Application of breast milk-derived plant lactobacillus to improvement of allergic diseases and regulation of intestinal flora

Abstract

The invention discloses an application of a breast milk-derived plant lactobacillus in improving allergic diseases and regulating intestinal flora, and belongs to the technical field of microorganisms. The plant lactobacillus GDMCC No: 63060 disclosed by the invention is obtained by being separated from breast milk. The strain provided by the invention has excellent gastrointestinal fluid tolerance, mucin adhesion and antibiotic sensitivity, can inhibit various pathogenic bacteria, can generate indole derivatives, effectively inhibits eczema mouse dermatitis symptoms, inhibits Th2 type immunoreaction hyperpolarization, and induces regulatory T cells. Therefore, the lactobacillus plantarum FN029 has a huge application prospect in preparation of products for inhibiting enteropathogenic bacteria, improving allergic diseases, regulating intestinal flora or promoting digestion function.

Description

Technical Field

[0001] The invention relates to application of a breast milk-derived Lactobacillus plantarum strain in improving allergic diseases and regulating intestinal flora, and belongs to the technical field of microorganisms. Background Art

[0002] In recent years, allergic diseases such as eczema, asthma, and food allergies have been increasing, especially in industrialized countries, and are considered by the World Allergy Organization (WAO) as a "public health issue of global concern." These allergic diseases not only seriously affect the quality of life of patients, but also bring huge medical burdens to society. It is estimated that by 2050, the global prevalence of allergic diseases will reach 4 billion, but there are currently no interventions to stop this trend. The occurrence of allergic diseases is related to the changes in lifestyle brought about by industrialization, such as improved sanitary conditions, excessive cleaning, abuse of antibiotics, and westernization of diet. More and more studies have shown that changes in intestinal diversity are the main cause of allergic processes, especially intestinal microecological disorders in early life can predict the occurrence and persistence of allergic diseases. Therefore, it is particularly important to develop a probiotic that can effectively regulate the immune system, relieve allergic symptoms, improve allergic diseases, and regulate intestinal flora.

[0003] It is well known that breastfeeding can provide short-term and long-term benefits for children's development, so the World Health Organization has proposed a strategy of exclusive breastfeeding for the first 6 months. Recent studies have shown that breast milk contains a series of bacteria, mainly bifidobacteria, streptococci, lactic acid bacteria and staphylococci, making it an ideal inoculum source for symbiotic bacteria in the intestinal tract of newborns and an important factor affecting the succession of intestinal flora. By breastfeeding infants, specific species of breast milk flora colonize in the infant's intestine through vertical transmission, which is crucial for promoting immune development and preventing allergic diseases. Studies have found that the proportion of mother-to-infant vertical transmission bacteria in breast milk flora is less than 0.0001% (Widespread vertical transmission of secretoryimmunoglobulin A coated trace bacterial variants from the mother to infantgut through breastfeeding. Food & Function. 2022Nov; 13(22): 11543-11554.), and traditional microbial isolation technology can only randomly isolate dominant species of breast milk, and it is difficult to effectively isolate such active vertically transmitted beneficial bacteria. The use of secretory immunoglobulin A (sIgA) magnetic bead enrichment technology combined with selective culture technology of the intended isolated bacterial species can accelerate the discovery of anti-allergic probiotics in breast milk.

[0004] In order to ensure that probiotics can survive and function in the human body, the strains must have good tolerance to gastrointestinal fluid. Due to the increasing bacterial resistance, it is also important to develop probiotics with antibiotic sensitivity. Single-functional strains may not be able to meet the needs of improving allergic diseases and inhibiting intestinal pathogens at the same time, or they may have insufficient tolerance in gastrointestinal fluid and cannot adhere well to the intestinal mucus layer. Therefore, the development of a multifunctional strain that integrates multiple functions can provide strong support for the prevention and treatment of allergic diseases, inhibition of intestinal pathogens, regulation of intestinal flora, improvement of antibiotic sensitivity, and development of new drugs in practical applications. Summary of the invention

[0005] The first object of the present invention is to provide a strain of Lactobacillus plantarum FN029, which is taxonomically named Lactiplantibacillus plantarum. The plant lactobacillus is deposited in the Guangdong Microbiological Culture Collection Center with a deposit number of GDMCC No: 63060 and a deposit date of December 20, 2022.

[0006] In one embodiment of the present invention, the Lactobacillus plantarum is derived from breast milk, sIgA immunomagnetic beads are used to enrich the bacteria in Tongwei breast milk samples, and a strain of Lactobacillus plantarum derived from breast milk is screened using Lactobacillus plantarum selective culture medium, and is named Lactiplantibacillus plantarum FN029.

[0007] In one embodiment of the present invention, the Lactobacillus plantarum GDMCC No: 63060 has the following characteristics:

[0008] (1) Morphological characteristics: Gram-positive bacteria, rod-shaped, non-spore-forming, cell width of about 0.60-0.65 μm, length of about 1.0-1.3 μm;

[0009] (2) Colony characteristics: After 48 h of culture in MRS medium, the colony has a diameter between 0.3 and 2 mm, a round shape from the front, a convex shape from the side, neat edges, milky white, opaque, and a moist and smooth surface;

[0010] (3) Growth characteristics: When cultured in MRS, it grows facultatively anaerobically, grows best at 30-37°C, the highest and lowest initial growth pH are 8.0 and 5.0, and the optimal initial growth pH is 6.8;

[0011] (4) It has excellent gastrointestinal fluid tolerance and mucin adhesion, can tolerate adverse digestive tract environments, and adheres well to the intestinal mucus layer;

[0012] (5) It can inhibit a variety of pathogenic bacteria, including but not limited to Clostridium perfringens, Escherichia coli, Clostridium difficile, and Staphylococcus aureus;

[0013] (6) sensitivity to antibiotics, including but not limited to vancomycin, chloramphenicol, penicillin, streptomycin, gentamicin, kanamycin, erythromycin, trimethoprim, and ampicillin;

[0014] (7) It can produce indole derivatives, with extremely high yields of indole-3-lactic acid and indole-3-acetic acid;

[0015] (8) Improve allergic diseases, relieve eczema, and regulate immunity;

[0016] (9) Regulate intestinal flora and restore intestinal flora disorders caused by allergic diseases;

[0017] (10) Promote digestion, improve host intestinal motility, and increase trypsin activity.

[0018] In one embodiment of the present invention, the allergic disease includes but is not limited to eczema, food allergy, allergic rhinitis, and asthma.

[0019] In one embodiment of the present invention, the relief of eczema includes but is not limited to:

[0020] (1) Reduced pathological changes such as epidermal and dermal swelling and severe inflammatory cell infiltration in the ears of mice with eczema;

[0021] (2) Reduce the number of eosinophils and mast cells in mice with eczema and alleviate allergic symptoms;

[0022] (3) Increase the proportion of regulatory T cells in the spleen of mice with eczema and inhibit inflammatory response;

[0023] (4) Reduce the levels of plasma cytokines IgE, IL-4, and plasma IL-4 / IFN-γ in eczema mice, increase the levels of IL-12 and IL-10, reduce the level of IL-33 in tissue homogenates, correct the Th1 / Th2 imbalance, enhance the cellular immune response, and inhibit excessive inflammatory response.

[0024] In one embodiment of the present invention, the restoration of intestinal flora disorder caused by allergic diseases includes increasing the Shannon index and uniformity caused by eczema, upregulating the abundance of Kineothrix alysoides and Alistipes dispar, and reducing the abundance of Akkermansia muciniphila.

[0025] The second object of the present invention is to provide a microbial agent containing the plant lactobacillus GDMCC No: 63060 of the present invention.

[0026] In one embodiment of the present invention, the microbial agent uses Lactobacillus plantarum GDMCC No: 63060 as a main microorganism.

[0027] In one embodiment of the present invention, the number of cells of GDMCC No: 63060 in the bacterial agent is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0028] In one embodiment of the present invention, the bacterial agent is prepared by preparing the seed solution of GDMCC No: 63060 and then expanding the culture.

[0029] In one embodiment of the present invention, the microbial agent contains living cells of the GDMCC No: 63060 strain of the present invention, dry cells of the GDMCC No: 63060 strain of the present invention obtained by freeze drying, immobilized cells of the GDMCC No: 63060 strain of the present invention, a liquid agent of the GDMCC No: 63060 strain of the present invention, a solid agent of the GDMCC No: 63060 strain of the present invention, or the GDMCC No: 63060 strain of the present invention in any other form.

[0030] In one embodiment of the present invention, the bacterial agent is a microbial preparation that tolerates adverse digestive tract environments, inhibits intestinal pathogens, has antibiotic sensitivity, produces indole derivatives, improves allergic diseases, regulates intestinal flora, and aids digestion.

[0031] 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.

[0032] In one embodiment of the present invention, the antibiotic is one or more of vancomycin, chloramphenicol, penicillin, streptomycin, gentamicin, kanamycin, erythromycin, trimethoprim, and ampicillin.

[0033] 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.

[0034] In one embodiment of the present invention, the allergic disease includes but is not limited to eczema, food allergy, allergic rhinitis, and asthma.

[0035] The third object of the present invention is to provide a product, which contains the Lactobacillus plantarum GDMCC No: 63060 or a bacterial agent containing the Lactobacillus plantarum GDMCC No: 63060.

[0036] In one embodiment of the present invention, the product is food, medicine or health care product.

[0037] In one embodiment of the present invention, the food is solid food, liquid food or semi-solid food.

[0038] In one embodiment of the present invention, the drug further contains a pharmaceutically acceptable excipient.

[0039] In one embodiment of the present invention, the auxiliary materials include any one of carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, antibacterial agents or buffers, or a combination of at least two of them.

[0040] In one embodiment of the present invention, the carrier includes but is not limited to liposomes and nanoparticles.

[0041] In one embodiment of the present invention, the excipient is one or more of mannitol, lactose, fatty acid, and polyethylene glycol.

[0042] In one embodiment of the present invention, the dosage form of the drug includes any one of suspension, granules, capsules, powders, tablets, emulsions, solutions, pills, injections, suppositories, enemas, aerosols, patches or drops.

[0043] In one embodiment of the present invention, the administration route of the drug includes oral administration, sublingual administration, skin mucosal administration, inhalation administration or injection administration.

[0044] In one embodiment of the present invention, the product has one or more functions of tolerating adverse digestive tract environment, inhibiting intestinal pathogens, having antibiotic sensitivity, producing indole derivatives, improving allergic diseases, regulating intestinal flora, and aiding digestion.

[0045] In one embodiment of the present invention, the number of cells of Lactobacillus plantarum GDMCC No: 63060 in the product is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0046] The fourth object of the present invention is to provide the use of the Lactobacillus plantarum GDMCC No: 63060 or a bacterial agent containing the Lactobacillus plantarum GDMCC No: 63060 in at least one of the following (1) to (6):

[0047] (1) Preparation of products that inhibit intestinal pathogens;

[0048] (2) preparing products containing indole derivatives as active ingredients;

[0049] (3) preparing products for preventing and / or treating eczema;

[0050] (4) Preparation of products for improving allergic diseases;

[0051] (5) Preparation of products for regulating intestinal flora;

[0052] (6) Prepare products with digestion-promoting function.

[0053] In one embodiment of the present invention, the product includes but is not limited to medicines, foods, functional foods, special medical purpose formula foods or health products.

[0054] In one embodiment of the present invention, the number of cells of Lactobacillus plantarum GDMCC No: 63060 in the product is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0055] 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.

[0056] In one embodiment of the present invention, the antibiotic is one or more of vancomycin, chloramphenicol, penicillin, streptomycin, gentamicin, kanamycin, erythromycin, trimethoprim, and ampicillin.

[0057] 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.

[0058] In one embodiment of the present invention, the prevention and / or treatment of eczema includes but is not limited to alleviating allergic symptoms, inhibiting inflammatory responses, and correcting Th1 / Th2 imbalance.

[0059] In one embodiment of the present invention, the allergic disease includes but is not limited to eczema, food allergy, allergic rhinitis, and asthma.

[0060] In one embodiment of the present invention, the regulation of intestinal flora includes but is not limited to restoring intestinal flora disorders caused by allergic diseases; optionally, it includes increasing the Shannon index and uniformity caused by eczema, upregulating the abundance of Kineothrix alysoides and Alistipes dispar, and reducing the abundance of Akkermansia muciniphila.

[0061] In one embodiment of the present invention, the digestion promotion includes but is not limited to improving the intestinal motility of the host and improving the activity of trypsin.

[0062] Deposit of biological materials:

[0063] Lactobacillus plantarum FN029, taxonomically named Lactiplantibacillus plantarum, was deposited in the Guangdong Microbiological Culture Collection Center on December 20, 2022, with the deposit number GDMCC No: 63060, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province.

[0064] Beneficial effects:

[0065] The plant lactobacillus FN029 isolated by the present invention is a multifunctional lactic acid bacterium with excellent gastrointestinal fluid tolerance and mucin adhesion, can tolerate adverse digestive tract environment, and adheres well to the intestinal mucus layer; the plant lactobacillus FN029 of the present invention can inhibit harmful pathogens in the intestine, including but not limited to Clostridium perfringens, Escherichia coli, Clostridium difficile, and Staphylococcus aureus, and has high sensitivity to antibiotics and good probiotic properties; in addition, the plant lactobacillus FN029 of the present invention can also produce indole derivatives, and the production of indole-3-lactic acid and indole-3-acetic acid is extremely high; the plant lactobacillus FN029 of the present invention also has good ability to improve allergic diseases and regulate immunity, can relieve eczema, reduce allergic symptoms, inhibit inflammatory response, and correct Th1 / Th2 imbalance; the plant lactobacillus FN029 of the present invention can restore the intestinal flora disorder caused by allergic diseases and regulate the intestinal flora; and can improve the host intestinal motility, improve the trypsin activity, and promote digestion.

[0066] In summary, the Lactobacillus plantarum FN029 of the present invention is a multifunctional strain with broad application prospects, which combines the functions of tolerating adverse digestive tract environment, inhibiting intestinal pathogens, being sensitive to antibiotics, producing indole derivatives, improving allergic diseases, regulating intestinal flora, and aiding digestion. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 Graph showing the corrected vertical transmission rate from breast milk to infant intestinal bacteria for samples from the population from which Lactobacillus plantarum FN029 was isolated.

[0068] Figure 2 The morphology of Lactobacillus plantarum FN029. (A) The bacterial liquid state of Lactobacillus plantarum FN029 after 24 hours of growth in MRS broth; (B) The colony of FN029 cultured on MRS agar for 48 hours; (C) The Gram staining morphology of FN029 (magnified 200 times); (D) The scanning electron microscope observation image of FN029.

[0069] Figure 3 This is the artificial gastric juice tolerance graph of Lactobacillus plantarum FN029. *p<0.05, **p<0.01, compared with 0h; ##p<0.01, comparison between end point 299v and FN029. 299v, Lactobacillus plantarum 299v; FN029, Lactobacillus plantarum FN029.

[0070] Figure 4 This is the artificial intestinal fluid tolerance graph of Lactobacillus plantarum FN029. **p<0.01, compared with 0h; #p<0.05, ##p<0.01, ###p<0.001, comparison between 299v and FN029 at different time points.

[0071] Figure 5 The effect of Lactobacillus plantarum FN029 on relieving eczema in mice. (A) Animal handling and eczema induction; (B) Eczema severity score, including the number of ear scratching, erythema / hemorrhage, rash, edema and scaly changes, with a score range of 0-10, and the higher the score, the more severe the eczema symptoms (n=10); (C) Increased ear thickness at the end of eczema induction relative to the ear thickness on day 0 (n=10); (D) Typical photos of mouse ear lesions. ##p<0.01, ###p<0.001, compared with the AD group. CON, healthy control; AD, eczema model control; 299v, Lactobacillus plantarum 299v treatment; FN029, Lactobacillus plantarum FN029 treatment; MC903+OVA, calcipotriol (MC903) and ovalbumin (OVA) were applied to the right ears of mice to induce eczema.

[0072] Figure 6 This is a graph showing the effect of Lactobacillus plantarum FN029 in improving neutrophil and mast cell infiltration in the ears of mice with eczema.

[0073] (A) Typical images of mouse ear slices; (B) Relative content of hypertrophic bacteria in mouse ear tissue slices; (C) Relative content of eosinophils in mouse ear tissue slices. **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; 299v, Lactobacillus plantarum 299v treatment; FN029, Lactobacillus plantarum FN029 treatment.

[0074] Figure 7 The effect of Lactobacillus plantarum FN029 on improving the spleen regulatory T cells in eczema model mice. (A) Flow cytometry analysis; (B) Relative content of spleen 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; 299v, Lactobacillus plantarum 299v treatment; FN029, Lactobacillus plantarum FN029 treatment.

[0075] Figure 8 This is the effect of Lactobacillus plantarum FN029 on improving plasma cytokines in eczema model mice. *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; 299v, Lactobacillus plantarum 299v treatment; FN029, Lactobacillus plantarum FN029 treatment.

[0076] Fig. 9 The effect diagram of the regulation of α and β diversity of intestinal flora caused by eczema by Lactobacillus plantarum FN029. (A) α diversity: Shannon, Shannon index; Phylogenetic diversity, species diversity; Evenness, uniformity; (B) β diversity: principal coordinate analysis (PCoA, left) of Bray-Curtis distance, non-metric multidimensional measurement 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; 299v, Lactobacillus plantarum 299v treatment; FN029, Lactobacillus plantarum FN029 treatment.

[0077] Fig.10 Figure 2 shows the relative content of bacteria species that were significantly altered in the intestinal flora of eczema dysbiosis regulated by Lactobacillus plantarum FN029 (%). *p<0.05, compared with the CON group; #p<0.05, compared with the AD group. CON, healthy control; AD, eczema model control; 299v, Lactobacillus plantarum 299v treatment; FN029, Lactobacillus plantarum FN029 treatment.

[0078] Fig.11 The effect diagram of Lactobacillus plantarum FN029 and 299v on the intestinal motility of mice. ***p<0.001, compared with the blank group; ###p<0.001, compared with the model group. Blank group, healthy control; model group, compound diphenoxylate modeling control; 299v, Lactobacillus plantarum 299v treatment; FN029, Lactobacillus plantarum FN029 treatment.

[0079] Fig.12 The effect of Lactobacillus plantarum FN029 and 299v on the body weight of mice. **p<0.01, compared with the blank group; #p<0.05, compared with 299v. Control group, healthy control; 299v, Lactobacillus plantarum 299v treatment; FN029, Lactobacillus plantarum FN029 treatment.

[0080] Fig.13 The effect diagram of Lactobacillus plantarum FN029 and 299v on the digestive enzyme activity of mouse intestine. **p<0.01, compared with the blank group; #p<0.05, compared with 299v. Control group, healthy control; 299v, Lactobacillus plantarum 299v treatment; FN029, Lactobacillus plantarum FN029 treatment. DETAILED DESCRIPTION

[0081] The present invention will be further described below in conjunction with the accompanying drawings and specific examples. These examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions in the art. Unless otherwise defined, all professional and scientific terms used in the text have the same meanings as those familiar to those skilled in the art.

[0082] Unless otherwise specified, the experimental materials and drugs used in the embodiments of the present invention can be obtained through conventional purchase.

[0083] The experimental materials involved in the following embodiments are:

[0084] Lactobacillus plantarum selective medium (1L): peptone (10g), beef extract (10g), yeast extract (5g), d-sorbitol (20g), ciprofloxacin (4mg), sodium acetate (5g), ammonium citrate (2g), potassium phosphate (2g), magnesium sulfate (0.1g), manganese sulfate (0.05g), bromocresol purple (0.02g) and agar (15g). The medium without ciprofloxacin was autoclaved at 121°C for 1min and cooled at 50°C. Ciprofloxacin was sterilized by filtration and added to the medium that had not solidified before cooling. The pH value of the medium was 6.0±0.1. After solidification, the medium was purple.

[0085] MRS broth medium (1L): 10g soy peptone, 5g beef extract, 5g yeast powder, 20g glucose, 1mL Tween-80, 2g sodium dihydrogen phosphate, 5g anhydrous sodium acetate, 2g triammine citrate, 0.02g manganese sulfate, 0.1g magnesium sulfate, 1L distilled water, adjust the pH to about 6.8, and sterilize at 121℃ for 15min.

[0086] MRS solid culture medium (MRS agar plate) (1L): soy peptone 10g, beef extract 5g, yeast powder 5g, glucose 20g, Tween-80 1mL, sodium dihydrogen phosphate 2g, anhydrous sodium acetate 5g, triammine citrate 2g, manganese sulfate 0.02g, magnesium sulfate 0.1g, distilled water 1L, adjust pH to about 6.8, agar 15g, sterilize at 121℃ for 15min.

[0087] Lactobacillus rhamnosus GG (deposit number ATCC 53013) and Clostridium difficile (deposit number ATCC 9689) were obtained from the American Type Culture Collection; Lactobacillus plantarum 299v (deposit number DSM9843) was obtained from the German Type Culture Collection; Staphylococcus aureus (deposit number ATCC 25923) and Escherichia coli (deposit number ATCC 8739) were obtained from the American Type Culture Collection; Clostridium perfringens (deposit number CMCC 64722) was obtained from the National Type Culture Collection.

[0088] Example 1: Isolation and identification of Lactobacillus plantarum FN029

[0089] 1. Analysis of vertically transmitted bacterial species in breast milk and paired infant feces

[0090] Thirty-five mother-infant pairs were recruited from Tongwei County and 28 mother-infant pairs were recruited from Wuxi City. Inclusion criteria were: (1) full-term vaginal delivery after 37 weeks of gestation; (2) exclusive breastfeeding from birth to the time of sample collection; and (3) no exposure to antibiotics or probiotics antenatally and during breastfeeding. Demographic and clinical data were collected, including maternal age and weight, mode of delivery, neonatal weight, lactation stage, and duration of pregnancy. All mothers had a gestation period of more than 38 weeks.

[0091] On postpartum day 42, breast milk and infant stool samples were collected. Before collection, hands were washed with soap and nipples were cleaned with chlorhexidine swabs, and then milk was expressed from breasts that had not been sucked before. Breast milk was systematically collected between 9:00 and 10:00 a.m. every morning using a sterile automatic breast pump equipped with a vacuum regulator. All collection bottles and suction funnels were sterilized by autoclave before use, and breast milk was quickly divided into equal portions under sterile conditions.

[0092] DNA was extracted from 1 mL of breast milk and 200 mg of fecal material using the TGuide S96 Magnetic Soil / Feces DNA Kit (Tiangen Biotechnology Co., Ltd., Beijing, China). Libraries were constructed according to the Illumina 16S metagenomic sequencing process, targeting the V3-V4 variable region of the 16S rRNA gene in the DNA sample. After the primary amplification, secondary PCR was performed using i5 forward and i7 reverse primers to integrate the Illumina Nextera barcode. The product was purified using the QIAquick PCR Purification Kit (Qiagen). The size and quality of the purified product were evaluated using the Bioanalyzer 2100 (Agilent). The purified amplicons were combined and sequenced using the Illumina MiSeq Sequencing System (Illumina). Amplicon sequence variants (ASVs) were extracted using the EasyAmplicon software. The ASVs were then annotated at the species level using the BLCA algorithm.

[0093] Univariate Fisher's exact test was used to compare the presence of ASV in breast milk and infant intestinal samples. The odds ratio (OR) of species transmission was calculated, and a one-sided p value was used to test the null hypothesis of OR = 1. The weighted transfer rate (WTR) was calculated using the R package randpermutationTransferStats.R, and a WTR greater than 1 indicated transmission.

[0094] like Figure 1 As shown, after analyzing the transmission rate of bacterial species from breast milk to the infant's intestine and correcting for potential biases, it was found that Plantarum was the main unique bacterial species vertically transmitted in Tongwei samples.

[0095] 2. Isolation and purification of strains

[0096] Take 1mL of breast milk and centrifuge it at 15000g for 5min, discard the supernatant, suspend the bacteria at 10% in peptone buffer, add 0.5% bovine serum albumin, add biotin-labeled rabbit anti-human immunoglobulin A serum, incubate for 15-30min, then add streptavidin-modified magnetic beads (0.5mg / mL), adsorb the bacteria with a magnet, wash twice with peptone buffer, and obtain sIgA-binding bacteria. Then take 1mL of the enrichment solution in the clean bench, dilute it tenfold with sterile saline, and select 10 -6 , 10 -7 , 10 -8Three dilution gradients, 100 μL of bacterial solution from each gradient was applied to the selective medium of Lactobacillus plantarum, and cultured anaerobically at 30°C for 48 hours. After the culture, a plate with 50-150 single colonies was selected from the culture medium, and colonies with different shapes, sizes and colors were picked, and they were streaked and purified on the MRS agar plate several times until the colony morphology on the entire plate was consistent, and a single colony was picked to be cultured in the MRS broth medium. The obtained strain was suspended in PBS containing 20% ​​glycerol and stored at -80°C.

[0097] Among them, yellow colonies with a diameter greater than 2 mm and surrounded by a yellow transparent circle were selected and transferred to MRS medium for 48 hours. The diameter was between 0.3-2 mm, the front shape was round, the side shape was convex, the edges were neat, milky white, opaque, and the surface was moist and smooth ( Figure 2 B). The strain grows facultatively anaerobically when cultured in MRS medium, grows best at 30-37°C, the highest and lowest initial growth pH are 8.0 and 5.0, the optimal initial growth pH is 6.8, and the culture state is turbid suspension ( Figure 2 A). The Gram stain of the colony smear is positive, the morphology is rod-shaped, no spore formation, the bacteria are about 0.60-0.65μm wide and 1.0-1.3μm 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).

[0098] 3. Lactobacillus specific primer amplification and 16S rDNA identification

[0099] The target strain genomic DNA was extracted using the Ezup column bacterial genomic DNA extraction kit, and the extracted lactic acid bacteria genomic DNA was used as a template for PCR amplification, and the PCR experiment was performed using lactobacillus-specific primers. Subsequently, the bacterial universal primers 27F and 1492R were used to perform a PCR experiment on 16S rDNA. After the PCR reaction amplification was completed, the PCR product was taken for agarose gel detection and photography. The length of the amplified fragment was about 1.2 kbp, and the PCR product was sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing.

[0100] The 16S rDNA sequencing result is shown in SEQ ID NO.1. EZCLOUD sequence alignment was performed on the NCBI website, and the results showed that the sequence had more than 99% homology with the 16S rDNA sequence of Lactiplantibacillus plantarum. The sequence alignment results of the strains were combined with the physiological and biochemical results to determine that the screened lactic acid bacteria was Lactiplantibacillus plantarum, named Lactiplantibacillus plantarum FN029, which was deposited in the Guangdong Microbiological Culture Collection Center with a deposit number of GDMCC No: 63060 and a deposit date of December 20, 2022.

[0101] SEQ ID NO.1:

[0102] TCGGGGGGGTGCTAATACATGCAAGTCGAACGAACTCTGGTATTGATTGGTGCTTGCATCA

[0103] TGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAA

[0104] GCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGT

[0105] CCGAGTTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAG

[0106] ATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGG

[0107] CCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTT

[0108] CCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCT

[0109] CGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGT

[0110] ATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGC

[0111] AAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGT

[0112] GAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAA

[0113] GAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAG

[0114] TGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCA

[0115] AACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGG

[0116] GTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCC

[0117] GCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGT

[0118] TTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAG

[0119] ATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGT

[0120] CGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATT

[0121] AAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGT

[0122] CAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGA

[0123] GTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGC

[0124] TGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTG

[0125] AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCA

[0126] AAGTCGGTGGGGTAACCTTTTAGGAACCAGCCGCTA

[0127] Example 2: Determination of digestive juice tolerance and mucin adhesion of Lactobacillus plantarum FN029 strain

[0128] 1. Research Methods

[0129] 1.1. Determination of tolerance to artificial gastric juice

[0130] After sterilizing PBS, add 3 g / L pepsin (purchased from Sigma, USA, product number: 9001-75-6, enzyme activity ≥2000 U / mg protein), 3 g / L NaCl, 1.1 g / L KCI, 0.15 g / L CaCl2, and 0.6 g / L NaHCO3, adjust the pH value to 3.0 with 1 mol / L HCl, and filter with a 0.22 μm microporous filter membrane for sterilization to prepare simulated gastric fluid.

[0131] The Lactobacillus plantarum FN029 and Lactobacillus plantarum 299v strains were activated and cultured in MRS medium for two generations, centrifuged and washed twice, and the cells were collected and suspended in 2 mL of simulated gastric fluid (concentration of 1×10 9 CFU / mL) and incubated at 37°C for 3 h. The viable bacterial concentration was determined by pouring MRS agar medium at 0, 1, and 2 h. Each sample was repeated 3 times and the average value was calculated.

[0132] 1.2. Determination of tolerance to artificial intestinal fluid

[0133] After sterilizing PBS, add 3g / L ox bile salt (purchased from Sigma, USA, product number: 48305), 0.1g / L lipase (purchased from Sigma, USA, product number: 9001-62-1, enzyme activity ≥20,000U / mg protein), 1g / L trypsin (purchased from Sigma, USA, product number: 9002-07-7, enzyme activity 1,000-2,000U / mg protein), 5g / L NaC1, 0.6g / L KC1, 0.3g / LCaCl2, and 0.6g / L NaHCO3, adjust the pH value to 8.0 with 0.1mol / L NaOH, and then filter and sterilize with a 0.22μm microporous filter membrane to prepare simulated intestinal fluid.

[0134] The Lactobacillus plantarum FN029 and Lactobacillus plantarum 299v were activated and cultured in MRS medium for two generations, centrifuged and washed twice, and the cells were collected and suspended in 2 mL of simulated intestinal fluid (concentration of 1×10 9 CFU / mL) and incubate at 37°C for 4h. Dilute the bacterial suspension and inoculate it on MRS agar medium and incubate it at 37°C for 36h. Count and determine the viable bacterial concentration by pouring MRS agar medium at 0, 1, 2, 3, and 4h. Repeat 3 times for each sample and calculate the average value.

[0135] 1.3. Determination of mucin adhesion

[0136] The bacteria were cultured in MRS broth containing 2% porcine gastric mucin for 24 h, washed twice, and suspended in PBS containing 0.05 Tween 20 to adjust the concentration of the bacterial suspension to 1×10 9 CFU / mL. Suspend 10 mg of freeze-dried porcine gastric mucin in 10 mL of 50 mM Tris-HCl (pH 9.0) and rotate gently at 4°C for 12 hours. Afterwards, centrifuge at 16,000 g for 10 minutes at 4°C, use the supernatant as the mucin solution, and store at 4°C. Add 100 μL of the mucin solution (1 mg / mL) to each well and store in a 4°C incubator for 12 hours. Discard the mucus and wash twice with 0.1% bovine serum albumin PBS. Add 200 μL of 2% bovine serum albumin PBS and incubate in a wet box at room temperature for 2 hours. Wash twice with 0.1% bovine serum albumin PBS. Add 100 μL of the bacterial suspension to each well and incubate overnight at 4°C. Wash the wells with PBST and check the binding with an inverted microscope. Pour the solution, wait for the wells to dry, and measure OD405 with an ELISA instrument. All measurements were repeated 3 times.

[0137] 1.4 Data Analysis

[0138] The data are expressed as mean ± standard deviation, and the differences between the two groups were analyzed using t-test.

[0139] 2. Results

[0140] The results of the artificial gastric acid resistance test of the strains are as follows Figure 3 As shown, after incubation in artificial gastric juice for 1 hour, the survival rate of FN029 was 96.43%, the survival rate of 299v was 89.77%, and the survival rate of FN029 was 6.66% higher than that of 299v; after incubation in artificial gastric juice for 2 hours, the survival rate of FN029 was 94.96%, the survival rate of 299v was 1.11%, and the survival rate of FN029 was 93.85% higher than that of 299v (Table 1).

[0141] Table 1 Artificial gastric juice tolerance of Lactobacillus plantarum

[0142]

[0143]

[0144] The results of the strain artificial intestinal fluid tolerance experiment are as follows Figure 4 As shown, 299v barely survived after 2 hours of incubation in artificial intestinal fluid (SIJ), while FN029 had a survival rate of 90.54% at the end of 4 hours of digestion in SIJ (Table 2). This indicates that 299v is susceptible to bile salts, the toxic effects of which can destroy the microbial membrane, while Lactobacillus plantarum FN029 is almost unaffected by bile salts and has extremely high tolerance to intestinal fluid.

[0145] Table 2 Tolerance of Lactobacillus plantarum to artificial intestinal fluid

[0146]

[0147] The results of the mucin adhesion test are shown in Table 3. The adhesion amount of Lactobacillus plantarum FN029 on the surface of pig gastric mucus matrix is ​​0.085 (OD405), which is 1.2 times the adhesion amount of Lactobacillus plantarum 299v on the surface of pig gastric mucus matrix.

[0148] Table 3 Mucin adhesion of Lactobacillus plantarum

[0149]

[0150] Note: The results are mean ± standard deviation, *p<0.05, the difference between the two groups was analyzed by t test.

[0151] From the above experimental results, it can be seen that the Lactobacillus plantarum FN029 provided by the present invention has excellent gastrointestinal fluid tolerance and mucin adhesion, can enter the human gastrointestinal tract in a living state, and survive in the gastrointestinal organs of animals including humans, exerting health effects.

[0152] Example 3: Evaluation of the antibacterial effect of Lactobacillus plantarum FN029

[0153] 1. Research Methods

[0154] 1.1. Preparation of strain fermentation broth

[0155] The strain stored in the glycerol tube was first activated by streaking on MRS solid medium for 3 times, and then a single colony was picked and expanded in MRS broth medium for 18 h (cultured under anaerobic conditions at 37 °C). The culture medium was adjusted with distilled water to a bacterial concentration of 1×10 9 CFU / mL, centrifuge at 4°C and 15000g for 5 min, collect the supernatant and adjust the pH to 7.0 to obtain the fermentation supernatant of the bacteria.

[0156] 1.2. Effect of strains in inhibiting pathogenic bacteria

[0157] The Oxford cup method was used to detect the inhibitory effect of Lactobacillus plantarum FN029 on several major intestinal pathogens (Clostridium perfringens CP01, Escherichia coli ATCC13932, Clostridium difficile CD01, Staphylococcus aureus ATCC25923). Specifically, the culture medium was incubated at 37°C for 16 h (approximately 1×10 9 CFU / mL) were spread on MRS solid medium, and the Oxford cup was carefully placed on the medium. 200 μL of the fermentation supernatant of the bacteria prepared in 1.1 above was added to each cup. After culturing at 37°C for 24 hours, the diameter of the inhibition zone was measured. Each sample was repeated 3 times and the average value was calculated.

[0158] 1.3 Statistical analysis

[0159] The data are expressed as mean ± standard deviation, and the differences between the two groups were analyzed using t-test.

[0160] 2. Results

[0161] The experimental results are shown in Table 4. The antibacterial effect of the fermentation supernatant of Lactobacillus plantarum FN029 screened by the present invention is better than that of Lactobacillus plantarum 299v. Among them, FN029 has the most significant inhibitory effect on Escherichia coli and Staphylococcus aureus, which are 1.25 times and 1.29 times that of 299v respectively.

[0162] Table 4 Antibacterial activity (diameter of inhibition zone, mm)

[0163]

[0164] Note: The results are mean ± standard deviation, *p<0.05, the difference between the two groups was analyzed by t test.

[0165] It can be seen from the above experimental results that the Lactobacillus plantarum FN029 provided by the present invention has a better effect of inhibiting pathogenic bacteria than Lactobacillus plantarum 299v.

[0166] Example 4: Sensitivity of Lactobacillus plantarum FN029 to antibiotics

[0167] 1. Research Methods

[0168] Nine antibiotic solutions (vancomycin, chloramphenicol, penicillin, streptomycin, gentamicin, kanamycin, erythromycin, trimethoprim and ampicillin) were prepared respectively, and Lactobacillus plantarum FN029 and Lactobacillus plantarum 299v were added to MRS broth medium containing different concentrations (2, 4, 8, 16, 32, 64, 128, 256, 512 and 1024 μg / mL) of antibiotics and cultured anaerobically at 37 °C for 24 h. The final concentrations of Lactobacillus plantarum FN029 and 299v in the system were both 1×10 7 CFU / mL. Each sample was repeated 3 times. After the culture was completed, the absorbance at 610nm of OD value in 96-well plate was measured. MIC (minimum inhibitory concentration) is considered as the lowest antibiotic concentration that can inhibit the growth of strains and is used to evaluate the antibiotic resistance of selected strains.

[0169] 2. Results Analysis

[0170] As shown in Table 5, the plant lactobacillus FN029 screened by the present invention has different degrees of sensitivity to vancomycin, chloramphenicol, penicillin, streptomycin, gentamicin, kanamycin, erythromycin, trimethoprim and ampicillin, and plant lactobacillus FN029 has significant resistance to gentamicin, streptomycin and trimethoprim. From the above experimental results, it can be seen that the plant lactobacillus FN029 provided by the present invention is more sensitive to the above antibiotics than plant lactobacillus 299v, indicating that plant lactobacillus FN029 has the potential to be used as a live bacteria product.

[0171] Table 5 The sensitivity of strains to different antibiotics

[0172]

[0173] Example 5: Analysis of indole derivatives produced by Lactobacillus plantarum FN029

[0174] 1. Research Methods

[0175] Lactobacillus plantarum FN029 and 299v were cultured in MRS broth medium for 24 hours, centrifuged (10000g, 5min) to remove the supernatant, and then suspended in physiological saline, and the OD value was adjusted to 1 at 600nm. 2mL of the bacterial suspension was inoculated into 18mL of MRS medium (MRS-Trp medium) to which L-tryptophan (Trp) (2g / L) was added. After 48 hours of cultivation, the culture was centrifuged at 10000g for 5 minutes and the supernatant was collected. 15mL of the supernatant or MRS-Trp medium was mixed with acetonitrile in a ratio of 1:1. Magnesium sulfate (0.2g / mL) and sodium acetate (0.05g / mL) were added, gently vortexed for 10 minutes, and then centrifuged at 3500rpm for 10 minutes.

[0176] Collect the organic phase (10 mL) and place it in a 50 mL centrifuge tube, and use liquid chromatography-mass spectrometry (LC-MS) to detect tryptophan metabolites. 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 mix for 30 seconds, and then perform machine analysis. Qualitative and quantitative determination was performed using liquid chromatography-electrospray ionization tandem mass spectrometry. The chromatographic conditions are as follows: Nexera series LC-40 liquid chromatograph, ACQUITY UPLC HSS T3 column (Waters), column temperature 40 ° C, injection volume 2 μL, mobile phase A (0.1% formic acid aqueous solution), mobile phase B (0.1% formic acid acetonitrile solution), chromatographic gradient is shown in Table 6. Mass spectrometry conditions: AB SCIEX QTRAP 6500+, positive and negative mode detection, curtain gas (CUR) 35, medium collision gas (CAD), ion spray voltage (IS) 5500 / -4500, temperature (TEM) 550. Ion source gas 1 (GS1) 50, ion source gas 2 (GS2) 50.

[0177] Table 6 Chromatographic gradient

[0178]

[0179] 2. Results Analysis

[0180] Indole derivatives are important active molecules produced by beneficial intestinal 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, indole-3-lactic acid and indole-3-acetic acid are significantly higher than the production of 299v, which are about 461 times and 6.7 times that of 299v, respectively. FN029 produces indole-3-lactic acid and indole-3-acetic acid, which is the basis for regulating immunity.

[0181] Table 7 Indole derivative production of Lactobacillus plantarum FN029 and 299v (ng / mL, n=3)

[0182]

[0183] Note: The results are mean ± standard deviation, *p<0.05, **p<0.01, and the differences between the two groups were analyzed by t-test.

[0184] Example 6: Effect of Lactobacillus plantarum FN029 on relieving eczema

[0185] 1. Experimental methods

[0186] 1.1 Animal experiments

[0187] Thirty 7-week-old female and 15 male BALB / c mice were fed adaptively for one week and co-housed overnight in a 2:1 ratio under clean conditions. The vaginal plug was checked the next morning, and the male mice were removed after the female mice were fertilized. The offspring were weaned 21 days after delivery and randomly divided into 4 groups (n=12):

[0188] 1) CON group (normal control group, intragastric administration of normal saline);

[0189] 2) AD group (eczema model control group, intragastric administration of normal saline);

[0190] 3) 299v group (eczema mice were gavaged with Lactobacillus plantarum 299v suspension, positive control group);

[0191] 4) FN029 group (eczema mice were gavaged with Lactobacillus plantarum FN029 bacterial suspension).

[0192] Preparation of bacterial suspension: Suspend Lactobacillus plantarum FN029 in PBS to obtain a concentration of 1×10 9 CFU / mL of bacterial suspension; Lactobacillus plantarum 299v was prepared with the same method as above to prepare a bacterial suspension of the same concentration.

[0193] The oral dose was 1×10 9CFU / day (100μL / day), and continued gavage for 40 days. Severe eczema was induced on the 31st to 40th day. 20μL 0.5nmol calcipotriol (MC903) and 20μL 0.2mg / mL ovalbumin (OVA) were applied to the right ear of the mice (AD group, 299v group, FN029 group), and anhydrous ethanol was applied to the left ear (CON group). The treatment was continued for 10 days, and the mice were euthanized on the 41st day. During the induction of eczema, the thickness of the right ear was measured with a vernier caliper every day to score the severity of eczema. After the experiment, the mice were euthanized. The eyeballs were removed and blood was collected in anticoagulant tubes. The whole blood was allowed to stand for one hour, and then centrifuged at 4℃, 5000×g for 15min to collect plasma. The terminal 1cm of the ileum was cut off, and then the contents were flushed out with 1mL PBS and frozen. The ear tissue was collected and cut into two parts, one of which was fixed in paraformaldehyde and the other was frozen. Tissues such as the ileum, cecum, colon, Peyer's patches, and spleen were collected and frozen for subsequent testing. Figure 5 As shown in A.

[0194] The severity of eczema was observed during the eczema modeling period and scored as follows:

[0195] (1) Number of scratching times (per minute): more than 80 times, 10 points; 50-80 times, 7 points; 20-50 times, 4 points; 10-20 times, 1 point; <10 times, 0 points.

[0196] (2) Ear erythema / hemorrhage: 10 points for an area exceeding 80% of the total ear skin area; 7 points for an area between 50% and 80%; 4 points for an area between 20% and 50%; 1 point for an area less than 20%; and 0 points for no erythema / hemorrhage.

[0197] (3) Ear rash: If the rash area is greater than 80% of the total ear skin area, 10 points will be awarded; if the area is between 50% and 80%, 7 points will be awarded; if the area is between 20% and 50%, 4 points will be awarded; if the area is less than 20%, 1 point will be awarded; and if there is no rash symptom, 0 points will be awarded.

[0198] (4) Ear edema: If the ears are dark red, uneven, and obviously swollen, they will be assigned 10 points; if the ears are light red, uneven, and swollen, they will be assigned 7 points; if the ear surface is slightly red and uneven, they will be assigned 4 points; if the ear surface is relatively smooth and close to the skin color of a normal mouse ear, they will be assigned 1 point; if there are no symptoms mentioned above, they will be assigned 0 points.

[0199] (5) Scaly changes in the ear: 10 points for an area exceeding 80% of the total skin area of ​​the ear; 7 points for an area between 50% and 80%; 4 points for an area between 20% and 50%; 1 point for an area less than 20%; and 0 points for no scaling.

[0200] The total score is expressed as the sum of the 5 items / 5.

[0201] 1.2. Ear histological examination

[0202] The ears of the mice were embedded in paraffin and fixed in paraformaldehyde, and sections were prepared. The sections were then dehydrated with different concentrations of alcohol, stained, decolorized, transparentized with xylene, and mounted to prepare pathological sections. The sections were stained with hematoxylin / eosin (H&E) to evaluate the infiltration of inflammatory cells in the ear tissues of the mice, and stained with Carbol 2R / hematoxylin and toluidine blue to evaluate the infiltration of eosinophils and mast cells, respectively. The number of eosinophils and mast cells in 10 random fields of view of the pathological sections of the ears of the mice was counted under an optical microscope.

[0203] 1.3. Mouse spleen regulatory T cells (CD4 + CD25 + Foxp3 + T) determination

[0204] The spleen of the mouse was taken out, cut into small pieces with sterile scissors, and placed in PBS; the spleen was ground with a syringe plunger and filtered through a 70 μm cell filter, and the filtrate was collected into a 15 mL centrifuge tube; centrifuged at 300 g for 5 min, and the supernatant was discarded; 2 mL of 1× red blood cell lysis buffer was added to resuspend the precipitate, and the lysis was carried out at room temperature for 2-3 min, and 10 mL of PBS was added to terminate the lysis reaction; centrifuged at 300 g for 5 min, and the supernatant was discarded to obtain a white cell precipitate, and the cells were resuspended with cell staining buffer, and the cell count was performed to adjust the cell concentration to 1×10 7 / mL. Dilute the fixative and membrane permeabilization agent for Foxp3 detection; take 100 μL of mouse spleen cells (1×10 6100 μL of cell staining buffer was added to resuspend the cells; 1 mL of fixative was added, vortexed and reacted at 4°C for 30 min; after the reaction was completed, centrifuged at 600 g for 5 min, and the supernatant was discarded; 2 mL of membrane permeabilization agent was added, vortexed and centrifuged at 600 g for 5 min, and the supernatant was discarded; 100 μL of membrane permeabilization agent was added to resuspend the cells, and PE Anti-Mouse CD4 (E-AB-F1097C) was added to the cell culture tube, and the mixture was mixed and incubated at 4°C for 30 min; after the reaction was completed, the mixture was centrifuged at 600 g for 5 min, and the supernatant was discarded; 100 μL of membrane permeabilization agent was added to resuspend the cells, and PE Anti-Mouse CD4 (E-AB-F1097C) was added to the cell culture tube, and the mixture was mixed and centrifuged at 600 g for 5 min, and the supernatant was discarded; 100 μL of membrane permeabilization agent was added to resuspend the cells, and PE Anti-Mouse CD4 (E-AB-F1102E) was added to the cell culture tube, and the mixture was mixed and centrifuged at 4°C for 30 min; after the reaction was completed, the mixture was centrifuged at 600 g for 5 min, and the supernatant was discarded; 100 μL of membrane permeabilization agent was added to resuspend the cells, and PE Anti-Mouse CD4 (E-AB-F1097C) was added to the cell culture tube, and the mixture was mixed and centrifuged at 600 g for 5 min, and the supernatant was discarded; 100 μL of membrane permeabilization agent was Foxp3 (E-AB-F1238D), mix well and incubate at room temperature for 30 minutes; add 2mL of membrane permeabilization agent, centrifuge at 600g for 5 minutes, and discard the supernatant; add 200μL of cell staining buffer, resuspend the cells, and then detect them on the machine. Anti-IgG1 was used as an isotype control. Treg was measured using a BD FACSVerseTM flow cytometer (CA, USA), and data analysis and processing were performed using FlowJo software version 10.1 (Tree Star, Ashland, OR, USA).

[0205] 1.4. Plasma cytokine determination

[0206] Plasma IgE, interleukin-4, interleukin-10, interleukin-12 (IL-4, IL-10, IL-12) and interferon gamma (IFN-γ) levels, as well as interleukin-33 (IL-33) levels in 10% homogenate of mouse ear tissue were measured by enzyme-linked immunosorbent assay. The specific measurement method was carried out according to the instructions of the corresponding enzyme-linked immunosorbent assay kit.

[0207] 1.5 Statistical analysis

[0208] Data are presented as mean ± standard error (SEM) or interquartile range (IQR). Data were analyzed using SPSS24 software. GraphPad Prism 9 was used to visualize the results. When the data variance was equal, one-way ANOVA and Tukey post hoc test were applied, otherwise, Kruskal-Wallis test was applied. The false discovery rate (FDR) was applied to correct the P value when appropriate, and the P value was adjusted for multiple comparisons using FDR. When the P value was <0.05, the difference was considered statistically significant. The relative proportions of immune cells between the two groups were compared by two-sided Welch t test and Benjamini Hochberg FDR correction.

[0209] 2. Research Results

[0210] 2.1 Effects of FN029 treatment on ear eczema symptoms and eosinophil and mast cell infiltration in eczematous mice

[0211] The mouse ears were fixed with paraformaldehyde, embedded in paraffin and prepared for pathological sections. The sections were stained with hematoxylin / eosin (H&E), Carbol2R / hematoxylin and toluidine blue. Under a microscope, the thickness of the dermis in the mouse ear sections was calculated, and the number of mast cells and eosinophils in the sections was counted.

[0212] On the fifth day of eczema induction, the relative thickness of the ears in the AD, 299v, and FN029 groups increased significantly compared with the normal control group, indicating the occurrence of eczema (p<0.05, Figure 5 B). The eczema severity score of the FN029-treated group (2.4±0.8) was significantly lower than that of the AD group (8.1±1.6) (p<0.05), and was approximately 29.6% of that of the AD group at the end point ( Figure 5 B). The thickness of the right ear dermis in the FN029 group (0.15±0.04) was significantly reduced by 55.89% compared with the AD group (0.34±0.15) (p<0.001) Figure 5 C). The ear epidermis and dermis of the mice in the AD group were swollen and inflammatory cells were severely infiltrated. The FN029 group could significantly alleviate these pathological changes ( Figure 5 D). The commercial strain 299v did not significantly improve atopic dermatitis (eczema) scores and ear thickness compared with the AD group.

[0213] like Figure 6 As shown in A, the ear sections of eczema mice were infiltrated with obvious mast cells and eosinophils. Only FN029 treatment significantly improved the infiltration degree, while 299v had no obvious effect. Figure 6 B. Figure 6 As shown in C, the number of eosinophils (421.3±87.6 cells / field) and mast cells (60.6±10.9 cells / field) in the ear tissue of mice in the AD group increased significantly (p<0.001). In contrast, the number of eosinophils (238.1±56.1 cells / field) and mast cells (35.4±7.95 cells / field) in the FN029 group was significantly lower than that in the AD group (p<0.001).

[0214] 2.2 Effect of FN029 treatment on the proportion of regulatory T cells in the spleen of mice with eczema

[0215] like Figure 7As shown, the proportion of regulatory T cells in the spleen of mice in the 299v group (6.2±0.6%) was about 1.2 times that of the AD group (5.0±0.4%), which was significantly higher than that of the AD group (p<0.001). There was no significant difference between the 299v and AD groups. Regulatory T cells are effector cells of immune tolerance, indicating that FN029 induced the development of immune tolerance.

[0216] 2.3 Effects of FN029 treatment on plasma cytokines in mice with eczema

[0217] like Figure 8 As shown, the plasma IL-4 (266.6±12.6 ng / mL) of FN029-treated mice was significantly reduced to 46% of the AD group (582.0±211.8 ng / mL), and the plasma total IgE (252.4±23.4 ng / mL) was significantly reduced to 75.4% of the eczema group (334.8±31.2 ng / mL). However, IL-12 (299.8±26.1 ng / mL) was significantly increased to 1.37 times that of 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 of the eczema group (199.1±9.3 ng / mL), and plasma IL-4 / IFN-γ (0.88±0.12) was reduced to 46% of the AD group (1.92±0.46). However, 299v could not significantly improve IL-4 (347.7±114.1ng / mL) and IL-4 / IFN-γ (1.46±0.51), and its effect on improving IgE (272.7±15.2ng / mL) was weaker than that of FN029. In addition, the IL-33 in ear homogenate was significantly reduced in FN029 treatment (160.6±22.4ng / mL) compared with the AD group (308.2±54.3ng / mL). Therefore, FN029 significantly inhibited the polarization of immune response to Th2 type and inhibited IgE-mediated allergic reaction.

[0218] Example 7: Effect of Lactobacillus plantarum FN029 on regulating intestinal flora

[0219] 1. Experimental methods

[0220] 1.1 Animal experiments and bacterial flora sequencing

[0221] Animal experiments were performed according to 1.1 in Example 6, and DNA from the cecal contents of mice was extracted after the animals were killed. The flora analysis was performed using a paired-end sequencing method based on a next-generation sequencing platform using 16S rDNA amplicon sequencing (v3-v4 region). An index adapter was added to the end of the 16S rDNA PCR product for next-generation sequencing (Illumina, San Diego, CA, USA). The second round of PCR amplicon enrichment was performed using the first round PCR product as a template (initial denaturation step 94°C 3min, 24 cycles, 94°C denaturation 5s, 57°C annealing 90s, 72°C extension 10s, and then 72°C extension 5min). PCR reactions were performed in triplicate using a 25μL mixture containing 2.5μL TransStart buffer, 2μL dNTPs, 1μL of each primer, and 20ng template DNA. The DNA library concentration was verified using a Qubit 3.0 fluorometer. The libraries were quantified to 10 nM according to the manufacturer's instructions, and then multiplexed and loaded onto an Illumina MiSeq instrument (Illumina, San Diego, CA, USA). Sequencing was performed using PE250 / 300 paired-end sequencing; image analysis and base calling were performed using MiSeq control software, and fastq files were obtained for quality control processing.

[0222] 1.2 Data Analysis

[0223] Amplicon sequence variation (ASV) analysis was performed using EasyAmplicon (v1.10). The -derep_fullength command in VSEARCH (v2.15) was used to splice the double-end sequences, and the -unoise3 algorithm in USEARCH (v10.0) was used to denoise the extended sequences to produce ASVs, and the -userarch_global command was used to create the ASVs table. The BLCA library was used for species-level annotation of ASVs (the reference database was the June 2024 version of the refseq 16S rRNA database). The vegan package in R was used for statistical analysis of alpha and beta diversity, and STAMP software was used for differential species analysis.

[0224] 2. Analysis results

[0225] 2.1. Regulatory effect of Lactobacillus plantarum FN029 on α and β diversity of intestinal flora caused by eczema

[0226] like Fig. 9As shown in the figure, the α diversity of the intestinal flora of eczema mice (Shannon index was 1.74±1.12; uniformity was 0.22±0.02) was significantly reduced compared with the CON group. After FN029 treatment, the Shannon index (2.42±0.83) and uniformity (0.43±0.11) were significantly improved. After principal coordinate analysis (PCoA) of Bray-Curtis distance, the flora of eczema mice deviated significantly from that of healthy mice, and the vertical coordinate explanation reached 19.53%. Through permutational multivariate analysis of variance (PERMANOVA), both FN029 and 299v treatments significantly changed the flora diversity (p<0.05), and there was no significant difference compared with the healthy control group. After non-metric multidimensional measurement (NDMS) analysis, the flora diversity of the FN029 treatment group deviated significantly from the AD group (p<0.05), while the 299v treatment group deviated significantly from both the AD group and the healthy control group (p<0.05).

[0227] 2.2. Lactobacillus plantarum FN029 regulates the relative content of bacterial species that significantly change the intestinal flora in eczema disorders

[0228] like Fig.10As shown, FN029 treatment significantly regulated the relative abundance of multiple bacteria, among which Kineothrix alysoides was the most dominant butyrate-producing bacterium in the intestine (Kineothrix alysoides, gen. nov., sp. nov., a saccharolytic butyrate-producer within the family Lachnospiraceae. Int. J. Syst. Evol. Microbiol. 67, 402–410). FN029 treatment (14.8±1.2%) increased the relative abundance of Kineothrix alysoides to 1.7 times that of the AD group (8.4±2.38%). Akkermansia muciniphila 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). The abundance of A. muciniphia in the AD group (2.93 ± 0.82%) was significantly increased compared with the CON group (0.02 ± 0.02%). After FN029 treatment, the abundance of A. muciniphia in the AD group (0.34 ± 0.02%) was significantly reduced (p < 0.05). Another branch of mycobacterium Alistipes dispar is a Gram-negative bacterium of the Bacteroidetes phylum that can produce indole, succinic acid, and a small amount of acetic acid and propionic acid. A. dispar (0.2±0.02%) in the AD group was significantly reduced to 1% of that in the CON group (2.0±0.98%) (p<0.05), and its abundance (0.93±0.13%) increased to 450 times that in the AD group after FN029 treatment. Fusimonas intestine, a species with unclear relationship with eczema, was significantly reduced in the AD group (1.8±1.1%) compared with the CON group (3.8±1.25%) (p<0.05), and its abundance (5.12±1.12%) was significantly increased after FN029 treatment compared with the AD group. Sangeribacter mnris is also a species with unclear association with eczema. Its abundance in the AD group (6.9±1.8%) increased slightly to 115% of that in the CON group (6.0±1.89%), and its abundance after FN029 treatment (0.73±0.28) was significantly reduced compared with the AD group (p<0.05).Except for Sangeribacter muris, 299v treatment did not significantly regulate the abundance of other bacterial species.

[0229] Example 8: Effect of Lactobacillus plantarum FN029 on promoting digestive function

[0230] 1. Research Methods

[0231] 1.1 Effects of probiotic treatment on intestinal motility in mice

[0232] Male 8-week-old 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 modeled with compound diphenoxylate. The experimental group was orally administered with Lactobacillus plantarum FN029 or 299v (1×10 9 CFU / day) for one week, which were FN029 treatment group and 299v treatment group. Before the end of the experiment, fasting but not water was allowed for 20 hours. On the day of the measurement, each experimental group and blank and model control group were given the test sample or distilled water again; 30 minutes later, each experimental group and model control group were given compound diphenoxylate 5-6 mg / kgbw, and the blank control group was given distilled water; 30 minutes later, each group was given an indicator (accurately weigh 100g of gum arabic, add 800mL of water, boil until the solution is transparent, weigh 100g of activated carbon powder and add it to the above solution and boil three times, wait for the solution to cool and adjust the volume to 1000mL to obtain the indicator, store it in a refrigerator at 4℃, and shake it before use); 25 minutes later, the animals were killed by dislocation of the neck, and the ink propulsion rate was used to indicate the intestinal motility of mice.

[0233] Calculate the ink advancement rate as follows:

[0234] Ink propagation rate (%) = ink propagation length (cm) / total length of small intestine (cm) × 100%.

[0235] 1.2. Determination of digestive enzymes

[0236] Six-week-old BALB / c mice were selected and divided into a blank control group, a Lactobacillus plantarum FN029 treatment group, and a Lactobacillus plantarum 299v treatment group. Sterile water, FN029 or 299v bacterial suspension (1×10 9 CFU / day) for 6 consecutive weeks. Fast for 24 hours, free access to water, and ether anesthesia for autopsy of all mice for sampling. Including gastric juice, duodenal juice, jejunum, ileum, liver, kidney, spleen, pancreas. The kits produced by Nanjing Jiancheng Bioengineering Institute were used to detect the activity of pepsin in mouse gastric juice, trypsin and amylase in duodenal juice, respectively, and the operation was performed according to the instructions of the kits.

[0237] 1.3 Statistical analysis

[0238] Data are presented as mean ± standard error (SEM) or interquartile range (IQR). Data were analyzed using SPSS24 software. GraphPad Prism 9 was used to visualize the results. When data variances were homogeneous, one-way ANOVA and Tukey post hoc test were applied, otherwise, Kruskal-Wallis test was applied.

[0239] 2. Results

[0240] 2.1 Effects of Lactobacillus plantarum FN029 on intestinal motility in mice

[0241] The effects of each group on the intestinal motility of mice are shown in Fig.11 . Compared with the blank control group, the model control group used compound diphenoxylate to artificially slow down intestinal motility, and oral administration of FN029 (72.0±0.9%) or 299v (67.1±1.2%) could significantly increase the ink propulsion rate of the mouse intestine (p<0.001). Compared with the model control group (50.0±1.7%), the ink propulsion rate of the FN029 group and the 299v group was significantly increased. Compound diphenoxylate can directly inhibit the intestinal mucosal receptors of smooth muscle, eliminate the peristaltic reflex of the local mucosa and weaken peristalsis, thereby prolonging the contact between intestinal contents and intestinal mucosa, allowing intestinal water absorption and dry stool. It is often used to model constipation mouse models. The results of this study showed that both FN029 and 299v can significantly improve the intestinal motility of mice and promote intestinal peristalsis to counteract the slowing of intestinal peristalsis caused by drugs, and the effect of FN029 is better than that of 299v.

[0242] 2.2 Effects of Lactobacillus plantarum FN029 on body weight and digestive enzyme activities in mice

[0243] The effects of each group on the body weight of mice are shown in Fig.12 Under 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±0.12g) and 299v (23.44±0.21g) was significantly higher than that of the control group (22.35±0.83g, p<0.05), and the average body weight gain rate of mice in the FN029 group (21.11±1.18%) and 299v group (18.52±1.13%) was significantly higher than that in the control group (11.17±2.00%, p<0.05) (Table 8).

[0244] Table 8 Effect of Lactobacillus plantarum on body weight of mice

[0245]

[0246] like Fig.13As shown, FN029 treatment can significantly increase trypsin activity (821.13±2.59U / mL), which is 128% (p<0.05) of the enzyme activity of the control group (638.88±11.44U / mL), and is also significantly higher than the enzyme activity of 299v (811.5±10.21U / mL, p<0.05).

[0247] In summary, Lactobacillus plantarum FN029 can promote intestinal motility, increase mouse body weight, and enhance trypsin activity, so it has the function of promoting intestinal digestion, and its effect is better than that of Lactobacillus plantarum 299v.

[0248] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Lactiplantibacillus plantarum FN029, characterized in that: The plant lactobacillus was deposited in the Guangdong Microbiological Culture Collection Center on December 20, 2022, with the deposit number GDMCC No: 63060.

2. A microbial agent, characterized in that: Contains the Lactobacillus plantarum FN029 according to claim 1.

3. The microbial agent according to claim 2, characterized in that: The bacterial agent uses GDMCC No: 63060 as the main microorganism; Optionally, the bacterial agent contains living cells of the GDMCC No: 63060 strain of claim 1, dry bacteria of the GDMCC No: 63060 strain obtained by freeze-drying, 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 bacterial agent is a microbial preparation that can tolerate adverse digestive tract environments, inhibit intestinal pathogenic bacteria, has antibiotic sensitivity, produces indole derivatives, improves allergic diseases, regulates intestinal flora, and aids digestion.

5. A product, characterized in that The product contains the Lactobacillus plantarum GDMCC No: 63060 described in claim 1 or a bacterial agent containing the Lactobacillus plantarum GDMCC No: 63060 described in claim 1.

6. The product according to claim 5, characterized in that The product is food, medicine or health product.

7. The product according to claim 6, characterized in that The food is solid food, liquid food or semi-solid food; the medicine contains pharmaceutically acceptable carriers or excipients.

8. The product according to claim 5, characterized in that The product has one or more functions of tolerating adverse digestive tract environments, inhibiting intestinal pathogens, being sensitive to antibiotics, producing indole derivatives, improving allergic diseases, regulating intestinal flora, and aiding digestion.

9. The product according to claim 5, characterized in that The number of cells of Lactobacillus plantarum GDMCC No: 63060 in the product is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

10. Use of the Lactobacillus plantarum GDMCC No: 63060 of claim 1 or a bacterial agent containing the Lactobacillus plantarum GDMCC No: 63060 of claim 1 in at least one of the following (1) to (6): (1) Preparation of products that inhibit intestinal pathogens; (2) preparing products containing indole derivatives as active ingredients; (3) preparing products for preventing and / or treating eczema; (4) Preparation of products for improving allergic diseases; (5) Preparation of products for regulating intestinal flora; (6) Prepare products with digestion-promoting function.

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