alleviate the inflammatory response of Artemisia argyi pollen-induced nasal mucosa.

[0068] Example 6 investigates the effects of *Pseudomonas diffusa* HGI-04 on the gut microbiota of AR mice. Establishment of the S1.AR mouse model: The specific method is the same as step S1 in Example 5.

[0069] S2. Metagenomic sequencing analysis of mouse gut microbiota: After the mice were euthanized at the end of the S1 model, their midsection feces were collected using sterile sampling tubes, flash-frozen in liquid nitrogen, and stored at -80°C.

[0070] DNA was extracted from fecal samples according to the instructions of the QIAamp Rapid DNA Fecal Extraction Kit. The extracted DNA was quality assessed by 1% agarose gel electrophoresis, requiring a DNA concentration >20 ng / μL and absorbance between 1.8 and 2.0 at 260 nm and 280 nm. DNA that passed the quality assessment was then processed according to NEBNext. ® Library construction was performed according to the Ultra DNA Library Construction Kit instructions. The 16-base random sequence was indexed using PCR. The prepared DNA library was sequenced by Novogene Corporation on the Illumina NovaSeq platform.

[0071] The KneadData pipeline was used to remove low-quality data and host-source sequences. Samples were assembled into contigs using the MEGAHIT tool. Contigs of appropriate length were binned using tools such as MetaBAT2, VAMB, and DAS TOOL, and then integrated using a custom script to obtain MAGs. The Check tool was used to classify MAGs according to their integrity and contamination rate; the dRep tool was used to obtain species-level genomes. The Kraken2 tool and the NCBI non-redundant protein sequence database were used to annotate the obtained SGBs. The Blastp module of the DIAMOND tool was used to align the SGBs with the UniProt database to search for and predict genes. The CoverM tool was used to obtain the final species abundance table.

[0072] The results are as follows Figures 14-17 As shown, compared with the control group, the α-diversity of the gut microbiota in the model group mice was reduced, and β-diversity analysis showed a significant change in community structure, suggesting that there is a gut microbiota imbalance in mice with Artemisia argyi pollen-induced allergic rhinitis. After intervention with Parabacterium difficile HGI-04, the α-diversity and community structure of the gut microbiota in mice were improved, indicating that HGI-04 has a role in improving the gut microbiota imbalance in mice with Artemisia argyi pollen-induced allergic rhinitis.

[0073] Example 7 investigated the effects of oral administration of *Pseudomonas difficile* HGI-04 on immune metabolites in AR mice. Establishment of the S1.AR mouse model: The specific method is the same as step S1 in Example 5.

[0074] S2. Sample Collection and Immune Metabolite Detection: Mice were sacrificed after S1 modeling and intervention, and serum samples were collected. Targeted metabolite detection methods were used to quantitatively analyze indole-3-acetic acid, tryptophan, propionic acid, and butyric acid in the samples. Results were expressed as peak area or relative abundance values. Control, model, and HGI-04 intervention groups were included in the detection process, and changes in the levels of these immune metabolites were compared based on the results of samples from each group.

[0075] Indole-3-acetic acid and tryptophan were used to evaluate changes related to tryptophan metabolism, while propionic acid and butyric acid were used to evaluate changes related to short-chain fatty acid metabolism. All four metabolites are closely related to gut microbiota metabolic activity and host mucosal immune regulation, and therefore can serve as important indicators for evaluating the HGI-04-mediated "gut microbiota-metabolism-immunity" regulatory role.

[0076] The results are as follows Figure 18As shown, the indole-3-acetic acid (IAA) levels in the model group were significantly higher than those in the control group (P<0.01), indicating that the AR model is accompanied by changes in indole metabolism. The IAA levels in the HGI-04 intervention group were higher than those in the model group, but the difference was not statistically significant (P>0.05). These results indicate that after HGI-04 intervention, IAA levels remained at a high level and showed a further increasing trend.

[0077] The tryptophan test results showed that the tryptophan level in the model group was significantly higher than that in the control group (P<0.05), indicating that there was abnormal tryptophan metabolism in the Artemisia argyi pollen-induced AR model. After HGI-04 intervention, the tryptophan level decreased significantly (P<0.001), and there was no significant difference between the HGI-04 group and the control group (P>0.05), suggesting that HGI-04 can reduce the abnormally elevated tryptophan level in the model state and bring it back to the control level.

[0078] Short-chain fatty acid (SCFA) assays showed no significant difference in propionic and butyric acid levels between the model group and the control group, indicating that the AR model itself did not cause a significant decrease in propionic and butyric acid levels under the experimental conditions. However, HGI-04 intervention significantly increased propionic and butyric acid levels, with the HGI-04 group showing significantly higher levels than the model group. This result suggests that HGI-04 does not simply "restore" the reduced SCFA levels in the model group, but rather significantly enhances propionic and butyric acid-related metabolic levels.

[0079] In summary, this invention has isolated and obtained a strain of *Pseudomonas difficile* HGI-04 (… Parabacteroides distasonis HGI-04 was tested and confirmed to have a good safety profile. Animal experiments showed that oral administration of HGI-04 reduced the frequency of sneezing and nose scratching in mice with Artemisia argyi pollen-induced allergic rhinitis, alleviated nasal mucosal inflammation, improved gut microbiota imbalance, and regulated the levels of immune metabolites such as indole-3-acetic acid, tryptophan, propionic acid, and butyric acid. Specifically, HGI-04 significantly reduced elevated tryptophan levels in model mice, significantly upregulated propionic and butyric acid levels, and maintained indole-3-acetic acid at a high level. This strain can serve as a functional strain resource for alleviating allergic rhinitis and can be used for the development of related drugs or animal feed.

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A strain of *Pseudomonas digiri*, characterized in that, The *Pseudomonas dignitaria* strain described is *Pseudomonas dignitaria* HGI-04, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38360, on April 22, 2026, and classified as follows: Parabacteroides distasonis .

2. The use of the strain of *Pseudomonas difficile* according to claim 1 in the preparation of a medicament for the prevention and / or relief of allergic rhinitis, wherein the medicament is a probiotic powder or a probiotic oral liquid.

3. The application according to claim 2, characterized in that: When applying, the viable count of *Pseudomonas dilataniae* should be no less than 1 × 10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

4. The application according to claim 2, characterized in that: Parabacterium difficile relieves allergic rhinitis by regulating immune metabolites associated with allergic rhinitis; the immune metabolites are one or more of indole-3-acetic acid, tryptophan, propionic acid and butyric acid.

5. A medicine for preventing and / or relieving allergic rhinitis, characterized in that: The active ingredient of the drug is *Pseudomonas difficile* as described in claim 1.

6. The drug according to claim 5, characterized in that: The viable count of *Pseudomonas difficile* is not less than 1 × 10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

7. A microbial preparation for preventing and / or allergic rhinitis, characterized in that: The microbial preparation contains *Pseudomonas difficile* as described in claim 1 and excipients; the viable count of *Pseudomonas difficile* in the microbial preparation is 1.0 × 10⁻⁶. 5 -1.0×10 12 CFU / mL or 1.0×10 5 -1.0×10 12 CFU / g.

8. The microbial preparation according to claim 7, characterized in that: The excipients include one or more of inulin, fructooligosaccharides, skim milk powder, lactoferrin, casein phosphopeptide, or hydrolyzed egg yolk powder.

9. The use of the microbial preparation according to claim 7 in the preparation of products for the prevention and / or relief of allergic rhinitis, characterized in that: The products include pharmaceuticals or animal feed.

10. The application according to claim 9, characterized in that: Parabacterium difficile relieves allergic rhinitis by regulating immune metabolites associated with allergic rhinitis; the immune metabolites are one or more of indole-3-acetic acid, tryptophan, propionic acid and butyric acid.

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