Lactobacillus amyloliquefaciens and application of lactobacillus amyloliquefaciens in intestinal diseases

Abstract

The invention relates to lactobacillus amylovorans and an application thereof in intestinal diseases, the lactobacillus amylovorans (LA) is named as lactobacillus amylovorans QC1H, is preserved in China Center for Type Culture Collection on March 28, 2023, and has a preservation number of CCTCC NO: M2023427, the lactobacillus amylovorans and / or a fermentation product thereof can significantly improve the ratio of mouse intestinal villus length to crypt depth, and the lactobacillus amylovorans and / or the fermentation product thereof can significantly improve the intestinal villus length and crypt depth of mice. The compound has the advantages of reducing mouse ileum enteritis factor expression, improving ileum tight junction protein expression and reducing intestinal permeability, and has important application significance and value in preparation of a therapeutic agent for preventing and treating enteritis caused by aflatoxin B1 (AFB1).

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a type of Lactobacillus amyloliquefaciens and its application in enteropathy. Background Technology

[0002] Aflatoxin is widely present in soil and can contaminate various feed crops for livestock and poultry, such as rice, corn, and peanuts. When animals consume feed contaminated with aflatoxin (AFB1), it can lead to liver and kidney damage, immunosuppression, increased susceptibility to disease, and decreased growth performance. At the same time, human health will be threatened if they consume animal-derived products with accumulated AFB1.

[0003] The gut, as the primary organ for nutrient absorption, is a crucial physical barrier against external pathogens and mycotoxins. Over 80% of AFB1 absorption occurs in the gut, making it a major target for AFB1 toxicity. AFB1 poisoning disrupts intestinal morphology, triggers inflammatory responses, undermines gut bacterial balance, and can even lead to cell apoptosis. Therefore, developing effective methods and strategies to reduce or completely eliminate aflatoxin contamination and restore the safety of food and feed is urgently needed. However, physical and chemical detoxification methods, while removing mycotoxin contamination, negatively impact the sensory characteristics and nutritional quality of grains, exhibiting problems such as low efficiency, high cost, and potential secondary contamination.

[0004] The gut microbiome is a complex collection of approximately 100 trillion microorganisms in the gastrointestinal tract. It plays a vital role in overall health by extracting nutrients and energy, regulating various physiological processes, eliminating toxins, and treating a range of disorders. Probiotics, in particular, form a natural barrier in the intestinal tissue, preventing the invasion of parasites, fungi, viruses, bacteria, and toxins. As a potential antibiotic alternative, probiotics can improve host immune function and reduce the incidence of intestinal diseases in livestock and poultry. Furthermore, probiotics can prevent or alleviate gastrointestinal infections by stimulating beneficial microorganisms or inhibiting the growth of pathogenic microorganisms, enhancing intestinal barrier function and the local immune system. However, the application of *Lactobacillus amyloliquefaciens* and its fermentation broth in improving AFB1-induced enteritis has not yet been reported.

[0005] Given the current situation where interventions for AFB1-induced enteritis are inefficient and costly, and there is a lack of probiotics for preventing and improving AFB1-induced enteritis, there is an urgent need to provide a safe, efficient, and natural method to improve AFB1-induced enteritis. Summary of the Invention

[0006] The purpose of this invention is to address at least one deficiency in the prior art by providing a *Lactobacillus amyloliquefaciens* and its application in enteropathy. By enhancing intestinal barrier function and inhibiting inflammatory responses, it can effectively alleviate the toxicity of AFB1 to the intestine and can be used for the prevention and treatment of enteropathy. This aims to improve the current situation where interventions for AFB1-induced enteritis are inefficient, costly, and lack probiotics for the prevention and improvement of AFB1-induced enteritis.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] The first aspect of the present invention is to provide a Lactobacillus amylovorus, named Lactobacillus amylovorus QC1H, which was deposited at the China Center for Type Culture Collection on March 28, 2023, with accession number CCTCC NO: M2023427.

[0009] Furthermore, the 16S rDNA sequence of the Lactobacillus amyloliquefaciens is shown in SEQ ID: 1.

[0010] Furthermore, the colonies of the Lactobacillus amyloliquefaciens on the MRS plate are round, white, with a raised, smooth surface, neat edges, and an opaque, single shape, with a diameter of about 2 to 3 mm.

[0011] Furthermore, the strain of *Lactobacillus amyloliquefaciens* exhibits a blue-purple color after Gram staining, and is rod-shaped with blunt, rounded ends, exists singly, and has no spores.

[0012] A second aspect of the present invention is to provide a therapeutic agent, the active ingredient of which comprises *Lactobacillus amyloliquefaciens* as described in the first aspect and / or the fermentation product of *Lactobacillus amyloliquefaciens*.

[0013] Furthermore, the concentration of the *Lactobacillus amyloliquefaciens* in the therapeutic agent is 10. 8 ~10 9 CFU / mL, preferably 10 8 CFU / mL.

[0014] Furthermore, when used on mice, the therapeutic agent is administered by gavage at a rate of 180–220 μL / day, preferably 200 μL / day.

[0015] Understandably, when changing the target population, the application method, dosage, and frequency of medication can be adjusted accordingly based on the characteristics of the target population (such as weight).

[0016] A third aspect of the invention is to provide an application of Lactobacillus amyloliquefaciens as described in the first aspect or a therapeutic agent as described in the second aspect, said application comprising at least one of the following applications: application in the preparation of a therapeutic agent for the prevention of enteropathy, and application in the preparation of a therapeutic agent for the treatment of enteropathy.

[0017] Furthermore, the enteropathy includes AFB1-induced enteropathy.

[0018] Furthermore, the AFB1-induced enteropathy includes intestinal damage, inflammatory lesions, and impaired intestinal barrier function.

[0019] Furthermore, the *Lactobacillus amyloliquefaciens* improves AFB1-induced inflammatory bowel injury by increasing intestinal length and villus length, reducing intestinal crypt depth, decreasing the expression level of intestinal inflammatory cytokines, increasing the expression level of intestinal tight junction proteins, and improving intestinal permeability.

[0020] Furthermore, the expression of the intestinal inflammatory cytokines includes the mRNA expression of inflammatory cytokines IL-6, IL-1β, and TNF-α.

[0021] Furthermore, the expression of the intestinal tight junction proteins includes the mRNA expression of tight junction proteins Zo-1, Occludin, and Claudin-1.

[0022] Compared with the prior art, the present invention, by adopting the above technical solution, has the following beneficial effects:

[0023] This invention isolates and screens *Lactobacillus amyloliquefaciens* QC1H from the feces of fresh piglets and sows, and prepares a 10% concentration of *Lactobacillus amyloliquefaciens* QC1H and / or its fermentation broth as active ingredients. 8 ~10 9 The therapeutic agent, with a concentration of CFU / mL, was demonstrated to have good therapeutic effects on AFB1-induced intestinal pathological damage and inflammatory lesions, as well as the expression of intestinal tight junction proteins. Furthermore, the therapeutic agent has the advantages of high concentration, high cost-effectiveness, safety, and low cost, and has good application prospects and significance in the prevention and treatment of intestinal diseases.

[0024] The *Lactobacillus amylovorus* involved in this invention is classified and named *Lactobacillus amylovorus*, specifically *Lactobacillus amylovorus* QC1H, and will be referred to as *Lactobacillus amylovorus* or LA below. It was deposited on March 28, 2023, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2023427, and the address of the depository is Wuhan University, Wuhan, China. Attached Figure Description

[0025] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are for illustrative purposes only, and do not constitute an undue limitation of the invention. In the drawings:

[0026] Figure 1 This is a schematic diagram of Lactobacillus amyloliquefaciens (LA) under a high-resolution scanning electron microscope in one embodiment of the present invention;

[0027] Figure 2 This is a figure showing the effect of Lactobacillus amyloliquefaciens (LA) on intestinal changes in AFB1-induced inflammatory bowel disease mice in one embodiment of the present invention;

[0028] Figure 3 This is a figure showing the effect of Lactobacillus amyloliquefaciens (LA) on the pathological changes of ileal tissue in mice with AFB1-induced inflammatory bowel disease in one embodiment of the present invention;

[0029] Figure 4 This is a figure showing the effect of Lactobacillus amyloliquefaciens (LA) on the gene expression levels of IL-1β, IL-6, and TNF-α in the ileum tissue of mice with AFB1-induced inflammatory bowel disease in one embodiment of the present invention;

[0030] Figure 5 This is a figure showing the effect of Lactobacillus amyloliquefaciens (LA) on the expression levels of Zo-1, Occludin, and Claudin-1 genes in the ileum tissue of AFB1-induced inflammatory bowel disease mice in one embodiment of the present invention.

[0031] Figure 6 This is a figure showing the effect of Lactobacillus amyloliquefaciens (LA) on changes in intestinal permeability in AFB1-induced inflammatory bowel disease mice in one embodiment of the present invention.

[0032] All experimental results in the above figures are expressed as mean ± SEM (n = 5). Different letters between data groups indicate significant differences between them (P < 0.05). Detailed Implementation

[0033] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental materials in the following embodiments that do not specify their source are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise stated, all parts are parts by weight, and all percentages are percentages by mass. Unless otherwise defined or stated, all professional and scientific terms used in the present invention have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the methods of the present invention.

[0034] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of the invention.

[0035] In this embodiment of the invention, the MRS broth composition is as follows: 10.0g peptone, 8.0g beef powder, 4.0g yeast powder, 20.0g glucose, 2.0g dipotassium hydrogen phosphate, 2.0g diamine hydrogen citrate, 5.0g sodium acetate, 0.2g magnesium sulfate, 0.04g manganese sulfate, 1.0mL Tween 80, and 1000mL distilled water. The broth is autoclaved at 118°C for 15 minutes before use.

[0036] Unless otherwise specified, all products in this invention can be obtained through commercially available purchases.

[0037] The present invention will be described by way of example below.

[0038] Example 1 - Isolation and Identification of Lactobacillus amylovorus (LA)

[0039] This embodiment relates to a specific method for isolating and identifying Lactobacillus amyloliquefaciens, which specifically includes the following steps:

[0040] (1) Isolation and screening of Lactobacillus amyloliquefaciens

[0041] Weigh 0.1g of fresh feces from piglets and sows, add 0.9mL of sterile physiological saline, mix thoroughly by pipetting, and serially dilute 10-fold with physiological saline. Spread 100μL of each dilution onto MRS plates and incubate upside down at 37℃ for 48h. Use an inoculation loop to pick up morphologically characteristic colonies of milky-white, round protrusions of lactic acid bacteria and streak them onto MRS plates. After streaking, incubate upside down at 37℃ for 48h. Repeat the above steps three times for isolation and purification until only a single morphological colony is observed on the plate, indicating successful purification. Gram staining results are positive.

[0042] The biological characteristics of the isolated and screened Lactobacillus amyloliquefaciens obtained by high-resolution scanning electron microscopy are as follows: the colonies on MRS plates are round, white, with a raised, smooth, and opaque surface, with neat edges, and a diameter of about 2-3 mm; the Gram staining result is positive, and the strain turns blue-purple after Gram staining. The morphology is rod-shaped, with blunt ends, and it exists singly without spores.

[0043] (2) Identification of Lactobacillus amyloliquefaciens

[0044] After activation by MRS plate and broth culture, the strain was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The 16S rDNA sequence (5'–3') of this strain is shown in SEQ ID NO: 1. After comparing the sequencing results with the NCBI GenBank database, strains with over 99% homology were identified. The species was then determined through comparison, showing 100% homology with the existing Lactobacillus amylovorus sequence on NCBI. The specific sequence of SEQ ID NO: 1 is shown below:

[0045]

[0046] Example 2 - Effect of Lactobacillus amyloliquefaciens on intestinal length in mice

[0047] This embodiment involves investigating the effect of *Lactobacillus amyloliquefaciens* isolated and screened in Example 1 on the intestinal length of mice, and further verifying its role in alleviating AFB1-induced intestinal damage. The specific steps include:

[0048] 1. Preparation of Lactobacillus amyloliquefaciens

[0049] After UV sterilization of the clean bench, the *Lactobacillus amyloliquefaciens* strain stored in the -80℃ freezer was revived. 100 μL of the bacterial suspension was spread onto an MRS plate and incubated at 37℃ for 24–48 h. Single colonies were then picked and inoculated into MRS broth. After incubation at 37℃ for 6 h, the colonies were transferred to the MRS broth at a 2% inoculum level and incubated at 37℃ for 24 h. The resulting bacterial suspension was then aliquoted into 50 mL centrifuge tubes and centrifuged at 8000×g for 10 min at 4℃. The supernatant was discarded, and a small amount of sterile PBS was added to the centrifuge tubes to agitate the precipitate. Plate counting was performed, and the culture was serially diluted with sterile PBS to prepare 1×10⁻⁶ plates. 8 CFU / mL Lactobacillus amyloliquefaciens bacterial solution, for later use.

[0050] 2. Experimental reagents, animal grouping and treatment

[0051] (1) Chemical reagents: aflatoxin B1 (Qingdao Pruibang Biotechnology Co., Ltd.), FITC-labeled dextran (Sigma-Aldrich, USA), AG RNAex Pro Reagent, reverse transcription kit and premixed qPCR kit (Hunan Aikerui Biotechnology Co., Ltd.).

[0052] (2) Animal grouping and treatment

[0053] Eighteen 7-week-old male C57BL / 6SPF mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. for the experiment. Six mice were housed in each cage in an SPF-grade environment (12-hour light, 12-hour dark alternation, free access to food and water, and constant temperature and humidity) for one week of acclimatization before the experiment began. The 18 mice were randomly divided into three groups: a control group, an AFB1 group, and an AFB1+LA group.

[0054] In this group, control mice consumed normal food and were administered physiological saline by gavage daily; AFB1 group mice were administered 0.75 mg / kg AFB1 by gavage daily; and AFB1+LA group mice, in addition to receiving 0.75 mg / kg AFB1 by gavage daily, were also administered 200 μL of 1×10⁻⁶ ozonated saline by gavage. 8 The experimental period was 28 days and the concentration of CFU / mL of Lactobacillus amyloliquefaciens was 28 days.

[0055] 3. Sample collection

[0056] Mice were fasted for 12 hours before sampling. Four hours before euthanasia, they were administered FD-4 by gavage. Four hours later, blood was collected by enucleation and left to stand in the dark for 2 hours. Serum was collected after centrifugation at 3500 rpm and 4°C for 15 minutes. The mice were then dissected, and their intestinal tissue was collected and measured. A portion of the tissue was fixed in 4% paraformaldehyde solution. The remaining tissue was flash-frozen in liquid nitrogen and stored at -80°C for subsequent molecular biological analysis.

[0057] 4. Results Analysis

[0058] The lengths of mice collected from different experimental groups were compared, and the changes in mouse intestinal length were as follows: Figure 2 As shown in the figure, compared with the blank control group, the AFB1-exposed mice had significantly shorter small intestines and more transparent intestinal walls. Gavage with Lactobacillus amyloliquefaciens LA significantly improved the shortened small intestines and thickened the intestinal wall tissue.

[0059] Example 3 - Effects of Lactobacillus amyloliquefaciens on pathological and histological changes in the ileum

[0060] This embodiment is based on the samples collected in Example 2, which are processed and analyzed to investigate the effect of Lactobacillus amyloliquefaciens on the pathological and histological changes of the ileum in mice with AFB1-induced intestinal injury, and to verify the alleviating effect of Lactobacillus amyloliquefaciens on AFB1-induced intestinal injury in mice. The steps include:

[0061] Hematoxylin-eosin staining was performed on intestinal tissues from mice in different experimental groups. Specifically, a ring was longitudinally cut from a fixed ileum and placed in a dehydration box for gradient dehydration. After dehydration, paraffin was melted three times at 65°C for 1 hour. The melted paraffin was placed in an embedding frame, and before it completely solidified, the dehydrated tissue was removed and embedded. The paraffin block was then placed in a paraffin microtome for 4μm thickness sectioning. The sectioned tissue was floated in warm water, then flattened with a glass slide and placed in an oven for baking. After the water and paraffin were dried, the sections were removed and placed at room temperature. The sections were then placed in xylene for 20 minutes, followed by 75% ethanol for 5 minutes. Excess ethanol was washed away with tap water. Staining then began: the sections were placed in hematoxylin staining solution for 3–5 minutes, rinsed with tap water, dehydrated, and then stained in eosin staining solution for 5 minutes. Finally, the sections were mounted. The sections were observed using a conventional optical microscope.

[0062] Pathological changes in the mouse ileum were observed and compared after hematoxylin-eosin staining. The staining results are as follows: Figure 3 As shown. From Figure 3It was found that the villi in the control group were intact and long, with shallow crypt depth and no obvious breakage. Compared with the control group, the villi in the AFB1-exposed group showed obvious breakage and were significantly shorter. Compared with the AFB1-exposed group, the villi in the LA group were relatively intact and significantly longer. By measuring the villi length / crypt depth ratio, it was found that the ratio was significantly decreased in the AFB1-exposed group (P<0.05), while the ratio was significantly increased after treatment with Lactobacillus amyloliquefaciens LA, indicating that the pathological changes in the ileum of mice were improved (P<0.05).

[0063] As can be seen from the above, Lactobacillus amyloliquefaciens can significantly increase intestinal length and intestinal villus length, reduce intestinal crypt depth, and improve pathological changes in the ileum of mice.

[0064] Example 4 - Detection of the relative expression levels of inflammatory factors and tight junction proteins in intestinal tissue by quantitative real-time PCR

[0065] This embodiment is based on samples from mice collected in each group in Example 2. The relative expression levels of inflammatory factors and tight junction proteins in intestinal tissue samples were detected by quantitative real-time PCR and analyzed to verify the alleviating effect of Lactobacillus amyloliquefaciens on AFB1-induced intestinal damage in mice.

[0066] Based on the target gene, its sequence was searched on the NCBI website, and primer specificity was verified by Primer blast. The delivered primer sequences were synthesized by Shanghai Sangon Biotech Co., Ltd., and the primer sequences used are shown in Table 1. Total intestinal RNA was extracted using Trizol reagent through grinding, extraction, precipitation, washing, and dissolution. Then, using a Nanodrop 2000 instrument, 1 μL of double-distilled water was added for zeroing. After zeroing, concentration measurements were performed, ensuring the sample purity A260 / 280 was within the range of 1.8–2.0 before use. After measurement, the sample was stored at -80℃ for use. Reverse transcription was performed using the Evo M-MLV reverse transcription premix kit from Hunan Aikerui Biotechnology Co., Ltd., following the instructions. After reverse transcription, using cDNA as a template, quantitative real-time PCR was performed using the SYBR Green Pro Taq HS Premix kit from Hunan Aikerui Biotechnology Co., Ltd., following the instructions.

[0067] Table 1 Primer sequence list

[0068]

[0069] Analysis of experimental results:

[0070] (1) Effects of Lactobacillus amyloliquefaciens on the gene expression levels of inflammatory cytokines IL-1β, IL-6 and TNF-α in the ileum

[0071] RNA was extracted from intestinal tissue for quantitative real-time PCR to analyze the expression levels of inflammation-related genes (IL-1β, IL-6, and TNF-α). The results are as follows: Figure 4 As shown, AFB1 exposure led to a significant increase in the expression levels of inflammatory cytokines in intestinal tissue (P<0.05), while the expression of inflammatory cytokines in the ileum of the LA group was significantly reduced compared with that of the AFB1 group (P<0.05).

[0072] (2) Effects of Lactobacillus amyloliquefaciens on the gene expression levels of ileal tight junction proteins ZO-1, Occludin, and Claudin-1

[0073] The test results are as follows Figure 5 As shown, compared with the control group, AFB1 exposure significantly downregulated the expression levels of tight junction protein genes ZO-1, Occludin, and Claudin-1 (P<0.05). Compared with the AFB1 group, Lactobacillus amyloliquefaciens LA treatment significantly reversed the above changes and significantly increased the expression of intestinal tight junction proteins (P<0.05).

[0074] As can be seen from the above, Lactobacillus amyloliquefaciens significantly reduced the expression levels of intestinal inflammatory cytokines and increased the expression levels of intestinal tight junction proteins.

[0075] Example 5 - Effect of Lactobacillus amyloliquefaciens on intestinal permeability in mice

[0076] This embodiment uses the serum collected from each group of mice in Example 2 for detection and analysis to explore the effect of Lactobacillus amyloliquefaciens on intestinal permeability in mice with AFB1-induced intestinal injury, and to verify the alleviating effect of Lactobacillus amyloliquefaciens on AFB1-induced intestinal injury in mice.

[0077] Intestinal permeability was measured using serum collected from mice in different experimental groups. Samples were added to light-protected ELISA plates, and FD4 was diluted with blank serum to set a standard curve. 10 μL of serum + 90 μL of PBS was added to the sample wells, and the fluorescence signal was measured using a fluorescence spectrometer under the conditions of excitation wavelength of 490 nm and emission wavelength of 535 nm.

[0078] Inflammation and barrier damage in the gut can also cause changes in intestinal permeability.

[0079] The results are as follows Figure 6As shown, compared with the blank control group, the serum FD4 concentration of mice in the AFB1 group was significantly increased (P<0.05). Compared with the AFB1 group, treatment with Lactobacillus amyloliquefaciens LA significantly reduced the serum FD4 concentration (P<0.05), indicating that Lactobacillus amyloliquefaciens LA significantly improved intestinal permeability in mice and effectively protected and repaired the intestinal barrier.

[0080] In summary, the *Lactobacillus amyloliquefaciens* isolated and screened in this invention can improve AFB1-induced inflammatory intestinal damage and repair the intestinal barrier in mice by increasing intestinal length and villus length, reducing intestinal crypt depth, decreasing the expression level of intestinal inflammatory cytokines, increasing the expression level of intestinal tight junction proteins, and improving intestinal permeability. Furthermore, this method is safe and highly effective. Therefore, the *Lactobacillus amyloliquefaciens* of this invention has significant application value and research value in the preparation of therapeutic agents for the treatment and prevention of intestinal diseases.

[0081] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A type of Lactobacillus amyloliquefaciens, characterized in that, The Lactobacillus amyloliquefaciens was named Lactobacillus amyloliquefaciens QC1H and was deposited at the China Center for Type Culture Collection on March 28, 2023, with accession number CCTCC NO: M2023427.

2. The *Lactobacillus amyloliquefaciens* according to claim 1, characterized in that, The 16S rDNA sequence of the Lactobacillus amyloliquefaciens is shown in SEQ ID:

1.

3. The *Lactobacillus amyloliquefaciens* according to claim 1, characterized in that, The colonies of *Lactobacillus amyloliquefaciens* on MRS plates are round, white, with a raised, smooth surface, neat edges, and an opaque, single shape, with a diameter of approximately 2–3 mm.

4. The *Lactobacillus amyloliquefaciens* according to claim 1, characterized in that, The strain of *Lactobacillus amyloliquefaciens*, after Gram staining, appears blue-purple, is rod-shaped with blunt ends, exists singly, and has no spores.

5. A therapeutic agent, characterized in that, The active ingredient of the therapeutic agent comprises Lactobacillus amyloliquefaciens as described in any one of claims 1 to 4 and / or the fermentation product of Lactobacillus amyloliquefaciens.

6. The therapeutic agent according to claim 5, characterized in that, The concentration of the Lactobacillus amyloliquefaciens in the therapeutic agent is 10. 8 ~10 9 CFU / mL.

7. The therapeutic agent according to claim 5, characterized in that, When used in mice, the therapeutic agent is administered by gavage at a rate of 180–220 μL / day.

8. The application of a *Lactobacillus amyloliquefaciens* as described in any one of claims 1 to 4 or a therapeutic agent as described in any one of claims 5 to 7, characterized in that, The application includes at least one of the following applications: application in the preparation of a therapeutic agent for the prevention of enteropathy, and application in the preparation of a therapeutic agent for the treatment of enteropathy.

9. The application according to claim 8, characterized in that, The enteropathy includes AFB1-induced enteropathy.

10. The application according to claim 9, characterized in that, The AFB1-induced enteropathy includes intestinal damage, inflammatory lesions, and impaired intestinal barrier function.

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