Use of lactobacillus plantarum in the preparation of a product for alleviating beta-lactoglobulin-induced food allergy

Abstract

The application discloses application of Lactobacillus plantarum KLDS1.0386 in preparation of a product for preventing beta-lactoglobulin-induced food allergy, wherein the Lactobacillus plantarum KLDS 1.0386 is preserved in the China General Microbiological Culture Collection Center on May 29, 2024, and the preservation number is CGMCC No. 30809; after intervention of the KLDS 1.0386, the levels of IL-4 and IL-17A in serum of mice are significantly reduced, and the levels of IFN-gamma and IL-10 are significantly increased; the KLDS 1.0386 inhibits IgE synthesis by down-regulating IL-4, makes T cell differentiation transform from Th2 predominance to Th1 / Th2 balance by up-regulating IFN-gamma, and enhances Treg activation by up-regulating IL-10, so that Th1 / Th2 imbalance is corrected, Th17-related inflammation is inhibited, and Treg-mediated immune regulation is enhanced.

Description

Technical Field

[0001] This invention relates to the field of Lactobacillus plantarum technology, and more particularly to the application of Lactobacillus plantarum in the preparation of products that alleviate β-lactoglobulin-induced food allergies. Background Technology

[0002] Food allergy (FA) refers to an immune response caused by food or food additives, which can lead to allergic reactions in the digestive system or the whole body. With the increasing incidence of food allergic diseases, food allergy has become a major public health concern worldwide. FA is the most common cause of anaphylactic shock in children, a potentially life-threatening severe allergic reaction that can affect multiple organ systems, including the respiratory tract, gastrointestinal tract, and skin. Currently, it is mainly treated with epinephrine injections. Studies by Pouessel et al. have shown that FA causes 37% of anaphylactic shock cases and 79% of recurrent anaphylactic shock cases. FA places a heavy burden on patients and their families, often requiring them to follow special dietary plans, continuously monitor for food allergens, increase medical resource consumption, and constantly worry about the occurrence of severe allergic symptoms. Studies have shown that fatty acids (FAs) are mainly caused by allergens stimulating CD4+. + T cells differentiate into Th2 cells, which produce cytokines such as IL-4, IL-5, and IL-13, thereby inducing B cells to produce antigen-specific IgE antibodies. After these antibodies bind to mast cells, they can stimulate mast cells to degranulate and release histamine, prostaglandins, and leukotrienes, thus triggering FA symptoms. Currently, the main treatments for food allergies (FA) include allergen avoidance, medications such as adrenaline, and oral immunotherapy. Oral immunotherapy primarily involves administering a fixed dose or a maintenance dose of the allergen orally to desensitize the patient to the allergen, thereby increasing the dose threshold required to trigger an allergic reaction. Although other treatment options such as sublingual immunotherapy and cutaneous immunotherapy exist, oral immunotherapy is considered the most effective. However, it is still accompanied by a high incidence of adverse reactions, including severe allergic reactions and eosinophilic esophagitis. Therefore, there is an urgent need for a highly effective and safe drug to alleviate food allergies. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides the following technical solution: Application of Lactobacillus plantarum KLDS1.0386 in the preparation of products for preventing β-lactoglobulin-induced food allergies. The Lactobacillus plantarum KLDS 1.0386 was deposited at the China General Microbiological Culture Collection Center on May 29, 2024, with accession number CGMCC No. 30809.

[0004] As an improvement to the above technical solution, the drug includes a live strain of Lactobacillus plantarum KLDS1.0386.

[0005] As an improvement to the above technical solution, Lactobacillus plantarum KLDS1.0386 is used to protect the intestinal barrier and intestinal mucosa.

[0006] As an improvement to the above technical solution, the application of Lactobacillus plantarum KLDS1.0386 in regulating immune response.

[0007] As an improvement to the above technical solution, the application of Lactobacillus plantarum KLDS1.0386 in regulating intestinal microbiota.

[0008] As an improvement to the above technical solution, the regulation of immune response includes: reducing the levels of β-LG-specific IgE, histamine and mMCP-1, downregulating the levels of IL-4 and IL-17A and upregulating the levels of IFN-γ and IL-10.

[0009] As an improvement to the above technical solution, according to the application described in claim 1, the product is characterized in that it is food, health product or medicine.

[0010] The beneficial effects of this invention are: Following KLDS 1.0386 intervention, serum levels of β-lactoglobulin-specific IgE, histamine, and mast cell protease-1 in mice were significantly reduced, returning to near-normal levels. KLDS 1.0386 inhibited mast cell degranulation and indirectly blocked it by reducing IgE. Crosslinking; After KLDS 1.0386 intervention, the serum levels of IL-4 and IL-17A in mice were significantly reduced, while the levels of IFN-γ and IL-10 were significantly increased. KLDS 1.0386 inhibited IgE synthesis by downregulating IL-4, upregulated IFN-γ to shift T cell differentiation from Th2 dominance to Th1 / Th2 balance, and enhanced IL-10 activation of Tregs, thereby correcting the Th1 / Th2 imbalance, inhibiting Th17-related inflammation, and enhancing Treg-mediated immunomodulation. After KLDS 1.0386 intervention, the pathological damage of the colon tissue in mice was significantly reduced, and the serum diamine oxidase and D-lactic acid levels were significantly decreased, effectively repairing the intestinal mucosal structure, reducing intestinal permeability, and thus alleviating the intestinal damage induced by β-lactoglobulin. After KLDS 1.0386 intervention, the diversity and richness of gut microbiota were significantly restored; at the phylum level, the abundance ratio of Firmicutes to Bacteroidetes returned to normal levels; at the genus level, beneficial bacteria such as Muribaclumintestinale and Lachnospiraceae_NK4A136_group were significantly enriched, while pro-inflammatory bacteria Lactobacillus murinus were selectively inhibited, thereby preventing gut microbiota dysbiosis induced by β-lactoglobulin. Attached Figure Description

[0011] Figure 1 shows the physiological index evaluation of the present invention, where (A) is the weight gain curve; and (B) is the change in colon length. Figure 2 This refers to the spleen index; Figure 3 The changes in mouse mast cells include (A) toluidine blue staining of mast cells in the ear; and (B) mast cell counting. Figure 4 Changes in allergy marker levels, including (A) changes in β-LG-specific IgE levels; (B) changes in histamine levels; and (C) changes in mMCP-1 levels. Figure 5 Serum cytokine levels, including (A) mouse IFN-γ level; (B) mouse IL-4 level; (C) mouse IL-17A level; (D) mouse IL-10 level; Figure 6 HE staining of mouse colon tissue; Figure 7 Changes in the levels of intestinal permeability markers in mouse serum; (A) DAO level; (B) D-LA level; Figure 8 Differences in the diversity of mouse gut microbiota were analyzed, including (A) the Chao1 index reflecting species richness; (B) the Shannon index reflecting both species richness and evenness; (C) the Simpson index reflecting community dominance; (D) the Venn diagram showing the composition of OTUs in each group; and (E) PCA analysis showing the overall differences in the microbiota structure of each group. Figure 9 Analysis of gut microbiota composition and differences, including (A) changes at the phylum level; (B) changes at the genus level; (C) phylogenetic clade plot of LEfSe analysis; and (D) LDA score plot. Detailed Implementation

[0012] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0013] In this invention, Lactobacillus plantarum KLDS 1.0386 is deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 29, 2024, with accession number CGMCC No. 30809.

[0014] 1. Materials and Methods 1.1 Experimental Materials 1.1.1 Test strains Lactobacillus plantarum KLDS 1.0386 was isolated from traditional fermented dairy products in Inner Mongolia; 1.1.2 Laboratory Animals The SPF-grade BALB / c mice used in the experiments were obtained from Liaoning Changsheng Biotechnology Co., Ltd., and all animal experiments were conducted in strict accordance with the Guidelines for the Care and Use of Laboratory Animals issued by the National Health Commission of China (NHC Publication No. 11, Rev. 2016). 1.1.3 Experimental Apparatus The Multiskan FC (IVD model) microplate reader was purchased from Thermo Fisher Scientific (China); the HPS-300 biochemical incubator was purchased from Harbin Donglian Electronic Technology Development Co., Ltd.; the JJ-12J dehydrator, JB-L5 freezing stage, and JB-P5 embedding machine were all purchased from Wuhan Junjie Electronic Co., Ltd.; the RM2016 pathological sectioner was purchased from Shanghai Leica Instruments Co., Ltd.; the KD-P tissue spreader was purchased from Zhejiang Jinhua Kedi Instrument Equipment Co., Ltd.; the GFL-230 oven was purchased from Tianjin Laiborui Instrument Equipment Co., Ltd.; and the Nikon Eclipse E100 upright optical microscope was purchased from Nikon Corporation, Japan.

[0015] 1.2 Experimental Methods 1.2.1 Strain screening and identification Aseptically, 1 mL of sample (fermented milk) was added to 9 mL of sterile physiological saline to form a 1:10 homogeneous dilution. This dilution process was repeated to obtain 10... -2 10 -3 10 -4 10 -5 and 10 -6 Diluent, use a sterile pipette tip to draw 100 μL of the above 10... -4 10 -5 and 10 -6Dilute the solution and spread it onto MRS agar plates using a spreader. Incubate at 37°C for 24–48 h in anaerobically or microaerophilically. Single colonies were picked based on colony morphology and repeatedly streaked on MRS plates for purification until a pure culture with consistent morphology was obtained. Subsequently, the isolates were identified by combining bacterial morphological observation, physiological and biochemical characteristics and 16S rRNA gene sequence analysis, and finally Lactobacillus plantarum KLDS1.0386 was obtained. 1.2.2 Animal Experiments 1.2.2.1 Animal Experiment Design Thirty 5-week-old SPF-grade female BALB / c mice were randomly divided into three groups of 10 mice each after one week of acclimatization: control group (NC), model group (MC), and KLDS 1.0386 intervention group (1.0386). From the start of the experiment (day 0), the control group (NC) was administered 200 μL of sterile saline by gavage daily, and an equal volume of sterile saline was injected intraperitoneally on days 0, 7, 14, and 21. The model group (MC) was administered 200 μL of sterile saline by gavage daily, and 200 μL of the sensitizer solution was injected intraperitoneally at the same time points. The Lactobacillus plantarum KLDS 1.0386 intervention group (1.0386) was administered 5 × 10⁵ gavage daily. 8 200 μL of bacterial suspension with CFU / mL was administered intraperitoneally as in the MC group. The sensitizer solution was prepared by mixing 1 mg β-LG with 100 μL of aluminum hydroxide adjuvant in 100 μL of physiological saline. On day 28, the MC group and the KLDS 1.0386 intervention group were administered 200 μL of β-LG challenge solution (prepared by mixing 20 mg β-LG and 200 μL of physiological saline) by gavage, while the NC group was administered an equal volume of physiological saline by gavage. Allergic symptoms were scored 30 min after challenge, and then blood was collected from the eyeballs. After blood collection, the mice were euthanized by cervical dislocation, and the target tissues such as the intestine and spleen were separated, flash-frozen in liquid nitrogen, and stored at -80℃.

[0016] 1.2.2.2 Allergy Symptom Score Thirty minutes after oral challenge with the allergen on day 28, the allergic symptoms of the mice were observed and scored to evaluate the effect of KLDS 1.0386 intervention on allergy symptoms. The scoring criteria are shown in Table 1. Table 1 1.2.2.3 Changes in body weight and colon length Starting from the day the formal experiment began (day 0), the mice's weight was monitored every 7 days to dynamically monitor their weight changes in order to assess the impact of the allergic reaction on the mice's overall physiological state. After euthanizing the mice by dislocation of the neck, the colon tissue was quickly removed, carefully stretched to maintain its natural extension, and the total length of the colon from the anus to the cecum junction was measured.

[0017] 1.2.2.4 Spleen Index Detection After euthanizing the mice by cervical dislocation, the abdomen was cut open from the side using clean dissecting scissors, and the spleen tissue was quickly removed. The adipose tissue around the spleen was carefully removed, and the spleen surface was gently rinsed with PBS solution to remove any residual blood. After processing, the sample was thoroughly dried with filter paper, weighed immediately, and the data was recorded. The calculation formula is as follows: 1.2.2.5 Toluidine blue staining for mast cell counting The left ear of a mouse was completely cut off with scissors and fixed by soaking in 4% paraformaldehyde. Then, it was embedded in paraffin and sectioned on a pathological microtome. After dewaxing, the paraffin sections were stained with toluidine blue for 5 minutes and then washed with water. Then, differentiation was performed with 1% glacial acetic acid. Finally, the reaction was terminated by washing with distilled water and the degree of differentiation was controlled under a microscope. After washing with tap water, the sections were dried in an oven and mast cells were counted under a microscope.

[0018] 1.2.2.6 Detection of allergy marker levels Mouse ocular blood was collected in 1.5 mL centrifuge tubes and allowed to stand for 1 hour. The tubes were then centrifuged at 3000×g at 4℃ for 15 minutes. The supernatant was carefully collected and stored at -80℃. Mouse ocular blood was also collected in 1.5 mL anticoagulant centrifuge tubes pre-filled with EDTA. After collection, the tubes were inverted 5-6 times to thoroughly mix the anticoagulant with the blood. The tubes were then centrifuged at 3000×g at 4℃ for 30 minutes. The supernatant was collected and stored at -80℃. The levels of β-LG-specific IgE, mMCP-1, and histamine in mouse serum were detected using enzyme-linked immunosorbent assay (ELISA).

[0019] 1.2.2.7 Detection of cytokine levels in serum The levels of inflammatory cytokines (IL-4, IFN-γ, IL-17, IL-10) in serum were detected using a mouse ELISA kit, and the specific operating procedures were performed according to the kit instructions.

[0020] 1.2.2.8 Histopathological evaluation of colon tissue After euthanizing mice by cervical dislocation, colonic tissue was collected. 2-3 cm sections of structurally intact colonic tissue, free of intestinal contents, were carefully cut and fixed in a 4% paraformaldehyde solution for subsequent HE staining. The prepared colonic tissue samples were placed in a dehydration chamber and sequentially immersed in alcohol, anhydrous ethanol, xylene, and paraffin wax for dehydration. The paraffin-soaked samples were then embedded into paraffin blocks using an embedding machine. These blocks were then cryo-sectioned to a thickness of 4 μm. The sections were then mounted on glass slides and dried in an oven at 60°C. The dried tissue sections were then sequentially immersed in xylene, anhydrous ethanol, and alcohol for dewaxing and hydration. The sections, washed with distilled water, were then immersed in hematoxylin for staining. After washing with distilled water, differentiation was performed using differentiation solution, followed by blueing with a blueing solution. Finally, the sections were washed with distilled water and immersed in eosin staining solution. The slides were dehydrated by soaking in anhydrous ethanol and xylene, then mounted with neutral resin. The mounted slides were then placed under a microscope for histopathological evaluation and image acquisition.

[0021] 1.2.2.9 Detection of serum intestinal barrier marker levels The levels of the intestinal barrier markers diamine oxidase (DAO) and D-lactic acid (D-LA) in serum were detected using a mouse ELISA kit, and the specific operating procedures were performed according to the kit instructions.

[0022] 1.2.2.10 Detection of intestinal microbiota After euthanizing mice, the colon was sterilized with 75% alcohol and aseptically isolated in a laminar flow hood. Its contents were placed in sterile cryovials, flash-frozen in liquid nitrogen, and stored at -80°C. Intestinal microbial DNA was extracted using the QIAamp DNA Mini Kit. PCR amplification was performed using primers specific to the V3-V4 region of the 16S rRNA gene. Amplification was then performed using NEB Q5 DNA high-fidelity polymerase, followed by quantitative mixing using the Quant-iT PicoGreen dsDNA Assay Kit. Library construction was then performed using the TruSeq Nano DNA LTLibrary Prep Kit. The library was quality-checked and quantified using the Agilent High Sensitivity DNA Kit and the Quant-iTPicoGreen dsDNA Assay Kit, respectively. The library was sequenced at 2×300 bp using the MiSeq platform. Sequencing data underwent splicing, quality control, OTU clustering (97% similarity), and species annotation. Alpha / Beta diversity analysis, phylogenetic tree construction, and LEfSe differential analysis were then performed.

[0023] 1.2.3 Data Processing Experimental data are expressed as mean ± standard deviation (mean ± SD). Graphs were generated using Origin 2021 software. SPSS 22.0 and Statistix 8.1 software were used for statistical analysis of the experimental data. One-way ANOVA and Duncan's multiple comparison test were employed. P < 0.05 was considered statistically significant, and P > 0.05 was considered not statistically significant. OTU clustering and diversity analysis were performed using QIIME v1.9.1 and uclust v1.2.22q software. Species were annotated in the Greengene database release 13.8. Data were joined using fastq-join software. Different superscript letters indicate whether there are significant differences between groups.

[0024] 2. Prevention of food allergy induced by target strains in mice with β-lactoglobulin 2.1 Allergy Symptom Score The allergy symptom scoring after oral challenge in allergic mice is a core indicator for assessing the severity of acute allergic reactions. The results directly reflect the intensity of the allergic reaction. The system quantifies the immediate physiological responses caused by the release of mediators such as histamine and leukotrienes, and shows a positive correlation with serum IgE levels and mast cell activation. Detailed scoring results are shown in Table 2. In the NC group, no allergic symptoms were observed in any mice. However, in the MC group, all mice exhibited allergic symptoms, mainly concentrated in the moderate to severe allergy range. Four mice scored 3 points, and three scored 4 points. Although no mice died, only one and two mice scored 1 and 2 points respectively, indicating that β-LG successfully induced a food allergy model. While no mice scored 0 points in the Lactobacillus plantarum KLDS 1.0386 intervention group, the symptom distribution shifted significantly towards milder symptoms compared to the MC group. The proportion of mice with mild symptoms (1 point) was 40%, significantly higher than in the MC group (10%); the proportion of mice with moderate to severe symptoms (3-4 points) was 30%, significantly lower than in the MC group (70%). These results indicate that Lactobacillus plantarum KLDS... 1.0386 significantly reduced the incidence of moderate to severe allergic symptoms while increasing the proportion of mild symptoms, indicating that it has a significant alleviating effect on β-LG-induced food allergies.

[0025] Table 2. Allergy Symptom Scores in Mice 2.2 Changes in mouse body weight and colon length Starting from day 0, the mice's weight was monitored every 7 days. Weight changes directly reflected their overall nutritional status and inflammation levels. The weight gain curve is shown below. Figure 1As shown in A, the body weight of the NC group mice showed a continuous and stable growth trend, with an average daily weight gain of 0.14 g, which is consistent with the normal growth pattern of SPF-grade BALB / c mice. In the MC group, the body weight was significantly lower than that of the NC group from day 0 to 7, indicating that β-LG may have induced intestinal barrier damage, leading to malabsorption. From day 7 to 28, the body weight slowly recovered, but on day 28, the body weight was still significantly lower than that of the NC group, indicating that the allergic state continued to inhibit the normal growth of the mice. Compared with the MC group, after intervention with Lactobacillus plantarum KLDS 1.0386, although the body weight was significantly lower from day 0 to 7, the body weight rapidly increased from the second week. By the end of the experiment, the body weight of the mice had approached that of the NC group, and they had basically returned to normal. This shows that intervention with Lactobacillus plantarum KLDS 1.0386 improved the malabsorption caused by food allergy. The colon is one of the main sites of damage after food allergens enter the body through the digestive tract. Food allergies induce intestinal inflammation, leading to characteristic pathological changes in the colonic mucosa and consequently altering colon length. Figure 1 B shows the changes in colon length in mice. The NC group had the longest colon length, with an average of 7.98 cm, while the MC group had a significantly shorter colon length (P<0.05), with an average of only 6.04 cm, indicating severe colon atrophy. After intervention with Lactobacillus plantarum KLDS 1.0386, the average colon length of mice was 7.76 cm, which was significantly higher than that of the MC group (P<0.05), and there was no significant difference between the MC and NC groups (P>0.05). 2.3 Spleen Index Detection In the FA model, the allergen-induced Th2 immune response leads to a massive proliferation of lymphocytes in the spleen, resulting in splenomegaly. Therefore, the spleen index can serve as an important indicator for assessing the effectiveness of FA intervention. The results are as follows: Figure 2 As shown, the spleen index of mice in the NC group was the lowest, with an average of only 5.36, indicating that there was no significant activation of the spleen. Compared with the NC group, the spleen index of mice in the MC group was significantly increased (P<0.05), with an average of 11.87, indicating that β-LG-induced FA caused significant spleen enlargement. After intervention with Lactobacillus plantarum KLDS 1.0386, the spleen index was only 7.06, which was significantly lower than that in the MC group (P<0.05), indicating that it could alleviate FA-induced spleen enlargement. 2.4 Changes in the number of mast cells in mice Mast cells are key effector cells in food allergies. The mediators they release upon activation, such as histamine, trypsin inhibitors, and leukotrienes, are the direct cause of allergy symptoms. Figure 3A shows the state of mouse ear tissue after toluidine blue staining. In the NC group, the ear structure of the mice was intact, with a clear boundary between the purple upper epidermis and the light blue lower dermis, and no obvious mast cell infiltration. In the MC group, a large number of mast cells were observed in the dermis of the ears (indicated by arrows), and some mast cells were degranulated, indicating that β-LG-induced food allergy led to a large-scale activation of mast cells. After intervention with *Lactobacillus plantarum* KLDS1.0386, the number of mast cells in the mouse ears decreased, indicating that *Lactobacillus plantarum* KLDS1.0386 alleviated mast cell infiltration and activation in the skin. Figure 3 B represents the mast cell count results. The NC group had the fewest mast cells, with an average of only 5 mast cells per field of view, and none of them showed degranulation. Compared with the NC group, the MC group had a significantly higher number of mast cells (P<0.05), and a large number of mast cells in a degranulated state could be clearly seen in the field of view. Although the number of mast cells in the 1.0386 group was also significantly higher than that in the NC group (P<0.05), it was significantly lower than that in the MC group (P<0.05), and the number of activated mast cells in the field of view was also significantly reduced.

[0026] 2.5 Detection of allergy marker levels β-LG-specific IgE, histamine, and mMCP-1 reflect the sensitization status, mast cell degranulation level, and mucosal mast cell activation degree, respectively, and are important indicators for evaluating β-LG-induced food allergy. In this study, ELISA was used to detect the serum levels of these indicators in mice after oral β-LG challenge. The results are as follows: Figure 4 As shown, compared with the NC group, the serum levels of β-LG-specific IgE, histamine, and mMCP-1 in the MC group were significantly increased (P<0.05), at 4.07 μg / L, 30.95 ng / mL, and 286.03 U / mL, respectively. This indicates that β-LG successfully induced a sensitization response in mice and activated mast cell and mucosal mast cell degranulation, exacerbating the allergic reaction. After intervention with Lactobacillus plantarum KLDS 1.0386, the serum levels of β-LG-specific IgE, histamine, and mMCP-1 in mice were 2.34 μg / L, 15.20 ng / mL, and 191.98 U / mL, respectively, all significantly lower than those in the MC group (P<0.05). Among these, the levels of β-LG-specific IgE and mMCP-1 showed no significant difference between the NC and MC groups (P>0.05), while the histamine level, although higher than that in the MC group, was significantly higher. The FA symptoms were significantly reduced in the control group, but still higher than those in the NC group (P<0.05). The results indicate that Lactobacillus plantarum KLDS 1.0386 can effectively inhibit β-LG-induced sensitization and reduce mast cell activation, thereby alleviating FA symptoms.

[0027] 2.5 Changes in serum cytokine levels Food allergies are closely related to immune imbalance, mainly manifested as Th1 / Th2 imbalance, enhanced Th17-related inflammation, and decreased Treg-mediated immune regulation. This study used ELISA to detect the serum levels of IFN-γ, IL-4, IL-17A, and IL-10 in mice after oral challenge with β-LG. The results are as follows: Figure 5 As shown, compared with the NC group, the serum IFN-γ and IL-10 levels in the MC group were 315.90 and 38.13 pg / mL, respectively, both significantly decreased (P<0.05), while the IL-4 and IL-17A levels were 47.75 and 61.63 pg / mL, respectively, both significantly increased (P<0.05). This indicates that β-LG sensitization resulted in significant Th1 / Th2 imbalance, Th17 cell overactivation, and Treg function suppression in mice. After KLDS 1.0386 intervention, the serum IFN-γ and IL-10 levels in mice were 509.30 and 79.21 pg / mL, respectively, both significantly higher than those in the MC group (P<0.05), while the IL-4 and IL-17A levels were significantly lower (P<0.05), at 33.61 and 42.14 pg / mL, respectively. pg / mL, among which, IL-17A level recovered to no significant difference from NC group (P>0.05), IL-4 level was significantly lower than MC group, but still higher than NC group (P<0.05); while IFN-γ level was significantly higher than NC group (P<0.05). The results indicate that KLDS 1.0386 can improve immune imbalance by enhancing Th1 and Treg-related immune responses and inhibiting Th2 and Th17-related inflammatory responses, thereby alleviating β-LG-induced FA; 2.6 Colon HE staining and pathological evaluation HE staining of the colon can directly reflect the integrity of its mucosal structure, the degree of inflammatory infiltration, and the damage to the intestinal barrier. This study evaluated the β-LG-induced intestinal damage and the intervention effect of KLDS 1.0386 by observing the villus morphology, crypt structure, inflammatory cell infiltration in the lamina propria, and epithelial integrity of the colon tissues of mice in each group. The results are shown in Figure 6. The colon tissue structure of mice in the NC group was intact, the villus arrangement was regular, the crypt morphology was normal, only a small number of lymphocytes were infiltrated in the lamina propria, the epithelial cells were tightly arranged, and the number of goblet cells was normal, indicating that the intestinal barrier function was good. Compared with the NC group, the colon tissue of the MC group showed obvious pathological damage, manifested as disordered villus arrangement, atrophy and even breakage, shallower crypts with abnormal morphology, a large number of inflammatory cells infiltrated in the lamina propria, partial shedding and necrosis of epithelial cells, and a reduction in goblet cells, indicating that β-LG-induced mouse colon caused obvious inflammatory damage and barrier disruption. After KLDS 1.0386 intervention, the villus arrangement of the mouse colon tended to be regular, and the villus length and crypt depth were better than those in the MC group. The group showed significant improvement, with reduced inflammatory cell infiltration in the lamina propria, more intact epithelial cell structure, restored goblet cell count, and significantly reduced overall pathological damage, approaching the level of the NC group. The results indicate that KLDS 1.0386 can effectively alleviate β-LG-induced colonic tissue damage.

[0028] 2.7 Changes in serum levels of intestinal permeability markers DAO and D-LA are important indicators for evaluating intestinal permeability and intestinal mucosal barrier function. DAO reflects intestinal epithelial integrity, while D-LA reflects changes in intestinal permeability. In this study, ELISA was used to detect the serum levels of DAO and D-LA in mice after oral challenge with β-LG. The results are as follows: Figure 7 As shown, compared with the NC group, the serum DAO and D-LA levels in the MC group mice were significantly increased (P<0.05), with average values ​​of 17.32 U / mL and 56.40 μmol / mL, respectively. This indicates that β-LG-induced allergic reactions lead to intestinal epithelial damage and increased intestinal permeability. After intervention with KLDS 1.0386, the serum DAO and D-LA levels in mice were 13.45 U / mL and 47.35 μmol / mL, respectively, both significantly lower than those in the MC group, but still significantly higher than those in the NC group (P<0.05). The results indicate that KLDS 1.0386 can repair β-LG-induced intestinal mucosal damage and increased intestinal permeability.

[0029] 2.8 Changes in gut microbiome structure 2.8.1 Analysis of gut microbial diversity To assess the effect of KLDS 1.0386 on the α-diversity of gut microbiota in β-LG-sensitized mice, the Chao1, Shannon, and Simpson indices were calculated based on 16S rDNA sequencing data. The results are as follows: Figure 8 As shown in AC, compared with the NC group, the Chao1, Shannon, and Simpson indices of mice in the MC group were significantly decreased (P<0.05), indicating that β-LG sensitization led to a decrease in the richness and diversity of gut microbiota and disordered community structure. After intervention with KLDS 1.0386, the Chao1 index was significantly higher than that in the MC group but still lower than that in the NC group (P<0.05), indicating that the richness of the microbiota was restored to a certain extent but had not yet reached the normal level. The Shannon index was slightly higher than that in the MC group, but there was no significant difference (P>0.05). The Simpson index was significantly higher than that in the MC group (P<0.05) and there was no significant difference compared with the NC group (P>0.05), indicating that the evenness of the microbiota was significantly improved. In conclusion, KLDS 1.0386 can alleviate β-LG-induced gut microbiota disorder to a certain extent and has a restorative effect on microbiota diversity. β-diversity is mainly used to reflect the overall differences in gut microbiota structure. This study combined Venn diagrams and principal component analysis (PCA) to assess the microbiota structure of each group. The results are shown in Figures 8D and 8E. Venn diagram analysis showed that there were 462 OTUs in the three groups, accounting for 51.3% of the total OTUs, constituting the core microbiota. Compared with the NC group, the number of unique OTUs in the MC group was significantly reduced, with only 77. After KLDS 1.0386 intervention, the number of unique OTUs recovered to a level similar to that of the NC group, indicating that the gut microbiota was damaged under sensitization and recovered to some extent after intervention. PCA analysis further showed that the NC group samples were concentrated and the microbiota structure was stable. The MC group was significantly separated from the NC group, indicating that β-LG sensitization significantly altered the gut microbiota structure. KLDS 1.0386 showed a separating trend from the MC group, with the overall distribution shifting towards the NC group, but still at a certain distance. The above results indicate that KLDS 1.0386 can repair the gut microbiota structure damaged by β-LG. 2.8.2 Analysis of Gut Microbiota Composition and Differential Taxonomy To investigate the regulatory effect of Lactobacillus plantarum KLDS1.0386 on the gut microbiota of β-LG sensitized mice, we systematically examined the colon contents of the NC group, MC group, and KLDS 1.0386 intervention group and analyzed in detail the changes in microbiota structure and functional associations. Figure 9A shows the changes in the gut microbiota at the phylum level in mice. Firmicutes and Bacteroidota are the core components of the gut microbiota in each group, accounting for over 85% of the total microbiota. The F / B ratio is a core indicator for assessing gut microbiota homeostasis, and its changes are closely related to intestinal barrier function, energy metabolism, immune regulation, and disease occurrence. Compared with the NC group, the MC group showed a significantly increased relative abundance of Firmicutes (P<0.05) and a significantly decreased relative abundance of Bacteroidota (P<0.05), resulting in an F / B ratio increase from 1.25 to 2.28 (P<0.05), indicating that -LG disrupted the core balance of the gut microbiota. In contrast, the 1.0386 group showed a significantly decreased relative level of Firmicutes and a significantly increased relative level of Bacteroidota (P<0.05) compared to the MC group, with the F / B ratio recovering to 1.44, showing no significant difference from the NC group (P>0.05), indicating that KLDS 1.0386 It effectively restored the phylum-level microbial community homeostasis; Figure 9 B shows changes at the genus level, with the dominant genera including Lactobacillus, Muribaculum, Lachnospiraceae_NK4A136_group, and Desulfovibrio. Compared with the NC group, the abundance of Lactobacillus was significantly increased in the MC group, while the abundance of Muribaculum and Lachnospiraceae_NK4A136_group was significantly decreased. In contrast, the abundance of Lactobacillus in the 1.0386 group was significantly decreased, while the abundance of Muribaculum and Lachnospiraceae_NK4A136_group was significantly restored, approaching the levels of the NC group. The LDA score graph of mouse gut microbiota is shown below. Figure 9As shown in Figure D, in the NC group, species mainly belonging to the family Muribacteriaceae (LDA=4.8) and its subclasses showed significant enrichment, including *Muribaculum* (LDA=4.7), *Muribaculum intestinale* (LDA=4.6), and *Lactobacillus vaginalis* (LDA=3.5). These species are the core dominant flora in the gut of healthy mice, and they participate in fiber degradation, short-chain fatty acid (SCFA) production, and intestinal barrier stability. Their enrichment reflects normal intestinal homeostasis. In contrast, the significantly enriched flora in the MC group mainly came from Firmicutes (LDA=4.9) and its subclasses, including Lactobacillaceae (LDA=4.7), Clostridia (LDA=4.6), *Lactobacillus* (LDA=4.5), and *Lactobacillus*. After intervention with KLDS 1.0386, the gut microbiota structure of *Murinus murinus* (LDA=4.4) significantly enriched towards a health-promoting direction. The core groups were all *Bacteroidetes* or beneficial lactobacilli, such as *Bacteroidales* (LDA=4.9) and *Bacteroidia* (LDA=4.8). Among them, the core group of *Bacteroidetes* can efficiently degrade dietary fiber, produce SCFAs such as acetic acid and propionic acid, and repair the intestinal barrier. Lactobacillus can inhibit the growth of pathogenic bacteria such as *Lactobacillus murinus* by producing bacteriocins, while regulating the immune response.

[0030] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it.

Claims

1. The application of *Lactobacillus plantarum* KLDS1.0386 in the preparation of products for preventing β-lactoglobulin-induced food allergies, characterized by: The Lactobacillus plantarum KLDS 1.0386 was deposited at the China General Microbiological Culture Collection Center on May 29, 2024, with accession number CGMCC No. 30809.

2. The application according to claim 1, characterized in that: The drug contains a live strain of Lactobacillus plantarum KLDS1.0386.

3. The application of Lactobacillus plantarum KLDS1.0386 according to claim 1 in protecting the intestinal barrier and intestinal mucosa.

4. The application of Lactobacillus plantarum KLDS1.0386 according to claim 1 in regulating immune response.

5. The application according to claim 4, characterized in that: The regulation of the immune response includes: reducing the levels of β-LG-specific IgE, histamine and mMCP-1, downregulating the levels of IL-4 and IL-17A and upregulating the levels of IFN-γ and IL-10.

6. The application of Lactobacillus plantarum KLDS1.0386 according to claim 1 in regulating intestinal microbiota.

7. The application according to claim 1, characterized in that, The product is food, health supplement, or medicine.

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