Application of lactobacillus plantarum pd01 and bifidobacterium breve bb08 in relieving microplastic inhalation-induced lung injury

Abstract

The present application relates to the application field of probiotics, and specifically discloses a combination of postbiotic bacterial agents of plantaricin PD01 and Bifidobacterium breve Bb08 and application thereof in relieving microplastic inhalation-induced lung injury. It is found in experiments that, at the cell level, the postbiotic of plantaricin PD01 can reduce downstream inflammatory response by inhibiting the response of lung epithelial A549 cells to TLR4 receptors of microplastics, and the postbiotic of Bifidobacterium breve Bb08 can prevent A549 cells from being damaged by microplastics and reduce the response of cGAS pathway and secretion of interferon. In mice exposed to microplastics, the combination of the postbiotic of plantaricin PD01 and the postbiotic of Bifidobacterium breve Bb08 has a better effect of relieving respiratory tract inflammation, and effectively relieves respiratory tract allergy and lung injury caused by microplastic exposure.

Description

Technical Field

[0001] This invention pertains to probiotic postbiotic preparations, specifically relating to a combination of Lactobacillus plantarum PD01 and Bifidobacterium breve Bb08 as a postbiotic and its application in alleviating respiratory allergies and lung damage induced by microplastic inhalation. Background Technology

[0002] Respiratory allergies are caused by an abnormal immune response to inhaled allergens and are a common immune system disorder. Toll-like receptors (TLRs), an important component of the innate immune system, can sense lipopolysaccharides and lipoproteins on the surface of exogenous bacteria and initiate downstream immune factor responses to defend against pathogen invasion. In allergic reactions, the activation of TLRs by allergens stimulates the release of downstream pro-inflammatory factors and reactive oxygen species, and recruits immune cells (macrophages, eosinophils, and neutrophils), leading to allergic inflammation and oxidative stress. The cGAS-STING signaling pathway also plays a crucial regulatory role in respiratory allergic reactions. Allergen-induced damage and apoptosis of respiratory epithelial cells result in the release of double-stranded DNA from the cytoplasm. When cGAS recognizes cytoplasmic dsDNA, it induces downstream interferon secretion through a cascade of signal transduction.

[0003] Existing research indicates that probiotics can inhibit the production of pro-inflammatory factors, regulate the balance of the immune system, reduce abnormal immune responses, and lower the risk of allergies.

[0004] Microplastics in the air enter the human body through respiration and accumulate in lung tissue. Multiple studies have reported that inhaling microplastics triggers inflammation, damages alveolar cells, induces oxidative stress, exacerbates respiratory allergies, rhinitis, and asthma symptoms, and increases the risk of pulmonary fibrosis and other respiratory diseases. Although current research has elucidated the harmful effects of microplastics on the respiratory system, effective means to alleviate allergies and lung damage caused by microplastic inhalation remain lacking. Summary of the Invention

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a metaphyte agent, said metaphyte agent being composed of *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum ) PD01 inactivated fermentation product and Bifidobacterium breve ( Bifidobacterium breve The mixture was prepared by mixing inactivated fermentation products of Bb08; wherein, *Lactobacillus plantarum* PD01 was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30386 and accession date of April 22, 2024; and the *Bifidobacterium breve* Bb08 was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33356 and accession date of January 9, 2025.

[0006] Preferably, the mixing ratio of the inactivated fermentation product of *Lactobacillus plantarum* PDO1 to the inactivated fermentation product of *Bifidobacterium breve* Bb08 is 1:1. In a specific embodiment of the present invention, the postbiotic agent prepared by mixing half a amount of inactivated fermentation product of *Lactobacillus plantarum* PDO1 and half a amount of inactivated fermentation product of *Bifidobacterium breve* Bb08 has a more positive and prominent effect in alleviating respiratory allergies and lung damage induced by microplastic inhalation compared to the full amount of postbiotic agent of *Lactobacillus plantarum* PDO1 and the full amount of postbiotic agent of *Bifidobacterium breve* Bb08.

[0007] The inactivation method used in the preparation of the probiotic agent from *Lactobacillus plantarum* PD01 and *Bifidobacterium breve* Bb08 is selected from heat inactivation, ultrasonic inactivation, or enzymatic inactivation. These three inactivation methods have been experimentally verified to have basically the same effect.

[0008] In a specific embodiment of the present invention, heat inactivation is used as a typical, non-limiting, representative example to illustrate the preparation method of the metabiotic agent, which specifically includes the following steps: *Lactobacillus plantarum* PD01 and *Bifidobacterium breve* Bb08 are inoculated separately into MRS liquid medium and cultured at 37°C for 24 hours to obtain a fermentation broth; the fermentation broth is counted using the dilution plating method; the fermentation broth is inactivated at 85°C for 20 minutes; the inactivated fermentation broth is diluted and plated onto MRS agar plates for culture to verify the absence of viable bacteria; the inactivated fermentation broth is diluted with sterile water to 1.0 × 10⁻⁶ based on the count results before inactivation. 9 One cell / mL was used to obtain *Lactobacillus plantarum* PD01 postbiotic agent and *Bifidobacterium breve* Bb08 postbiotic agent, respectively; the two postbiotic agents were mixed to obtain the postbiotic agent.

[0009] Secondly, the present invention provides the application of the metabiotic agent in any of the following aspects: To prepare products for relieving respiratory inflammation induced by microplastic inhalation; To develop products for relieving respiratory allergies induced by microplastic inhalation; To develop products for alleviating lung injury induced by microplastic inhalation.

[0010] Furthermore, the metabiotic agent works by simultaneously inhibiting the TLR4 signaling pathway and the cGAS signaling pathway.

[0011] Specifically, the PD01 postbiotic inhibits the response of the TLR4 receptor in lung epithelial cells to microplastics, thereby reducing the expression levels of downstream pro-inflammatory cytokines TNF-α, IL-1β, and IL-8. The Bb08 postbiotic protects mitochondrial integrity, reduces cytoplasmic double-stranded DNA release, and inhibits the cGAS-STING pathway response and downstream interferon IFN-β secretion. The combined use of these two postbiotics can simultaneously inhibit both the TLR4 and cGAS pathways, achieving a synergistic relief of respiratory inflammation and allergic reactions induced by microplastic exposure.

[0012] Furthermore, the relief of respiratory inflammation is manifested by reducing the number of macrophages, eosinophils, neutrophils and lymphocytes in bronchoalveolar lavage fluid, and / or reducing the levels of TNF-α, IL-1β, IL-8 and IFN-β.

[0013] Furthermore, the relief of respiratory allergies is manifested by reducing serum levels of IgE and / or histamine.

[0014] Furthermore, the relief of lung injury is manifested by inhibiting the increase of MDA in lung tissue and / or promoting the recovery of SOD levels.

[0015] Furthermore, the metabiotic agent also has an anti-inflammatory effect on microplastic-exposed A549 lung epithelial cells in vitro.

[0016] The products described in this invention include, but are not limited to, food, pharmaceuticals, feed additives, or inhaled preparations.

[0017] The microplastics described in this invention are mixtures of two or more selected from PET, PE, PMMA, PP, PS, PU, ​​and PVC.

[0018] The beneficial effects of this invention are as follows: The combined postbiotic of *Lactobacillus plantarum* PD01 and *Bifidobacterium breve* Bb08 provided by this invention exhibits a significantly better synergistic effect than a single postbiotic in alleviating respiratory allergies and lung damage induced by microplastic inhalation.

[0019] In a microplastic-exposed lung epithelial A549 cell model, PD01 postbiotic significantly inhibited the expression of TLR4 receptor and downstream pro-inflammatory factors TNF-α, IL-1β and IL-8, while Bb08 postbiotic significantly inhibited the expression of cGAS receptor and downstream interferon IFN-β. The mixed postbiotic showed significant inhibitory effects on both the TLR4 and cGAS pathways, achieving simultaneous blockade of the two inflammatory pathways activated by microplastics.

[0020] In a mouse model of microplastic nasal exposure, the combined postbiotic intervention reduced the number of macrophages in bronchoalveolar lavage fluid from 37.2 × 10⁻⁶ in the model group. 4The number of cells / mL decreased to 12.0 × 10⁻⁶. 4 The number of cells / mL decreased by 67.7%; the eosinophil count decreased from 3.4 × 10⁻⁶. 4 The number of cells / mL decreased to 1.0 × 10⁻⁶ 4 The number of cells / mL decreased by 70.6%; the neutrophil count decreased from 2.3 × 10⁻⁶. 4 The number of cells / mL decreased to 0.54 × 10⁻⁶. 4 The number of cells / mL decreased by 76.5%; the lymphocyte count decreased from 5.2 × 10⁻⁶. 4 The number of cells / mL decreased to 1.6 × 10⁻⁶ 4 The number of cells / mL decreased by 69.2%. All four indicators were significantly lower than those in the PD01 monotherapy group and the Bb08 monotherapy group. Among them, the number of macrophages and neutrophils decreased by 48.3% and 55.0% respectively compared with the PD01 monotherapy group, and the number of lymphocytes decreased by 36.0% compared with the Bb08 monotherapy group.

[0021] In terms of allergy relief, the serum IgE level in the mixed postbiotic group was significantly lower than that in the PD01 and Bb08 single groups, and the histamine level was also the lowest among all groups, indicating that the mixed postbiotic has a synergistic effect in relieving allergic reactions caused by microplastic exposure that cannot be achieved by a single postbiotic.

[0022] In terms of lung injury protection, the mixed postbiotic intervention significantly reduced the MDA level in lung tissue compared to the model group, and the SOD level rebounded better than either single postbiotic group, indicating that the mixed postbiotic can effectively reduce oxidative stress damage to lung tissue caused by microplastic exposure.

[0023] The above results indicate that the combined postbiotic of the present invention, through the synergistic inhibition of the TLR4 and cGAS dual pathways, has a synergistic effect in alleviating respiratory inflammation, allergic reactions and lung injury induced by microplastic inhalation, providing a safe, stable and efficient intervention for respiratory damage caused by microplastic inhalation. Attached Figure Description

[0024] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the invention.

[0025] Figure 1 This is a colony morphology diagram of Bifidobacterium breve Bb08.

[0026] Figure 2 Gram staining image of Bifidobacterium breve Bb08;

[0027] Figure 3 Particle size distribution of mixed microplastics

[0028] Figure 4Detection of immune factor expression levels in lung epithelial A549 cells under different treatments;

[0029] Figure 5 Detection of mtDNA release in lung epithelial A549 cells under different treatments;

[0030] Figure 6 The level of double-stranded DNA in bronchoalveolar lavage fluid in different treatment groups. Detailed Implementation

[0031] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0033] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0034] Example 1: Isolation and identification of *Lactobacillus plantarum* PD01 and *Bifidobacterium breve* Bb08

[0035] For detailed information on Lactobacillus plantarum PD01, please refer to ZL 202411547090.6.

[0036] This invention uses MRS medium to culture *Lactobacillus plantarum* PD01 and *Bifidobacterium breve* Bb08. The MRS medium formulation is as follows: peptone 10.0 g / L, beef meal 8.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1.0 g / L, and pH adjusted to 5.5-6.0. The solid medium is prepared by adding 15.0 g / L agar to the above formulation. The medium is autoclaved at 121°C for 15 minutes before use.

[0037] Bifidobacterium breve Bb08 was isolated from breast milk. Breast milk samples were added to MRS broth medium and anaerobically enriched at 37°C for 24 hours. The culture was then diluted and spread onto MRS agar plates, and incubated anaerobically at 37°C for 24-48 hours. After colony growth, single colonies were picked based on morphological characteristics and purified by streak plating three times on MRS agar plates to obtain pure cultures. The purified strain was inoculated into liquid MRS medium and anaerobically incubated at 37°C for 24 hours for later use.

[0038] The isolated *Bifidobacterium breve* Bb08 was cultured under the following conditions: MRS medium, anaerobic incubation at 37°C for 24 hours. After culturing on MRS agar plates, *Bifidobacterium breve* Bb08 formed milky-white, round colonies with smooth, raised edges and intact margins. (See...) Figure 1 .

[0039] Morphological identification: The isolated and purified strain was inoculated onto MRS agar plates and anaerobically cultured at 37°C for 24 hours. Colony morphology was then observed. Figure 1 As shown, this strain forms milky-white, round colonies on MRS agar plates. The colonies are raised, smooth, and have intact edges. Fresh cultures were Gram-stained, and the bacterial morphology was observed under a light microscope. Figure 2 As shown, this strain is Gram-positive, and the cells are short rod-shaped, arranged singly or in pairs.

[0040] Sequencing and identification: Using bacterial culture as a template, PCR amplification was performed using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') (SEQ ID No. 2) and 1492R (5'-GGTTACCTTGTTACGACTT-3') (SEQ ID No. 3) for the 16S rRNA gene. The PCR reaction conditions were: 94℃ pre-denaturation for 5 minutes; 94℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 90 seconds, for a total of 30 cycles; final extension at 72℃ for 10 minutes. The PCR amplification products were sent to a sequencing company for sequencing, and the 16S rRNA gene sequence was obtained as shown in SEQ ID No. 1. The sequencing results were submitted to the NCBI database for BLAST comparison. The results showed that this strain is related to *Bifidobacterium breve* (…). Bifidobacterium breve The type strain of the bacterium showed over 99.8% sequence homology, confirming that the strain was *Bifidobacterium breve*. Bifidobacterium breve The strain was named Bifidobacterium breve Bb08.

[0041] The aforementioned *Bifidobacterium breve* Bb08 was deposited on January 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China. The accession number is CGMCC No. 33356, and the classification name is *Bifidobacterium breve* (…). Bifidobacterium breve ).

[0042] Example 2 Preparation of postbiotic inoculant

[0043] Lactobacillus plantarum PD01 and Bifidobacterium breve Bb08 were inoculated into MRS liquid medium and cultured at 37°C for 24 hours to obtain the fermentation broth. The fermentation broth was counted using the dilution plating method.

[0044] The fermentation broth was inactivated at 85°C for 20 minutes. The inactivated fermentation broth was then diluted and plated onto MRS agar plates for incubation to verify the absence of viable bacteria.

[0045] After inactivation, the fermentation broth was diluted with sterile water to a concentration of 1.0 × 10⁻⁶ based on the pre-inactivation count. 9 The *Lactobacillus plantarum* PD01 and *Bifidobacterium breve* Bb08 postbiotic inoculants were obtained by mixing the two inoculants at a 1:1 ratio to obtain 5.0 × 10⁶ cells / mL. 8 A mixed probiotic agent containing 1 cell / mL.

[0046] Commercially available strains of *Bifidobacterium breve* M-16V and *Lactobacillus plantarum* LP115 were purchased as control strains. Using the same MRS liquid medium, postbiotic agents of *Bifidobacterium breve* M-16V and *Lactobacillus plantarum* LP115 were prepared at the same concentrations according to the same method.

[0047] Example 3: Co-incubation treatment of microplastic-exposed lung epithelium A549 with metabiotic agent

[0048] Seven types of microplastic powders detected in the respiratory tract were purchased: PET, PE, PMMA, PP, PS, PU, ​​and PVC. These were mixed in equal proportions to prepare a mixed microplastic sample. The microplastic particle size distribution is shown in [link to microplastic sample]. Figure 3 The median particle size (D50) was 38 µm. Microplastic samples were prepared into a 100 mg / mL suspension using PBS, dispersed by sonication, and then sterilized for later use.

[0049] Human alveolar epithelial cells A549 were purchased from ATCC. A549 cells were cultured in DMEM medium containing 1% streptomycin / penicillin and 10% fetal bovine serum in a 37°C, 5% CO2 incubator. The seeding density was 5.0 × 10⁶ cells / year. 4 A549 cells / mL were grown to subconfluence, and the culture medium was removed. The experimental group was then treated with fresh culture medium containing 100 µg / mL mixed microplastics. Subsequently, the metagenic solution from Example 2 (final concentration 1.0 × 10⁻⁶) was added to the culture medium at a 1:10 ratio. 8 A549 cells / mL were incubated for 24 hours. The control group received the mixed microplastics followed by an equal volume of sterile PBS. The blank group received no mixed microplastics, only an equal volume of fresh culture medium and an equal volume of sterile PBS. After incubation, cells were collected by blowing and centrifugation, the supernatant was discarded, and the cells were collected after co-incubation with the metaphytic agent.

[0050] Example 4: Regulation of immune response by metabiotic agents in microplastic-exposed lung epithelial A549 cells.

[0051] A549 cells from each group treated in Example 3 were used. After RNA extraction and reverse transcription, the expression of immune genes was detected using real-time quantitative PCR. Figure 4 It can be seen that, compared with the blank group, microplastic exposure in the control group induced the upregulation of Toll-like receptor TLR4, pro-inflammatory cytokines TNF-α, IL-1β, and IL-8 expression in A549 lung epithelial cells; it also promoted the upregulation of cGAS receptor and interferon IFN-α / β expression. This indicates that microplastic exposure induced an inflammatory response in A549 cells.

[0052] In the postbiotic treatment group, *Lactobacillus plantarum* PD01 significantly inhibited the expression response of the TLR4 receptor induced by microplastics and reduced the expression levels of downstream pro-inflammatory factors TNF-α, IL-1β, and IL-8. The control strain *Lactobacillus plantarum* LP115 did not significantly inhibit the TLR4 receptor or downstream responses. *Bifidobacterium breve* Bb08 postbiotic showed more significant inhibition of cGAS receptor expression and significantly suppressed the expression level of its downstream interferon IFN-β. The control strain *Bifidobacterium breve* M-16V had no inhibitory effect on the cGAS receptor, and the expression level of downstream interferon IFN-β was not significantly different from that in the microplastic exposure group. Treatment with a mixed postbiotic agent of *Lactobacillus plantarum* PD01 and *Bifidobacterium breve* Bb08 showed significant inhibitory effects on both the TLR4 and cGAS pathways.

[0053] Example 5: Protective effect of metabiotic agents against mitochondrial damage in microplastic-exposed lung epithelial A549 cells.

[0054] To further confirm the inhibitory effect of the metabiotic agent on the cGAS pathway, cells treated in Example 3 were taken and subjected to 1.0 × 10⁻⁶ cells. 6Each cell was permeabilized with 250 µL of digitonin buffer (150 mM NaCl, 50 mM HEPES, 25 µg / mL digitonin, pH 7.4) at 50 rpm for 20 min at room temperature. Cells and supernatant were then separated by centrifugation at 16,000 g for 10 min at 4°C. DNA was extracted from the supernatant using a DNA extraction kit, and mtDNA copy number was detected by real-time quantitative PCR using mitochondrial DNA (mtDNA) specific primers (F: 5'-CATCTGGTTCCTACTTCAGGG-3', R: 5'-CCGTGAGTGGTTAATAGGGTG-3') (SEQ ID No. 4 and SEQ ID No. 5). PCR reaction conditions were: 95°C pre-denaturation for 30 seconds; 95°C denaturation for 10 seconds; 60°C annealing extension for 30 seconds, for a total of 40 cycles. Total DNA was extracted from the centrifuged cells using a DNA extraction kit. The total DNA copy number was detected by real-time quantitative PCR using GAPDH primers (F: 5'-GTCAGCCGCATCTTCTTTTG-3', R: 5'-GCGCCCAATACGACCAAATC-3') (SEQ ID No. 6 and SEQ ID No. 7). The amount of mtDNA released from the cytoplasm was then normalized by dividing the number of mtDNA copies in the cytoplasm by the total DNA copy number.

[0055] from Figure 5 It can be seen that microplastic exposure in the control group caused mitochondrial damage in A549 cells, resulting in a large amount of mitochondrial DNA leakage into the cytoplasm. In the Bb08 treatment group and the mixed post-biotic treatment group, the amount of mtDNA released was significantly reduced. Figure 4 The results show that both Bb08 postbiotic and mixed postbiotic inoculants can effectively alleviate mitochondrial damage caused by microplastics, reduce double-stranded DNA release, and thus reduce the response of the cGAS pathway.

[0056] Example 6: Alleviating respiratory inflammatory response of metabiotics in mice exposed to microplastics.

[0057] Six-week-old SPF-grade male Balb / C mice were purchased and, after one week of acclimatization, randomly divided into five groups: control group, model group, PD01 group, Bb08 group, and mixed probiotic group (n=6 per group). Mice in the control group received 10 µL of sterile saline intranasally every 3 days, while the other groups received 10 µL of the aforementioned mixed microplastic suspension prepared with sterile saline intranasally every 3 days. The microplastic dosage was 0.75 mg / kg per infusion. The infusion period lasted 60 days. Starting from day 31, all groups were administered gavage intervention once daily. The control and model groups received 100 µL of sterile saline via gavage, while the PD01, Bb08, and mixed probiotic groups received 100 µL of their respective probiotic preparations via gavage. The gavage intervention lasted for 30 days. Twelve hours after the last gavage, the mice were euthanized, and bronchoalveolar lavage fluid was collected: 0.8 mL of sterile PBS solution was injected into the right lung through the trachea using a syringe with a tubing, and the bronchoalveolar lavage fluid was aspirated and removed. Subsequently, bronchoalveolar lavage fluid was collected from the left lung in the same manner, and the samples were combined for analysis.

[0058] After counting the total number of cells in the bronchoalveolar lavage fluid (BALF) using a hemocytometer, the BALF was added to a glass slide, stained with Giemsa stain, and observed under an oil immersion microscope. The proportions of four types of immune cells—macrophages, eosinophils, neutrophils, and lymphocytes—were counted and analyzed. Based on the total number and proportion, the number of each type of immune cell was calculated. The results are shown in Table 1. Intranasal exposure to mixed microplastics significantly increased the number of inflammatory cells. Postbiotic intervention significantly reduced the number of inflammatory cells in the BLF. Among them, PD01 postbiotic intervention significantly reduced the number of macrophages, eosinophils, and neutrophils, while Bb08 postbiotic intervention significantly reduced the number of lymphocytes. After mixed postbiotic intervention, the number of all four types of inflammatory cells in the BLF was significantly reduced, and lower than the number of cells after single postbiotic intervention.

[0059] The levels of inflammatory factors in bronchoalveolar lavage fluid were detected using the corresponding ELISA kits. Compared with the model group, PD01 post-biotic intervention significantly reduced the levels of three pro-inflammatory factors: TNF-α, IL-1β, and IL-8, while Bb08 post-biotic intervention significantly reduced the level of interferon IFN-β. In the mixed post-biotic intervention group, the levels of all four inflammatory factors were significantly lower than those in the model group.

[0060] Table 1. Number of inflammatory cells and levels of inflammatory factors in mice of each group

[0061]

[0062] # indicates a comparison with the model group, P<0.05.

[0063] The level of double-stranded DNA in bronchoalveolar lavage fluid was detected using a double-stranded DNA quantification kit. Compared with the model group, Bb08 postbiotic and mixed postbiotic interventions significantly reduced the release of double-stranded DNA in the bronchoalveolar lavage fluid. Figure 6 ).

[0064] In summary, the results indicate that intranasal microplastic exposure in mice induced a respiratory tract inflammatory response, leading to the infiltration of inflammatory cells and the release of pro-inflammatory factors. This also damaged epithelial cells, resulting in the release of double-stranded DNA, activation of the cGAS signaling pathway, and stimulation of interferon secretion, further exacerbating the inflammatory response. The PD01 postbiotic showed better efficacy in reducing inflammatory cell infiltration and the release of pro-inflammatory factors. The Bb08 postbiotic exhibited a protective effect against interferon secretion. Combined intervention with PD01 and Bb08 postbiotics simultaneously reduced responses in different inflammatory pathways, effectively alleviating respiratory tract inflammatory damage caused by microplastic exposure.

[0065] Example 7: Alleviating respiratory allergies in mice exposed to microplastics by metabiotic agents.

[0066] Serum samples were collected from mice in each group in Example 6, and IgE and histamine levels were detected using ELISA. Table 2 shows that although single postbiotic intervention reduced IgE levels, mixed postbiotic intervention was more effective than single postbiotic intervention in reducing IgE. The mixed postbiotic also significantly reduced blood histamine levels. These results indicate that the mixed postbiotic significantly alleviated the allergic reaction caused by microplastic exposure.

[0067] Table 2 Serum IgE and histamine levels in each group of mice

[0068]

[0069] # indicates a comparison with the model group, P<0.05.

[0070] Example 8: Alleviating lung injury in mice exposed to microplastics by metabiotic agents.

[0071] Lung tissue from mice in Example 6 was homogenized with physiological saline at a 1:10 ratio, centrifuged, and the supernatant was collected. MDA and SOD levels were detected using a kit. Protein levels were detected using a BCA kit for normalized concentration calculation. Compared to the control group, the MDA content in the lung tissue of mice exposed to microplastics was significantly increased, while the SOD level was significantly decreased, indicating oxidative stress damage to the lung tissue. Single metabiotic intervention improved the decrease in SOD levels; the combined use of two metabiotic interventions significantly inhibited the increase in MDA, and SOD levels also showed a greater recovery (see Table 3). This indicates that the mixed metabiotic significantly alleviated lung damage caused by microplastic exposure.

[0072] Table 3. MDA and SOD levels in lung tissue of mice in each group

[0073]

[0074] # indicates a comparison with the model group, P<0.05.

[0075] It should be noted that, in this document, the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0076] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments described herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A metaphyte agent, characterized in that, The postbiotic agent is composed of *Lactobacillus plantarum* (… Lactiplantibacillus plantarum ) PD01 inactivated fermentation product and Bifidobacterium breve ( Bifidobacterium breve The mixture was prepared by mixing inactivated fermentation products of Bb08; wherein, *Lactobacillus plantarum* PD01 was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30386 and accession date of April 22, 2024; and the *Bifidobacterium breve* Bb08 was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33356 and accession date of January 9, 2025.

2. The metaphyte agent according to claim 1, characterized in that, The inactivated fermentation product of *Lactobacillus plantarum* PD01 and the inactivated fermentation product of *Bifidobacterium breve* Bb08 are mixed in a 1:1 ratio.

3. The metaphytic agent according to claim 1 or 2, characterized in that, The preparation method of the metabiotic agent includes the following steps: *Lactobacillus plantarum* PD01 and *Bifidobacterium breve* Bb08 are inoculated separately into MRS liquid medium and cultured at 37°C for 24 hours to obtain a fermentation broth; the fermentation broth is counted using the dilution plating method; the fermentation broth is inactivated at 85°C for 20 minutes; the inactivated fermentation broth is diluted and plated onto MRS agar plates for culture to verify the absence of viable bacteria; the inactivated fermentation broth is diluted with sterile water to 1.0 × 10⁻⁶ based on the count results before inactivation. 9 One cell / mL was used to obtain *Lactobacillus plantarum* PD01 postbiotic agent and *Bifidobacterium breve* Bb08 postbiotic agent, respectively; the two postbiotic agents were mixed to obtain the postbiotic agent.

4. The use of the metabiotic agent according to any one of claims 1-3 in the preparation of products for relieving respiratory inflammation induced by microplastic inhalation.

5. The use of the metabiotic agent according to any one of claims 1-3 in the preparation of products for relieving respiratory allergies induced by microplastic inhalation.

6. The use of the metabiotic agent according to any one of claims 1-3 in the preparation of products for alleviating lung injury induced by microplastic inhalation.

7. The application according to any one of claims 4-6, characterized in that, The metabiotic agent works by inhibiting the TLR4 signaling pathway and / or the cGAS signaling pathway.

8. The application according to claim 4, characterized in that, The relief of microplastic inhalation-induced respiratory inflammation is manifested by reducing the number of macrophages, eosinophils, neutrophils and lymphocytes in bronchoalveolar lavage fluid, and / or reducing the levels of TNF-α, IL-1β, IL-8 and IFN-β.

9. The application according to claim 5, characterized in that, The relief of microplastic inhalation-induced respiratory allergies is manifested by reducing serum IgE and / or histamine levels.

10. The application according to claim 6, characterized in that, The relief of microplastic inhalation-induced lung injury is manifested by inhibiting the increase of MDA in lung tissue and / or promoting the recovery of SOD levels.

Images