Application of a breast milk-derived plantarum lactis strain for improving allergic diseases and regulating intestinal flora

Abstract

The application discloses application of a breast milk-derived plant lactobacillus to improving allergic diseases and regulating intestinal flora, and belongs to the technical field of microorganisms. The plant lactobacillus GDMCC No: 63060 is isolated from breast milk. The strain has excellent gastrointestinal fluid tolerance, mucus adhesion and antibiotic sensitivity, can inhibit various pathogenic bacteria, can produce indole derivatives, effectively inhibit the dermatitis symptoms of eczema mice, inhibit excessive polarization of Th2 type immune response, and induce regulatory T cells. Therefore, the plant lactobacillus FN029 has great application prospect in preparation of products for inhibiting intestinal pathogenic bacteria, improving allergic diseases, regulating intestinal flora or helping digestive function.

Description

Technical Field

[0001] This invention relates to the application of a human milk-derived plant Lactobacillus to improve allergic diseases and regulate intestinal flora, belonging to the field of microbial technology. Background Technology

[0002] In recent years, allergic diseases such as eczema, asthma, and food allergies have been on the rise, especially in industrialized countries, and are considered a "public health concern worldwide" by the World Allergy Organization (WAO). These allergic diseases not only severely impact patients' quality of life but also impose a huge medical burden on society. It is estimated that by 2050, the global prevalence of allergic diseases will reach 4 billion, but currently there are no interventions to stop this trend. The occurrence of allergic diseases is related to lifestyle changes brought about by industrialization, such as improved sanitation, excessive cleaning, antibiotic overuse, and Westernized diets. Increasing research demonstrates that changes in gut diversity are a major cause of allergic processes, especially early-life gut microbiota dysbiosis, which can predict the occurrence and persistence of allergic diseases. Therefore, developing probiotics that can effectively regulate the immune system, alleviate allergy symptoms, improve allergic diseases, and regulate gut microbiota is particularly important.

[0003] It is well known that breastfeeding provides both short-term and long-term benefits for children's development, hence the World Health Organization's strategy of exclusive breastfeeding for the first six months. Recent studies have shown that breast milk contains a range of bacteria, primarily Bifidobacteria, Streptococci, Lactobacilli, and Staphylococci, making it an ideal source of inoculation for commensal bacteria in the newborn's gut and a significant factor influencing gut microbiota succession. Through breastfeeding, specific strains of breast milk bacteria colonize the infant's gut via vertical transmission, which is crucial for promoting immune development and preventing allergic diseases. Studies have found that the proportion of vertically transmitted bacteria in breast milk is less than 0.0001% (Widespread vertical transmission of secretory immunoglobulin A coated trace bacterial variants from the mother to infantgut through breastfeeding. Food & Function. 2022 Nov;13(22):11543-11554.). Traditional microbial isolation techniques can only randomly isolate dominant breast milk bacteria, making it difficult to effectively isolate these active, vertically transmitted beneficial bacteria. By using magnetic bead enrichment technology for secretory immunoglobulin A (sIgA) combined with selective culture technology for the target bacterial strain, the discovery of anti-allergy probiotics in breast milk can be accelerated.

[0004] For probiotics to survive and function effectively in the human body, strains must possess good tolerance to gastrointestinal fluids. Due to increasing bacterial resistance, developing antibiotic-sensitive probiotics is also crucial. Single-function strains may not simultaneously meet the needs of improving allergic diseases and inhibiting pathogenic gut bacteria, or they may lack sufficient tolerance in gastrointestinal fluids and fail to adhere well to the intestinal mucus layer. Therefore, developing multifunctional probiotic strains can provide strong support for the prevention and treatment of allergic diseases, the inhibition of pathogenic gut bacteria, the regulation of gut microbiota, the improvement of antibiotic sensitivity, and the development of novel drugs in practical applications. Summary of the Invention

[0005] The first objective of this invention is to provide a strain of *Lactobacillus plantarum* FN029, taxonomically named... Lactiplantibacillus plantarum The *Lactobacillus plantarum* was deposited at the Guangdong Microbial Culture Collection Center, with accession number GDMCC No: 63060, and the deposit date was December 20, 2022.

[0006] In one embodiment of the present invention, the *Lactobacillus plantarum* is derived from breast milk. Bacteria from a breast milk sample from Tongwei City are enriched using sIgA immunomagnetic beads, and a strain of *Lactobacillus plantarum* derived from breast milk is screened using a selective culture medium for *Lactobacillus plantarum*, and named *Lactobacillus plantarum*. Lactiplantibacillus plantarum FN029.

[0007] In one embodiment of the present invention, the *Lactobacillus plantarum* GDMCC No: 63060 has the following characteristics: (1) Morphological characteristics: Gram-positive bacteria, rod-shaped, without spore formation, cell width is about 0.60~0.65µm, and length is about 1.0~1.3µm; (2) Colony characteristics: After 48 h of culture on MRS medium, the diameter is between 0.3 and 2 mm. The front is round and the side is convex. The edges are neat, milky white, opaque and moist. (3) Growth characteristics: It grows facultatively in MRS culture, with the best growth at a temperature of 30-37℃. The highest and lowest initial growth pH is 8.0 and 5.0, respectively, and the optimal initial growth pH is 6.8. (4) It has excellent tolerance to gastrointestinal fluids and mucin adhesion, and can tolerate adverse digestive tract environments and adhere well to the intestinal mucus layer; (5) It can inhibit a variety of pathogenic bacteria, including but not limited to Clostridium perfringens, Escherichia coli, Clostridium difficile, and Staphylococcus aureus; (6) Sensitive to antibiotics, including but not limited to vancomycin, chloramphenicol, penicillin, streptomycin, gentamicin, kanamycin, erythromycin, trimethoprim, and ampicillin; (7) It can produce indole derivatives, with extremely high yields of indole-3-lactic acid and indole-3-acetic acid; (8) Improves allergic diseases, relieves eczema, and regulates immunity; (9) Regulate the intestinal flora and restore the intestinal flora disorder caused by allergic diseases; (10) Promotes digestion, improves host intestinal motility, and increases trypsin activity.

[0008] In one embodiment of the present invention, the allergic diseases include, but are not limited to, eczema, food allergies, allergic rhinitis, and asthma.

[0009] In one embodiment of the present invention, the relief of eczema includes, but is not limited to: (1) Reduces pathological changes such as swelling of the epidermis and dermis of the ears and severe infiltration of inflammatory cells in mice with eczema; (2) Reduces the number of eosinophils and mast cells in eczema mice, alleviating allergy symptoms; (3) Increase the proportion of regulatory T cells in the spleen of eczema mice and inhibit the inflammatory response; (4) Reduce the levels of plasma cytokines IgE, IL-4, and plasma IL-4 / IFN-γ in eczematous mice, increase the levels of IL-12 and IL-10, reduce the level of IL-33 in tissue homogenate, correct the Th1 / Th2 imbalance, thereby enhancing the cellular immune response and inhibiting the excessive inflammatory response.

[0010] In one embodiment of the present invention, the restoration of intestinal flora disorder caused by allergic diseases includes increasing the Shannon index and evenness reduction caused by eczema, upregulating the abundance of Kineothrix alysoides and Alistipes dispar, and reducing the abundance of Akkermansia muciniphila.

[0011] The second objective of this invention is to provide a microbial inoculant containing *Lactobacillus plantarum* GDMCC No: 63060 of this invention.

[0012] In one embodiment of the present invention, the microbial agent uses Lactobacillus plantarum GDMCC No: 63060 as the main microorganism.

[0013] In one embodiment of the present invention, the number of GDMCC No: 63060 cells in the bacterial agent is not less than 1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0014] In one embodiment of the present invention, the microbial agent is obtained by preparing a seed culture of GDMCC No: 63060 and then expanding it.

[0015] In one embodiment of the present invention, the microbial agent contains live cells of the GDMCC No. 63060 strain of the present invention, freeze-dried dried cells of the GDMCC No. 63060 strain of the present invention, immobilized cells of the GDMCC No. 63060 strain of the present invention, liquid agent of the GDMCC No. 63060 strain of the present invention, solid agent of the GDMCC No. 63060 strain of the present invention, or the GDMCC No. 63060 strain of the present invention existing in any other form.

[0016] In one embodiment of the present invention, the microbial agent is a microbial preparation that is tolerant to adverse digestive tract environments, inhibits intestinal pathogens, is sensitive to antibiotics, produces indole derivatives, improves allergic diseases, regulates intestinal flora, and aids digestion.

[0017] In one embodiment of the present invention, the pathogenic bacteria include, but are not limited to, Clostridium perfringens, Escherichia coli, Clostridium difficile, and Staphylococcus aureus.

[0018] In one embodiment of the present invention, the antibiotic is one or more of vancomycin, chloramphenicol, penicillin, streptomycin, gentamicin, kanamycin, erythromycin, trimethoprim, and ampicillin.

[0019] In one embodiment of the present invention, the indole derivative is one or more of indole-3-acetamide, indole-3-lactic acid, indole-3-carboxylic acid, indole-3-carboxaldehyde, indole-3-acetic acid, 3-indoleacrylic acid, and 3-indolepropionic acid.

[0020] In one embodiment of the present invention, the allergic diseases include, but are not limited to, eczema, food allergies, allergic rhinitis, and asthma.

[0021] A third object of the present invention is to provide a product containing *Lactobacillus plantarum* GDMCC No: 63060 or a bacterial agent containing *Lactobacillus plantarum* GDMCC No: 63060.

[0022] In one embodiment of the present invention, the product is food, medicine, or health product.

[0023] In one embodiment of the present invention, the food is a solid food, a liquid food, or a semi-solid food.

[0024] In one embodiment of the present invention, the pharmaceutical product further contains pharmaceutically acceptable excipients.

[0025] In one embodiment of the present invention, the excipients include any one or a combination of at least two of the following: carrier, diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

[0026] In one embodiment of the present invention, the carrier includes, but is not limited to, liposomes and nanoparticles.

[0027] In one embodiment of the present invention, the excipient is one or more of mannitol, lactose, fatty acids, and polyethylene glycol.

[0028] In one embodiment of the present invention, the dosage form of the medicine includes any one of suspension, granules, capsules, powders, tablets, emulsions, solutions, pellets, injections, suppositories, enemas, aerosols, patches, or drops.

[0029] In one embodiment of the present invention, the route of administration of the drug includes oral administration, sublingual administration, skin and mucous membrane administration, inhalation administration, or injection administration.

[0030] In one embodiment of the present invention, the number of *Lactobacillus plantarum* GDMCC No: 63060 cells in the product is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0031] A fourth object of the present invention is to provide the use of *Lactobacillus plantarum* GDMCC No: 63060 or a bacterial agent containing *Lactobacillus plantarum* GDMCC No: 63060 in the preparation of products containing indole derivative active ingredients.

[0032] In one embodiment of the present invention, the product includes, but is not limited to, pharmaceuticals, food, functional food, special medical purpose formula food, or health products.

[0033] In one embodiment of the present invention, the number of *Lactobacillus plantarum* GDMCC No: 63060 cells in the product is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0034] In one embodiment of the present invention, the pathogenic bacteria include, but are not limited to, Clostridium perfringens, Escherichia coli, Clostridium difficile, and Staphylococcus aureus.

[0035] In one embodiment of the present invention, the antibiotic is one or more of vancomycin, chloramphenicol, penicillin, streptomycin, gentamicin, kanamycin, erythromycin, trimethoprim, and ampicillin.

[0036] In one embodiment of the present invention, the indole derivative is one or more of indole-3-acetamide, indole-3-lactic acid, indole-3-carboxylic acid, indole-3-carboxaldehyde, indole-3-acetic acid, 3-indoleacrylic acid, and 3-indolepropionic acid.

[0037] In one embodiment of the present invention, the prevention and / or treatment of eczema includes, but is not limited to, alleviating allergy symptoms, suppressing inflammatory responses, and correcting Th1 / Th2 imbalance.

[0038] In one embodiment of the present invention, the allergic diseases include, but are not limited to, eczema, food allergies, allergic rhinitis, and asthma.

[0039] In one embodiment of the present invention, the regulation of intestinal flora includes, but is not limited to, restoring intestinal flora imbalance caused by allergic diseases; optionally, it includes increasing the Shannon index and evenness reduction caused by eczema, and upregulating *Kinetinobacterium arachnoideum* (…). Kineothrix alysoides ) and other branches ( Alistipes dispar Abundance of Akermansia muciniphila ( ) and reduction of Akermansia muciniphila ( ) Akkermansia muciniphila Abundance.

[0040] In one embodiment of the present invention, the digestive enhancement includes, but is not limited to, increasing host intestinal motility and increasing trypsin activity.

[0041] Preservation of biological materials: Lactobacillus plantarum ( Lactiplantibacillus plantarum )FN029, taxonomically named Lactiplantibacillus plantarum It was deposited at the Guangdong Microbial Culture Collection Center on December 20, 2022, with accession number GDMCC No: 63060, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province.

[0042] Beneficial effects: The *Lactobacillus plantarum* FN029 isolated in this invention is a multifunctional lactic acid bacterium with excellent tolerance to gastrointestinal fluids and mucin adhesion, enabling it to tolerate adverse digestive tract environments and adhere well to the intestinal mucus layer. *Lactobacillus plantarum* FN029 of this invention can inhibit harmful pathogens in the intestine, including but not limited to *Clostridium perfringens*, *Escherichia coli*, *Clostridium difficile*, and *Staphylococcus aureus*, and exhibits high sensitivity to antibiotics, demonstrating good probiotic properties. Furthermore, *Lactobacillus plantarum* FN029 of this invention can also produce indole derivatives, with extremely high yields of indole-3-lactic acid and indole-3-acetic acid. *Lactobacillus plantarum* FN029 of this invention also has good ability to improve allergic diseases and regulate immunity, relieving eczema, reducing allergy symptoms, inhibiting inflammatory responses, and correcting Th1 / Th2 imbalance. *Lactobacillus plantarum* FN029 of this invention can restore intestinal flora imbalance caused by allergic diseases and regulate intestinal flora; it can also improve host intestinal motility, increase trypsin activity, and promote digestion.

[0043] In summary, the *Lactobacillus plantarum* FN029 strain of this invention integrates the functions of tolerating adverse digestive tract environments, inhibiting intestinal pathogens, possessing antibiotic sensitivity, producing indole derivatives, improving allergic diseases, regulating intestinal flora, and aiding digestion. It is a multifunctional strain with broad application prospects. Attached Figure Description

[0044] Figure 1 Vertical transfer rate of gut microbiota corrected for human samples from breast milk to infants for Lactobacillus plantarum FN029 isolation.

[0045] Figure 2 Morphological images of *Lactobacillus plantarum* FN029. (A) State of *Lactobacillus plantarum* FN029 after 24 hours of growth in MRS broth; (B) Colony of FN029 after 48 hours of culture on MRS agar; (C) Gram staining morphology of FN029 (magnified 200 times); (D) Scanning electron micrograph of FN029.

[0046] Figure 3 This is a graph showing the artificial gastric juice tolerance of Lactobacillus plantarum FN029. p<0.05 p<0.01, compared with 0 h; ## p<0.01, endpoint 299v compared with FN029. 299v, Lactobacillus plantarum 299v; FN029, Lactobacillus plantarum FN029.

[0047] Figure 4 Figure showing the artificial intestinal fluid tolerance of Lactobacillus plantarum FN029. p<0.01, compared with 0 h; # p<0.05, ## p<0.01, ### p<0.001, compared with FN029 at different time points.

[0048] Figure 5 The effect of *Lactobacillus plantarum* FN029 on eczema in mice. (A) Animal treatment and eczema induction; (B) Eczema severity score, including the number of ear scratching, erythema / hemoptysis, rash, edema, and scaly changes, ranging from 0 to 10, with higher scores indicating more severe eczema symptoms (n = 10); (C) Increase in ear thickness at the induced eczema endpoint relative to ear thickness on day 0 (n = 10); (D) Typical photographs of mouse ear lesions. ## p<0.01, ### p<0.001, compared with the AD group. CON, healthy control; AD, eczema model control; LGG, *Lactobacillus rhamnosus* GG treatment; FN029, *Lactobacillus plantarum* FN029 treatment; MC903+OVA, calcipotriol (MC903) and ovalbumin (OVA) were applied to the right ear of mice to induce eczema.

[0049] Figure 6 Images showing the effect of *Lactobacillus plantarum* FN029 on improving neutrophil and mast cell infiltration in the ears of mice with eczema. (A) Typical image of a mouse ear section; (B) Relative content of mast bacteria in a mouse ear tissue section; (C) Relative content of eosinophils in a mouse ear tissue section. p<0.01 p<0.001, compared with the CON group; ### p<0.001, compared with the AD group. CON, healthy control; AD, eczema model control; LGG, Lactobacillus rhamnosus GG treatment; FN029, Lactobacillus plantarum FN029 treatment.

[0050] Figure 7 Graphs showing the effect of *Lactobacillus plantarum* FN029 on increasing splenic regulatory T cells in a mouse eczema model. (A) Flow cytometry analysis; (B) Relative content of splenic regulatory T cells. p<0.001, compared with the CON group; ### p<0.001, compared with the AD group. CON, healthy control; AD, eczema model control; LGG, Lactobacillus rhamnosus GG treatment; FN029, Lactobacillus plantarum FN029 treatment.

[0051] Figure 8 The effect of Lactobacillus plantarum FN029 on improving plasma cytokines in a mouse model of eczema. p<0.05 p<0.01 p<0.001, compared with the CON group; # p<0.05, ## p<0.01, ### p<0.001, compared with the AD group. CON, healthy control; AD, eczema model control; LGG, Lactobacillus rhamnosus GG treatment; FN029, Lactobacillus plantarum FN029 treatment.

[0052] Figure 9 The regulatory effect of *Lactobacillus plantarum* FN029 on the α and β diversity of gut microbiota dysbiosis caused by eczema is shown in the figure. (A) α diversity: Shannon index; Phylogenetic diversity; Evenness; (B) β diversity: principal coordinate analysis of Bray-Curtis distance (PCoA, left) and nonmetric multidimensional measure analysis (NDMS, right). p<0.05, compared with the CON group; # p<0.05, compared with the AD group. CON, healthy control; AD, eczema model control; LGG, Lactobacillus rhamnosus GG treatment; FN029, Lactobacillus plantarum FN029 treatment.

[0053] Figure 10 The relative content (%) of bacteria species that significantly altered the gut microbiota in patients with eczema dysbiosis due to Lactobacillus plantarum FN029 regulation. p<0.05, compared with the CON group; # p<0.05, compared with the AD group. CON, healthy control; AD, eczema model control; LGG, Lactobacillus rhamnosus GG treatment; FN029, Lactobacillus plantarum FN029 treatment.

[0054] Figure 11 The effect of Lactobacillus plantarum FN029 and 299v on intestinal motility in mice is shown in the figure. p<0.001, compared with the control group; ### p<0.001, compared with the model group. Control group: healthy controls; Model group: compound diphenoxylate model control; 299v: *Lactobacillus plantarum* 299v treatment; FN029: *Lactobacillus plantarum* FN029 treatment.

[0055] Figure 12 The effect of Lactobacillus plantarum FN029 and 299v on mouse body weight is shown in the figure. p<0.01, compared with the control group; # p<0.05, compared with 299v. Control group: healthy controls; 299v: *Lactobacillus plantarum* 299v treatment; FN029: *Lactobacillus plantarum* FN029 treatment.

[0056] Figure 13 The effect of Lactobacillus plantarum FN029 and 299v on the activity of intestinal digestive enzymes in mice is shown in the figure. p<0.01, compared with the control group; # p<0.05, compared with 299v. Control group: healthy controls; 299v: *Lactobacillus plantarum* 299v treatment; FN029: *Lactobacillus plantarum* FN029 treatment. Detailed Implementation

[0057] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, where specific conditions are not specified, are generally performed under conventional conditions in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar with the art.

[0058] Unless otherwise specified, the experimental materials and reagents used in the embodiments of this invention can be obtained through conventional purchase.

[0059] The experimental materials involved in the following embodiments: Selective culture medium for *Lactobacillus plantarum* (1 L): peptone (10 g), beef extract (10 g), yeast extract (5 g), d-sorbitol (20 g), ciprofloxacin (4 mg), sodium acetate (5 g), ammonium citrate (2 g), potassium phosphate (2 g), magnesium sulfate (0.1 g), manganese sulfate (0.05 g), bromocresol purple (0.02 g), and agar (15 g). The ciprofloxacin-free medium was autoclaved at 121 °C for 1 min and cooled at 50 °C. Ciprofloxacin was filtered and sterilized before being added to the unsolidified medium. The pH of the medium was 6.0 ± 0.1. After solidification, the medium turned purple.

[0060] MRS broth medium (1 L): 10 g soybean peptone, 5 g beef extract, 5 g yeast powder, 20 g glucose, 1 mL Tween-80, 2 g sodium dihydrogen phosphate, 5 g anhydrous sodium acetate, 2 g triamine citrate, 0.02 g manganese sulfate, 0.1 g magnesium sulfate, 1 L distilled water. Adjust pH to approximately 6.8 and sterilize at 121 °C for 15 min.

[0061] MRS solid medium (MRS agar plate) (1 L): 10 g soybean peptone, 5 g beef extract, 5 g yeast powder, 20 g glucose, 1 mL Tween-80, 2 g sodium dihydrogen phosphate, 5 g anhydrous sodium acetate, 2 g triamine citrate, 0.02 g manganese sulfate, 0.1 g magnesium sulfate, 1 L distilled water, adjust pH to approximately 6.8, 15 g agar, sterilize at 121 ℃ for 15 min.

[0062] Lactobacillus rhamnosus GG (accession number ATCC 53013) and Clostridium difficile (accession number ATCC 9689) were obtained from the U.S. Culture Collection; Lactobacillus plantarum 299v (accession number DSM9843) was obtained from the German Microbiological Culture Collection; Staphylococcus aureus (accession number ATCC 25923) and Escherichia coli (accession number ATCC 8739) were obtained from the American Center for Type Culture Collection; and Clostridium perfringens (accession number CMCC 64722) was obtained from the National Microbiological Culture Collection Center.

[0063] Example 1: Isolation and identification of Lactobacillus plantarum FN029 1. Analysis of vertically transferred bacterial species in fecal microbiota of breast milk and paired infants Thirty-five mother-infant pairs were recruited from Tongwei County and 28 mother-infant pairs from Wuxi City. Inclusion criteria: (1) full-term vaginal delivery after 37 weeks of gestation; (2) exclusive breastfeeding from birth to sample collection; (3) no exposure to antibiotics or probiotics before delivery and during breastfeeding. Demographic and clinical data, including maternal age and weight, mode of delivery, neonatal weight, breastfeeding stage, and gestational length, were collected. All mothers had pregnancies exceeding 38 weeks.

[0064] On day 42 postpartum, collect breast milk and infant stool samples. Wash hands with soap before collection and clean nipples with a chlorhexidine swab, then express milk from breasts that have not been previously suckled. Systematically collect breast milk every morning between 9:00 and 10:00, using a sterile automatic breast pump equipped with a vacuum regulator. All collection bottles and suction funnels are autoclaved before use, and breast milk is quickly aliquoted under aseptic conditions.

[0065] DNA was extracted from 1 mL of breast milk and 200 mg of fecal matter using the TGuide S96 Magnetic Soil / Fecal DNA Kit (Tiangen Biotech Co., Ltd., Beijing, China). Libraries were constructed according to the Illumina 16S metagenomic sequencing pipeline, targeting the V3-V4 variable region of the 16S rRNA gene in the DNA samples for amplification. After the initial amplification, a second PCR was performed using i5 forward and i7 reverse primers to integrate the Illumina Nextera barcode. The products were purified using the QIAquick PCR Purification Kit (Qiagen). The size and quality of the purified products were assessed using a Bioanalyzer 2100 (Agilent). The purified amplicon sequences were pooled and sequenced using the Illumina MiSeq sequencing system (Illumina). Amplicon sequence variants (ASVs) were extracted using EasyAmplicon software. ASV species-level annotation was then performed using the BLCA algorithm.

[0066] The univariate Fisher's exact test was used to compare the presence of ASV in breast milk and infant gut samples. The odds ratio (OR) for strain transfer was calculated, and the null hypothesis of OR=1 was tested using a one-sided p-value. The weighted transfer rate (WTR) was calculated using the R package randpermutationTransferStats.R, and a WTR greater than 1 indicated that transfer had occurred.

[0067] like Figure 1 As shown, after analyzing the bacterial species transmission rate from breast milk to the infant's intestines and correcting for potential biases, it was found that *Lactobacillus plantarum* was the main unique bacterial species vertically transmitted in the Tongwei samples.

[0068] 2. Isolation and purification of strains Centrifuge 1 mL of breast milk at 15000 g for 5 min, discard the supernatant, and resuspend the bacterial cells at 10% in peptone buffer. Add 0.5% bovine serum albumin and biotin-labeled rabbit anti-human immunoglobulin A serum, incubate for 15-30 min, then add streptavidin-modified magnetic beads (0.5 mg / mL), adsorb the bacterial cells with a magnet, wash twice with peptone buffer, and obtain sIgA-binding bacteria. Then, in a clean bench, take 1 mL of the enrichment solution and perform a tenfold serial dilution with sterile physiological saline, selecting 10... -6 10 -7 10 -8Three dilution gradients were used, with 100 μL of bacterial culture from each gradient plated onto selective medium for *Lactobacillus plantarum* and anaerobic incubation at 30 °C for 48 h. After incubation, plates with 50-150 single colonies were selected from the medium. Colonies with different morphologies, sizes, and colors were picked and repeatedly streaked on MRS agar plates for purification until the colonies on the entire plate had a uniform morphology. Single colonies were then picked and enriched in MRS broth. The resulting strain was suspended in PBS containing 20% ​​glycerol and stored frozen at -80 °C.

[0069] Among them, yellow colonies with a diameter greater than 2 mm and a yellow transparent ring around them were selected and transferred to MRS medium for 48 h of culture. The colonies were 0.3-2 mm in diameter, round on the front, convex on the side, with neat edges, milky white, opaque, and moist and smooth. Figure 2 B). The strain exhibits facultative anaerobic growth in MRS medium, with optimal growth at 30-37 ℃. The highest and lowest initial growth pH values ​​are 8.0 and 5.0, respectively, with the optimal initial growth pH being 6.8. The culture state is a turbid suspension. Figure 2 A). The colony smears are Gram-positive, rod-shaped, without spore formation, and the bacterial cells are approximately 0.60-0.65 µm wide and 1.0-1.3 µm long. Figure 2 C). Scanning electron microscopy showed that FN029 cells were approximately 0.60 to 0.65 µm wide and 1.0 to 1.3 µm long. Figure 2 D).

[0070] 3. Lactobacillus-specific primer amplification and 16S rDNA identification Genomic DNA of the target strain was extracted using the Ezup column-based bacterial genomic DNA extraction kit. The extracted lactic acid bacteria genomic DNA was used as a template for PCR amplification, and PCR experiments were performed using lactobacillus-specific primers. Subsequently, PCR experiments on 16S rDNA were conducted using universal bacterial primers 27F and 1492R. After the PCR reaction was completed, the PCR products were examined and photographed on an agarose gel. The amplified fragment length was approximately 1.2 kbp. The primers were then used to send the PCR products to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0071] The 16S rDNA sequencing results are shown in SEQ ID NO.1. EZCLOUD sequence alignment was performed on the NCBI website, and the results showed that this sequence is similar to *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum The 16S rDNA sequence of the strain showed over 99% homology. Combining sequence alignment results with physiological and biochemical results, the screened lactic acid bacteria were identified as *Lactobacillus plantarum*, and named... Lactiplantibacillus plantarumFN029 was deposited at the Guangdong Microbial Culture Collection Center, with accession number GDMCC No: 63060, on December 20, 2022.

[0072] SEQ ID NO.1: Example 2: Determination of digestive fluid tolerance and mucin adhesion of Lactobacillus plantarum strain FN029 1. Research Methods 1.1 Determination of tolerance to artificial gastric juice After sterilizing PBS, add 3 g / L pepsin (purchased from Sigma-Aldrich, catalog number: 9001-75-6, enzyme activity ≥2000 U / mg protein), 3 g / L NaCl, 1.1 g / L KCl, 0.15 g / L CaCl2, and 0.6 g / L NaHCO3. Adjust the pH to 3.0 with 1 mol / L HCl, and filter sterilize using a 0.22 μm microporous membrane to prepare simulated gastric juice.

[0073] The *Lactobacillus plantarum* FN029 and *Lactobacillus plantarum* 299v strains were activated and cultured for two generations in MRS medium, washed twice by centrifugation, and the bacterial cells were collected and suspended in 2 mL of simulated gastric fluid (concentration 1×10⁻⁶). 9 The sample was incubated at 37°C for 3 hours (CFU / mL). Viable bacterial concentrations were determined by pouring MRS agar at 0, 1, and 2 hours. Each sample was tested three times, and the average value was calculated.

[0074] 1.2 Determination of tolerance to artificial intestinal fluid After sterilizing PBS, add 3 g / L ox bile salt (purchased from Sigma-Aldrich, catalog number: 48305), 0.1 g / L lipase (purchased from Sigma-Aldrich, catalog number: 9001-62-1, enzyme activity ≥20,000 U / mg protein), 1 g / L trypsin (purchased from Sigma-Aldrich, catalog number: 9002-07-7, enzyme activity 1,000-2,000 U / mg protein), 5 g / L NaCl, 0.6 g / L KCl, 0.3 g / L CaCl2, and 0.6 g / L NaHCO3. Adjust the pH to 8.0 with 0.1 mol / L NaOH, and then filter through a 0.22 μm microporous membrane for sterilization to prepare a simulated intestinal fluid.

[0075] The *Lactobacillus plantarum* FN029 and *Lactobacillus plantarum* 299v were activated and cultured in MRS medium for two generations, followed by centrifugation and washing twice. The bacterial cells were collected and suspended in 2 mL of simulated intestinal fluid (concentration 1×10⁻⁶). 9 The bacterial suspension was incubated at 37 °C for 4 h (CFU / mL). The diluted suspension was then inoculated onto MRS agar medium and cultured anaerobically at 37 °C for 36–48 h. Viable cell concentrations were determined at 0, 1, 2, 3, and 4 h using the MRS agar pour method. Each sample was tested three times, and the average value was calculated.

[0076] 1.3 Determination of mucin adhesiveness The bacteria were cultured in MRS broth containing 2% porcine gastric mucin for 24 h, washed twice, and then resuspended in PBS containing 0.05 Tween 20 to adjust the bacterial suspension concentration to 1×10⁻⁶. 9 CFU / mL. 10 mg of lyophilized porcine gastric mucin was suspended in 10 mL of 50 mM Tris-HCl (pH 9.0) and gently rotated at 4 °C for 12 h. Afterward, it was centrifuged at 16000 g for 10 min at 4 °C, and the supernatant was used as the mucin solution and stored at 4 °C. 100 μL (1 mg / mL) of the mucin solution was added to each well and stored at 4 °C for 12 h. The mucin was discarded, and the sample was washed twice with 0.1% bovine serum albumin PBS. 200 μL of 2% bovine serum albumin PBS was added, and the sample was incubated at room temperature for 2 h. The sample was washed twice with 0.1% bovine serum albumin PBS. 100 μL of bacterial suspension was added to each well and incubated overnight at 4 °C. The wells were washed with PBST, and binding was examined under an inverted microscope. The solution was poured out, and after the wells dried, OD405 was measured using an ELISA instrument. All measurements were repeated three times.

[0077] 1.4 Data Analysis The data are expressed as mean ± standard deviation, using t The differences between the two groups were analyzed.

[0078] 2. Results Results of the acid resistance test of the strain in artificial gastric juice are as follows: Figure 3 As shown, after 1 h of incubation in artificial gastric fluid, the survival rate of FN029 was 96.43%, while that of 299v was 89.77%, with FN029 having a survival rate 6.66% higher than 299v; after 2 h of incubation in artificial gastric fluid, the survival rate of FN029 was 94.96%, while that of 299v was 1.11%, with FN029 having a survival rate 93.85% higher than 299v.

[0079] Table 1. Tolerance of *Lactobacillus plantarum* to artificial gastric juice

[0080] Results of the strain's artificial intestinal fluid tolerance test are as follows: Figure 4 As shown, 299v showed almost no survival after 2 hours of incubation in artificial intestinal fluid (SIJ), while FN029 only decreased by 0.60 logs at the end of 4 hours of digestion in SIJ, with a survival rate of 90.54% (Table 2). This indicates that 299v is susceptible to the effects of bile salts, whose toxicity can disrupt the microbial membrane, while *Lactobacillus plantarum* FN029 is almost unaffected by bile salts and exhibits extremely high intestinal fluid tolerance.

[0081] Table 2. Artificial intestinal fluid tolerance of *Lactobacillus plantarum*

[0082] The results of the mucin adhesion experiment are shown in Table 3. The amount of *Lactobacillus plantarum* FN029 adhering to the surface of the porcine gastric mucus matrix was 0.085 (OD405), which is 1.2 times that of *Lactobacillus plantarum* 299v adhering to the surface of the porcine gastric mucus matrix.

[0083] Table 3. Mucin Adhesion of *Lactobacillus plantarum*

[0084] Note: Results are mean ± standard deviation. p<0.05, t The differences between the two groups were analyzed.

[0085] As can be seen from the above experimental results, the *Lactobacillus plantarum* FN029 provided by the present invention has excellent gastrointestinal fluid tolerance and mucin adhesion, and can enter the human gastrointestinal tract in a viable state and survive in the gastrointestinal organs of animals, including humans, to exert health benefits.

[0086] Example 3: Evaluation of the antibacterial effect of Lactobacillus plantarum FN029 1. Research Methods 1.1 Preparation of Fermentation Broth for Strains The strain preserved in glycerol tubes was first streaked three times on MRS solid medium for activation, then single colonies were picked and cultured in MRS broth for 18 h (under anaerobic conditions at 37 °C). The culture medium was then adjusted to a cell concentration of 1 × 10⁻⁶ cells / mL with distilled water. 9 Centrifuge at 15000 g for 5 min at 4 ℃ with CFU / mL, and collect the supernatant. Adjust the pH to 7.0 to obtain the fermentation supernatant.

[0087] 1.2. Effect of the strain on inhibiting pathogenic bacteria The inhibitory effect of *Lactobacillus plantarum* FN029 on several major enteropathogenic bacteria (*Clostridium perfringens* CP01, *Escherichia coli* ATCC 8739, *Clostridium difficile* CD01, and *Staphylococcus aureus* ATCC25923) was detected using the Oxford cup method. Specifically, the bacteria were incubated at 37 °C for 16 h (approximately 1 × 10⁻⁶). 9 Different pathogenic bacteria (CFU / mL) were spread onto MRS solid medium. Oxford cups were carefully placed on the medium, and 200 μL of the fermentation supernatant of the bacteria prepared in 1.1 was added to each cup. After incubation at 37 ℃ for 24 h, the diameter of the inhibition zone was measured. Each sample was repeated three times, and the average value was calculated.

[0088] 1.3 Statistical Analysis The data are expressed as mean ± standard deviation, using t The differences between the two groups were analyzed.

[0089] 2. Results The experimental results are shown in Table 4. The antibacterial effect of the fermentation supernatant of *Lactobacillus plantarum* FN029 obtained by this invention is better than that of *Lactobacillus plantarum* 299v. Among them, FN029 showed the most significant inhibitory effect on *Escherichia coli* and *Staphylococcus aureus*, with inhibitory effects 1.25 times and 1.29 times that of 299v, respectively.

[0090] Table 4 Antibacterial activity (diameter of inhibition zone, mm)

[0091] Note: Results are mean ± standard deviation. p<0.05, t The differences between the two groups were analyzed.

[0092] The experimental results above show that the *Lactobacillus plantarum* FN029 provided by this invention has a better inhibitory effect on pathogenic bacteria than *Lactobacillus plantarum* 299v.

[0093] Example 4: Antibiotic sensitivity of Lactobacillus plantarum FN029 1. Research Methods Nine antibiotic solutions (vancomycin, chloramphenicol, penicillin, streptomycin, gentamicin, kanamycin, erythromycin, trimethoprim, and ampicillin) were prepared separately. *Lactobacillus plantarum* FN029 and *Lactobacillus plantarum* 299v were added to MRS broth containing different concentrations (2, 4, 8, 16, 32, 64, 128, 256, 512, and 1024 μg / mL) of antibiotics and anaerobically cultured at 37 °C for 24 h. The final concentration of *Lactobacillus plantarum* FN029 and 299v in the system was 1 × 10⁻⁶. 7 CFU / mL. Each sample was repeated three times. After incubation, the absorbance at 610 nm (OD value) was measured in the 96-well plate. MIC (Minimum Inhibitory Concentration) is considered the lowest antibiotic concentration that can inhibit the growth of a bacterial strain and is used to assess the antibiotic resistance of selected strains.

[0094] Table 5. Susceptibility results of strains to different antibiotics

[0095] 2. Results As shown in Table 5, the *Lactobacillus plantarum* FN029 screened in this invention exhibits varying degrees of sensitivity to vancomycin, chloramphenicol, penicillin, streptomycin, gentamicin, kanamycin, erythromycin, trimethoprim, and ampicillin, while *Lactobacillus plantarum* 299v shows significant resistance to gentamicin, streptomycin, and trimethoprim. The above experimental results indicate that *Lactobacillus plantarum* FN029 provided by this invention is more sensitive to the above antibiotics than *Lactobacillus plantarum* 299v, suggesting that *Lactobacillus plantarum* FN029 has the potential to be used as a live bacterial product.

[0096] Example 5: Analysis of indole derivatives produced by Lactobacillus plantarum FN029 1. Research Methods *Lactobacillus plantarum* FN029 and 299v were cultured in MRS broth for 24 h, centrifuged (10000 g, 5 min) to remove the supernatant, and then resuspended in physiological saline, adjusting the OD value to 1 at 600 nm. 2 mL of the bacterial suspension was inoculated into 18 mL of MRS medium supplemented with L-tryptophan (Trp) (2 g / L) (MRS-Trp medium). After 48 hours of culture, the culture was centrifuged at 10000 g for 5 min, and the supernatant was collected. 15 mL of the supernatant or MRS-Trp medium was mixed with acetonitrile at a 1:1 ratio. Magnesium sulfate (0.2 g / mL) and sodium acetate (0.05 g / mL) were added, and the mixture was gently vortexed for 10 min, followed by centrifugation at 3500 rpm for 10 min.

[0097] Collect the organic phase (10 mL) and transfer it to a 50 mL centrifuge tube. Detect tryptophan metabolites using liquid chromatography-mass spectrometry (LC-MS). Take 50 μL of acetonitrile extract, add 10 μL of internal standard working solution (4000 ng / mL), then add 40 μL of 1% acetonitrile-water (containing 0.1% formic acid), vortex for 30 seconds, and then perform machine analysis. Qualitative and quantitative determinations were performed using liquid chromatography-electrospray ionization tandem mass spectrometry. Chromatographic conditions were as follows: Nexera series LC-40 liquid chromatograph, ACQUITY UPLC HSS T3 column (Waters), column temperature 40 ℃, injection volume 2 μL, mobile phase A (0.1% formic acid aqueous solution), mobile phase B (0.1% formic acid acetonitrile solution), and chromatographic gradients are shown in Table 6. Mass spectrometry conditions: AB SCIEX QTRAP 6500+, positive and negative mode detection, curtain gas (CUR) 35, collisional medium gas (CAD), ion spray voltage (IS) 5500 / -4500, temperature (TEM) 550. Ion source gas 1 (GS1) 50, ion source gas 2 (GS2) 50.

[0098] Table 6 Chromatographic gradient

[0099] 2. Statistical Analysis The data are expressed as mean ± standard deviation, using t The differences between the two groups were analyzed.

[0100] 3. Results Analysis Indole derivatives are important active molecules produced by beneficial gut bacteria that participate in immune regulation and inhibit allergic diseases. As shown in Table 7, FN029 mainly produces four indole derivatives: indole-3-lactic acid, indole-3-acetic acid, indole-3-carboxaldehyde, and 3-indolepropionic acid. Among them, the production of indole-3-lactic acid and indole-3-acetic acid is significantly higher than that of 299v, approximately 461 times and 6.7 times that of 299v, respectively. The production of indole-3-lactic acid and indole-3-acetic acid by FN029 is the basis for its immune-regulating effects.

[0101] Table 7. Yields of indole derivatives from Lactobacillus plantarum FN029 and 299v (ng / mL, n=3)

[0102] Note: Results are mean ± standard deviation. p<0.05, p<0.01, t The differences between the two groups were analyzed.

[0103] Example 6: The effect of Lactobacillus plantarum FN029 in relieving eczema 1. Experimental Methods 1.1 Animal Experiments Thirty 7-week-old female and 15 male BALB / c mice were acclimatized for one week and housed together overnight in a 2:1 female-to-male ratio under clean conditions. The vaginal plugs were checked the following morning; after fertilization, the male mice were removed. The offspring were weaned 21 days postpartum and randomly divided into 4 groups (n=12): 1) CON group (normal control group, administered physiological saline by gavage); 2) AD group (eczema model control group, administered physiological saline by gavage); 3) LGG group (eczema mice were administered Lactobacillus rhamnosus GG suspension by gavage, positive control group); 4) FN029 group (eczema mice were given a suspension of Bacillus plantarum FN029 by gavage).

[0104] Preparation of bacterial suspension: *Lactobacillus plantarum* FN029 was suspended in PBS to obtain a concentration of 1×10⁻⁶. 9 CFU / mL bacterial suspension; LGG was prepared into bacterial suspension of the same concentration using the same method described above.

[0105] The gavage dose was 1×109 CFU / day (100 μL / day) was administered by gavage for 40 consecutive days. Severe eczema was induced on days 31-40. 20 μL of 0.5 nmol calcipotriol (MC903) and 20 μL of 0.2 mg / mL ovalbumin (OVA) were applied to the right ear of offspring mice (AD group, LGG group, FN029 group), while anhydrous ethanol was applied to the left ear (CON group). This treatment was repeated for 10 days, and the offspring mice were euthanized on day 41. During eczema induction, the thickness of the right ear was measured daily using calipers, and the severity of eczema was scored. Offspring mice were euthanized after the experiment. Blood was collected from the eyeballs in anticoagulant tubes. Whole blood was allowed to stand for one hour, then centrifuged at 5000×g for 15 min at 4 ℃, and plasma was collected. The terminal ileum (1 cm) was cut off, and the contents were flushed out with 1 mL PBS and frozen. Ear tissue was collected, cut into two parts, one part was fixed in paraformaldehyde, and the other was frozen. Tissues from the ileum, cecum, colon, Peyer's nodes, and spleen were collected and cryopreserved for subsequent measurements. The experimental procedure is as follows: Figure 5 As shown in Figure A.

[0106] During the eczema modeling process, the severity of eczema was observed and scored as follows: (1) Number of scratches (per minute): 10 points for more than 80 scratches, 7 points for 50-80 scratches, 4 points for 20-50 scratches, 1 point for 10-20 scratches, and 0 points for <10 scratches.

[0107] (2) Ear erythema / bleeding: 10 points for areas covering more than 80% of the total skin area of ​​the ear; 7 points for areas between 50% and 80%; 4 points for areas between 20% and 50%; 1 point for areas less than 20%; 0 points for no erythema / bleeding.

[0108] (3) Ear rash: 10 points are awarded if the rash area is greater than 80% of the total skin area of ​​the ear; 7 points are awarded if the area is between 50% and 80%; 4 points are awarded if the area is between 20% and 50%; 1 point is awarded if the area is less than 20%; 0 points are awarded if there are no rash symptoms.

[0109] (4) Ear edema: Dark red, uneven, and obviously swollen ears get 10 points; light red, uneven, and edematous ears get 7 points; slightly red and uneven ear surface gets 4 points; relatively smooth ear surface and skin color close to normal mouse ears get 1 point; no above symptoms get 0 points.

[0110] (5) Scaly changes on the ear: 10 points for areas covering more than 80% of the total skin area of ​​the ear; 7 points for areas between 50% and 80%; 4 points for areas between 20% and 50%; 1 point for areas less than 20%; 0 points for no scaly changes.

[0111] The total score is expressed as the sum of the five items divided by 5.

[0112] 1.2. Ear histological examination Mouse ears were embedded and fixed in paraffin-embedded paraformaldehyde and sectioned. The sections were then subjected to a series of steps including dehydration, staining, destaining, xylene clearing, and mounting with different concentrations of alcohol to prepare pathological sections. Hematoxylin / eosin (H&E) staining was used to assess inflammatory cell infiltration in the mouse ear tissue; Carbol 2R / hematoxylin and toluidine blue staining were used to assess eosinophil and mast cell infiltration. The number of eosinophils and mast cells in 10 random fields of view of the mouse ear pathological sections was counted under an optical microscope.

[0113] 1.3. Mouse spleen regulatory T cells (CD4) + CD25 + FoxP3 + T) Measurement Mouse spleens were harvested and cut into small pieces using sterile scissors, then placed in PBS. The spleen was ground using a syringe plunger and filtered through a 70 μm cell filter. The filtrate was collected into a 15 mL centrifuge tube. The tube was centrifuged at 300 g for 5 min, and the supernatant was discarded. 2 mL of 1× erythrocyte lysis buffer was added to resuspend the pellet, and lysis was performed at room temperature for 2–3 min. 10 mL of PBS was then added to terminate the lysis reaction. The tube was centrifuged at 300 g for 5 min, and the supernatant was discarded to obtain a white cell pellet. The cells were resuspended in cell staining buffer, and cell counts were performed. The cell concentration was adjusted to 1×10⁻⁶ cells / mL. 7 / mL. Dilute the fixative and membrane-permeable agent specifically for Foxp3 detection; take 100 μL of mouse spleen cells (1×10⁶ cells / mL). 6Place 100 cells in a flow cytometry tube, add 1 part FITC Anti-Mouse CD4 (E-AB-F1097C) and 1 part APC Anti-Mouse CD25 (E-AB-F1102E), mix well, and incubate at 4 °C for 30 min. After incubation, add 1 mL of cell staining buffer, centrifuge at 300 g for 5 min, discard the supernatant, add 100 μL of cell staining buffer, and resuspend the cells. Add 1 mL of fixative, vortex to mix, and react at 4 °C for 30 min. After reaction, centrifuge at 600 g for 5 min, discard the supernatant. Add 2 mL of permeabilizing agent, vortex to mix, centrifuge at 600 g for 5 min, discard the supernatant. Add 100 μL of permeabilizing agent, resuspend the cells, add PE Anti-Mouse Foxp3 (E-AB-F1238D), mix well, and incubate at room temperature for 30 min. Add 2 mL of permeabilizing agent, centrifuge at 600 g for 5 min. After 1 minute, discard the supernatant; add 200 μL of cell staining buffer, resuspend the cells, and they are ready for flow cytometry analysis. Use anti-IgG1 as an isotype control. Measure Tregs using a BD FACSVerse™ flow cytometer (CA, USA), and perform data analysis and processing using FlowJo software version 10.1 (Tree Star, Ashland, OR, USA).

[0114] 1.4 Plasma cytokine assay The levels of plasma IgE, interleukin-4, interleukin-10, interleukin-12 (IL-4, IL-10, IL-12), and interferon-γ (IFN-γ), as well as the level of interleukin-33 (IL-33) in 10% homogenate of mouse ear tissue, were determined using enzyme-linked immunosorbent assay (ELISA). Specific assay methods were performed according to the instructions of the corresponding ELISA kits.

[0115] 1.5 Statistical Analysis Data are expressed as mean ± standard error (SEM) or interquartile range (IQR). SPSS 24 software was used for data analysis. GraphPad Prism 9 was used for visualization of results. One-way ANOVA and Tukey's post-hoc test were applied when the variances were homogeneous; otherwise, the Kruskal-Wallis test was applied. False discovery rate (FDR) corrected p-values ​​were applied where appropriate. P-values ​​were adjusted for multiple comparisons using FDR. A p-value < 0.05 was considered statistically significant. Two-tailed Welch... t Tests and Benjamini Hochberg FDR corrections were used to compare the relative proportions of immune cells between the two groups.

[0116] 2. Research Results 2.1 Effects of FN029 treatment on eczema symptoms in the ears of eczema mice and on eosinophil and mast cell infiltration. Mouse ears were fixed with paraformaldehyde, embedded in paraffin, and pathological sections were prepared. The sections were stained with hematoxylin / eosin (H&E), Carbol 2R / hematoxylin, and toluidine blue. Under a microscope, the dermal thickness in the mouse ear sections was calculated, and mast cells and eosinophils were counted.

[0117] On the fifth day of eczema induction, compared with the normal control group, the relative ear thickness of the AD group, LGG group, and FN029 group was significantly increased, indicating the occurrence of eczema (p<0.05). Figure 5 B). The eczema severity score in the FN029 treatment group (2.4±0.8) was significantly lower than that in the AD group (mean ± SD: 8.1±1.6) (p<0.05), and was approximately 29.6% of that in the AD group at the endpoint. Figure 5 B). The dermal thickness of the right ear in group FN029 (mean ± SD: 0.15 ± 0.04) was significantly reduced by 55.89% compared with group AD (mean ± SD: 0.34 ± 0.15) (p < 0.001). Figure 5 C). In the AD group mice, the epidermis and dermis of the ears were swollen, and inflammatory cell infiltration was severe. The FN029 treatment significantly alleviated these pathological changes. Figure 5 D). Compared with the AD group, the commercial strain LGG did not significantly improve atopic dermatitis (eczema) scores and ear thickness.

[0118] like Figure 6 As shown in Figure A, ear sections from eczema-affected mice showed significant infiltration of mast cells and eosinophils. Treatment with FN029 alone significantly improved the degree of infiltration, while LGG had no significant effect. Figure 6 As shown in B and C, the number of eosinophils (mean ± SD: 421.3 ± 87.6 cells / field) and mast cells (mean ± SD: 60.6 ± 10.9 cells / field) in the ear tissue of AD group mice was significantly increased (p < 0.001). In contrast, the number of eosinophils (mean ± SD: 238.1 ± 56.1 cells / field) and mast cells (mean ± SD: 35.4 ± 7.95 cells / field) in FN029 group was significantly lower than that in AD group (p < 0.001).

[0119] 2.2 Effect of FN029 treatment on the proportion of regulatory T cells in the spleen of eczema mice like Figure 7As shown, the proportion of regulatory T cells in the spleen of mice in the LGG group (6.2±0.6%) was approximately 1.2 times that of the AD group (5.0±0.4%), significantly higher than that in the AD group (p<0.001). There was no significant difference between the LGG and AD groups. Regulatory T cells are effector cells of immune tolerance, indicating that FN029 induces the development of immune tolerance.

[0120] 2.3 Effects of FN029 treatment on plasma cytokines in eczematous mice like Figure 8 As shown, in FN029-treated mice, plasma IL-4 (266.6±12.6 ng / mL) was significantly reduced to 45.7% of that in the AD group (582.0±211.8 ng / mL), plasma total IgE (252.4±23.4 ng / mL) was significantly reduced to 75.4% of that in the eczema group (334.8±31.2 ng / mL), while IL-12 (299.8±26.1 ng / mL) was significantly increased to 1.37 times that in the eczema group (218.6±15.5 ng / mL), IL-10 (340.4±57.9 ng / mL) was significantly increased to 1.71 times that in the eczema group (199.1±9.3 ng / mL), and plasma IL-4 / IFN-γ (0.88±0.12) was reduced to 46% of that in the AD group (1.92±0.46). LGG did not significantly improve IL-4 (347.7±114.1 ng / mL) and IL-4 / IFN-γ (1.46±0.51), and its effect on improving IgE (272.7±15.2 ng / mL) was weaker than that of FN029. Furthermore, FN029 significantly reduced IL-33 in ear homogenate (160.6±22.4 ng / mL) to 51.9% of that in eczema (308.2±54.3 ng / mL). Therefore, FN029 significantly inhibited the Th2-polarized immune response and suppressed IgE-mediated allergic reactions.

[0121] Example 7: The effect of Lactobacillus plantarum FN029 on regulating intestinal flora 1. Experimental Methods 1.1 Animal experiments and microbial sequencing Animal experiments were conducted as described in Example 6, Section 1.1. After euthanizing the animals, DNA was extracted from the cecal contents of mice. Microbial community analysis was performed using paired-end sequencing (v3-v4 region) on a next-generation sequencing platform with 16S rDNA amplicon sequencing. An index adapter was added to the ends of the 16S rDNA PCR products for next-generation sequencing (Illumina, San Diego, CA, USA). A second round of PCR amplicon enrichment was performed using the first-round PCR products as templates (initial denaturation step: 94 °C for 3 min, 24 cycles; denaturation at 94 °C for 5 s, annealing at 57 °C for 90 s, extension at 72 °C for 10 s, and final extension at 72 °C for 5 min). PCR reactions were performed in triplicate using 25 μL mixtures containing 2.5 μL TransStart buffer, 2 μL dNTPs, 1 μL of each primer, and 20 ng template DNA. DNA library concentration was verified using a Qubit 3.0 fluorometer. Following the manufacturer's instructions, the library was quantized to 10 nM, then multiplexed and loaded onto an Illumina MiSeq instrument (Illumina, San Diego, CA, USA). Sequencing was performed using PE250 / 300 paired ends; image analysis and base retrieval were performed using MiSeq control software to obtain a fastq file for quality control.

[0122] 1.2 Data Analysis Amplicon sequence variant (ASV) analysis was performed using EasyAmplicon (v1.10). VSEARCH (v2.15) was used for the analysis. -derep_fullength The command concatenates paired sequences using the - in USEARCH (v10.0). unoise3 The algorithm performs extended sequence denoising to produce ASVs, using - userarch_global The command creates an ASVs table. Species-level annotation of ASVs is performed using the BLCA library (reference database: refseq 16S rRNA database, June 2024 version). α and β diversity statistical analysis is performed using the vegan package in R, and differential species analysis is performed using STAMP software.

[0123] 2. Analysis Results 2.1 The regulatory effect of Lactobacillus plantarum FN029 on the α and β diversity of gut microbiota dysbiosis caused by eczema. like Figure 9As shown, the α-diversity (Shannon index, species diversity index, and evenness) of the gut microbiota in eczema mice was significantly reduced. After FN029 treatment, the Shannon index (2.42±0.83) and evenness (0.43±0.11) were significantly increased, reaching 139% and 131% of those in the eczema model group (Shannon index 1.74±1.12; evenness 0.22±0.02), respectively. Principal coordinate analysis (PCoA) using Bray-Curtis distance showed that the gut microbiota of eczema mice deviated significantly from that of healthy mice, with the ordinate explaining 19.53%. Permutation multivariate analysis of variance (PERMANOVA) showed that both FN029 and LGG treatments significantly altered the microbiota diversity (p<0.05), with no significant difference compared to the healthy control group. Nonmetric multidimensional analysis (NDMS) showed that the microbial diversity of the FN029 treatment group was significantly different from that of the AD group (p<0.05), while the LGG treatment group was significantly different from both the AD group and the healthy control group (p<0.05). 2.2. The relative content of bacterial species that significantly altered the intestinal flora in patients with eczema dysbiosis regulated by *Lactobacillus plantarum* FN029. like Figure 10 As shown, FN029 treatment significantly modulated the relative abundance of various bacteria, including *Azolotria edulis* (…). Kineothrix alysoides (Kineothrix alysoides, gen. nov., sp. nov., a saccharolytic butyrate-producer within the family) is the most dominant butyrate-producing bacterium in the gut. Lachnospiraceae Int. J. Syst. Evol. Microbiol. 67, 402–410), treatment with FN029 (14.8 ± 1.2%) increased the relative abundance of *Akkermansia myxophila* to 1.7 times that of the AD group (8.4 ± 2.38%). Akkermansiamuciniphil a) is a Gram-negative bacterium that can cause infantile eczema (Imbalance of Gut). Streptococcus , Clostridium , and Akkermansia Determines The Natural Course of Atopic Dermatitis in Infant. Allergy, asthma & immunology research, 2019, 12(2): 322-337, AD group (2.93 ± 0.82%) compared with CON group (0.02 ± 0.02%) A. muciniphia The abundance increased significantly by 144 times in the FN029 treatment group. A. muciniphiaThe abundance (0.34±0.02%) decreased to 11.82% in the AD group (p<0.05). Alternariae Alistipes dispar It is a Gram-negative bacterium belonging to the Bacteroidetes phylum that produces indole, succinic acid, and small amounts of acetic acid and propionic acid. AD group mice... A. dispar The abundance of the fungus in the group AD was significantly reduced (0.2±0.02%), representing 1% of the abundance in the CON group (2.0±0.98%) (p<0.05). After treatment with FN029, its abundance increased to 450 times that of the AD group (0.93±0.13%). This fungus, with an unclear relationship to eczema, is a bacterial species. Fusimonas intestine The abundance of FN029 decreased from 1.8±1.1% in the AD group to 31.6% in the CON group (3.8±1.25%) (p<0.05), while the abundance after FN029 treatment increased by 134.18% in the AD group (5.12±1.12%). Sangeribacter mnris This fungus, whose association with eczema is unclear, showed a slight increase in abundance in the AD group (6.9±1.8%) to 115% of that in the CON group (6.0±1.89%), while its abundance after FN029 treatment (0.73±0.28%) decreased to 1 / 11 of that in eczema (p<0.05). Except... Sangeribacter muris In addition, LGG treatment did not significantly regulate the abundance of other bacterial species.

[0124] Example 8: The effect of Lactobacillus plantarum FN029 on promoting digestive function 1. Research Methods 1.1 Effects of probiotic treatment on intestinal motility in mice Eight-week-old male BALB / c mice were selected. The control group was divided into a blank control group and a model control group. The model control group was induced using compound diphenoxylate. The experimental groups were orally administered *Lactobacillus plantarum* FN029 or 299v (1×10⁻⁶). 9 The test was administered for one week using CFU / day to the FN029 treatment group and the 299v treatment group. Before the end of the experiment, animals were fasted for 20 hours but allowed free water. On the day of measurement, each experimental group, as well as the blank and model control groups, were given the test sample or distilled water again. 30 minutes later, each experimental group and the model control group were given compound diphenoxylate 5-6 mg / kgbw, while the blank control group was given distilled water. 30 minutes later, each group was given an indicator (accurately weigh 100 g of gum arabic, add 800 mL of water, boil until the solution is clear, weigh 100 g of activated carbon powder and add to the above solution, boil three times, and after the solution cools, dilute to 1000 mL to obtain the indicator, store in a refrigerator at 4 ℃, and shake well before use). 25 minutes later, the animals were euthanized by cervical dislocation, and the ink propulsion rate was calculated.

[0125] Calculate the ink propulsion rate using the following formula: Ink propulsion rate (%) = Ink propulsion length (cm) / Total length of small intestine (cm) × 100%.

[0126] 1.2 Determination of digestive enzymes Six-week-old BALB / c mice were selected. They were divided into a blank control group, a *Lactobacillus plantarum* FN029 treatment group, and a *Lactobacillus plantarum* 299v treatment group, which were orally administered sterile water, FN029, or 299v bacterial suspension (1×10⁻⁶). 9 CFU / day for 6 consecutive weeks. Mice were fasted for 24 hours but allowed free access to water. All mice were anesthetized with ether and then necropsed for sampling. Samples were collected from gastric juice, duodenal juice, jejunum, ileum, liver, kidneys, spleen, and pancreas. The activities of pepsin in gastric juice and trypsin and amylase in duodenal juice were measured using kits produced by Nanjing Jiancheng Bioengineering Institute, following the kit instructions.

[0127] 1.3 Statistical Analysis Data are expressed as mean ± standard error (SEM) or interquartile range (IQR). SPSS 24 software was used for data analysis. GraphPad Prism 9 was used for visualization of the results. One-way ANOVA and Tukey's post-hoc test were applied when the data variances were homogeneous; otherwise, the Kruskal-Wallis test was applied.

[0128] 2. Results 2.1 Effects of Lactobacillus plantarum FN029 on intestinal motility in mice The effects of each group on intestinal motility in mice are shown in the figure. Figure 11 In the model control group, intestinal motility was artificially slowed down by compound diphenoxylate. Gavage administration of FN029 (72.0 ± 0.9%) and 299v (67.1 ± 1.2%) significantly increased the ink propulsion rate in the mouse intestine (p<0.001). Compared with the model control group (50 ± 1.7%), FN029 and 299v increased the ink propulsion rate by 44% and 32.67%, respectively. Compound diphenoxylate can directly inhibit the intestinal mucosal receptors of smooth muscle, eliminate the peristaltic reflex of the local mucosa, and thus weaken peristalsis, thereby prolonging the contact between intestinal contents and intestinal mucosa, promoting intestinal water absorption, and drying out the stool. It is often used to establish a mouse model of constipation. The results of this study indicate that both FN029 and 299v can significantly improve intestinal motility in mice and promote intestinal peristalsis to counteract drug-induced slowing of intestinal motility, with FN029 showing a superior effect compared to 299v.

[0129] 2.2 Effects of *Lactobacillus plantarum* FN029 on mouse body weight and digestive enzyme activity The effects of each group on mouse body weight are shown in the figure. Figure 12Under the premise that there was no significant difference in initial body weight, after 5 weeks of probiotic gavage treatment, the average final body weight of mice gavaged with FN029 (23.96 g) and 299v (23.44 g) was significantly higher than that of the control group (22.35 g, p<0.05), and the average body weight gain rate of mice in the FN029 group (21.11%) and 299v group (18.52%) was significantly higher than that of the control group (11.17%, p<0.05) (Table 8).

[0130] Table 8. Effects of *Lactobacillus plantarum* on mouse body weight.

[0131] like Figure 13 As shown, FN029 treatment significantly increased trypsin activity (821.13±2.59 U / mL), which was 128% of the enzyme activity of the control group (638.88±11.44 U / mL) (p<0.05), and was also significantly higher than the enzyme activity of 299v (811.5±10.21 U / mL, p<0.05).

[0132] In summary, *Lactobacillus plantarum* FN029 can promote intestinal motility, increase mouse body weight, and enhance trypsin activity, thus possessing the function of promoting intestinal digestion, and its effect is superior to that of *Lactobacillus plantarum* 299v.

[0133] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of *Lactobacillus plantarum* ( Lactiplantibacillus plantarum FN029, characterized in that, The *Lactobacillus plantarum* was deposited at the Guangdong Microbial Culture Collection Center on December 20, 2022, with accession number GDMCC No: 63060.

2. A microbial inoculant, characterized in that, Contains *Lactobacillus plantarum* FN029 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The bacterial agent uses GDMCC No: 63060 as the main microorganism; The bacterial agent contains live cells of the GDMCC No: 63060 strain of claim 1, freeze-dried dried cells of the GDMCC No: 63060 strain, immobilized cells of the GDMCC No: 63060 strain, liquid bacterial agent of the GDMCC No: 63060 strain, solid bacterial agent of the GDMCC No: 63060 strain, or the GDMCC No: 63060 strain in any other form.

4. The microbial agent according to claim 2, characterized in that, The microbial agent is a microbial preparation that is resistant to adverse digestive tract environments, inhibits intestinal pathogens, is sensitive to antibiotics, produces indole derivatives, improves allergic diseases, and aids digestion.

5. A product characterized in that, The product contains *Lactobacillus plantarum* GDMCC No: 63060 as described in claim 1 or a bacterial agent containing *Lactobacillus plantarum* GDMCC No: 63060 as described in claim 1; the product is food, medicine, or health product.

6. The product according to claim 5, characterized in that, The food is a solid food, liquid food, or semi-solid food; the medicine contains a pharmaceutically acceptable carrier or excipient.

7. The product according to claim 5, characterized in that, The product contains at least 1×10⁻⁶ Lactobacillus plantarum GDMCC No.: 63060 cells. 6 CFU / mL or 1×10 6 CFU / g.

8. The use of *Lactobacillus plantarum* GDMCC No: 63060 of claim 1 or a bacterial agent containing *Lactobacillus plantarum* GDMCC No: 63060 of claim 1 in the preparation of products containing indole derivative active ingredients, wherein, The active ingredient containing indole derivatives is one or more of the following: indole-3-acetamide, indole-3-lactic acid, indole-3-carboxylic acid, indole-3-formaldehyde, indole-3-acetic acid, 3-indole acrylic acid, 3-indole propionic acid, and 3-indole acetonitrile.

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