Lactobacillus reuteri YZ-1 and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus reuteri YZ-1 and application thereof. The lactobacillus reuteri YZ-1 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.34974, and the lactobacillus reuteri YZ-1 is separated from the intestinal tract of an aquatic animal zebra fish. The strain can relieve fish liver and intestine injury induced by high-iron feed and reduce the content of total iron and ferrous iron in the liver; the fermentation supernate inhibits streptococcus dysgalactiae, but thalli do not inhibit streptococcus dysgalactiae; the survival rate of the zebrafish infected by the edwardsiella tarda is improved; expression of inflammatory factors is inhibited, and intestinal inflammation is improved. The invention also provides a microbial inoculum containing the strain and a functional fish feed. The strain disclosed by the invention has the functions of relieving iron overload, resisting infection and resisting inflammation, is unique in antibacterial mode and is suitable for being applied to aquaculture.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Lactobacillus reuteri YZ-1 and its applications. Background Technology

[0002] The intensive development of aquaculture has led to increasingly prominent disease problems. Bacterial diseases, such as those caused by Edwardsiella tarda, are a significant concern. (Edwardsiella tarda) Caused by Edwardsiella pneumoniae, Vibrio anguillarum (Vibrio anguillarum) Vibrio infection and Streptococcus pyogenes caused by it (Staphylococcus dysgalactiae) Streptococcal diseases and other illnesses caused by these diseases often result in significant economic losses for the aquaculture industry.

[0003] Meanwhile, with increasing environmental requirements and cost pressures, the proportion of plant protein sources replacing fishmeal in aquatic feeds is constantly rising. This substitution strategy often leads to an increase in anti-nutritional factors in the feed and an imbalance in the addition of trace elements (especially iron), which in turn causes intestinal dysfunction and metabolic abnormalities in aquatic animals. Studies have shown that excessive iron intake leads to the accumulation of iron ions in the liver and intestinal tissues, inducing oxidative stress, causing hepatocyte damage and impaired intestinal barrier function. This hepato-intestinal damage induced by high-iron feed has become a new health problem restricting the sustainable development of aquaculture.

[0004] In addition, various stress factors in the aquaculture environment (such as chemical stimulation and water quality fluctuations) often lead to excessive intestinal inflammatory response in aquatic animals, manifested as intestinal tissue damage and upregulation of inflammatory factors (such as TNF-α, IL-1β, IL-6, and IL-8), which further weakens the body's immunity and increases the risk of secondary infections.

[0005] Probiotics, as a green and safe biological control method, can promote the health of aquatic animals by regulating the intestinal microecology, competitively excluding pathogens, and enhancing immunity. *Lactobacillus reuteri* (Limosilactobacillus) reuteri) It is a recognized probiotic, widely used in humans and terrestrial animals. However, current technologies regarding the application of *Lactobacillus reuteri* in aquaculture primarily focus on promoting growth or broad-spectrum antibacterial activity, which has the following shortcomings: 1. No effective solutions have been found for the liver and gut damage induced by high-iron feed, a problem unique to aquaculture. Although some studies have focused on micronutrient metabolism, no probiotic strains have been reported that can actively reduce liver iron accumulation and alleviate tissue damage caused by high iron intake. 2. For specific aquatic pathogens such as Streptococcus dysgalactiae, the inhibitory effect of probiotics currently relies mainly on bacterial competition, while strains that can exert antibacterial function by secreting metabolites and have a unique antibacterial mode are still scarce. 3. There are no existing reports on comprehensive probiotics that can simultaneously address multiple health issues such as pathogen infection, chemical enteritis, and nutritional iron overload. In particular, there is a lack of indigenous probiotic strains derived from aquatic animals that possess all of the above-mentioned multiple functions.

[0006] Therefore, screening a strain of *Lactobacillus reuteri* isolated from aquatic animals that can alleviate hepatobiliary damage induced by high-iron feed, has a unique antibacterial mode against *Streptococcus faecium*, and can simultaneously resist pathogen infection and intestinal inflammation has important practical significance and application value. Summary of the Invention

[0007] This invention addresses the current lack of comprehensive probiotics capable of simultaneously resolving multiple health problems in aquaculture, such as liver and intestinal damage induced by high-iron feed, Streptococcus pyogenes infection, and bacterial enteritis. It provides a strain of Lactobacillus reuteri YZ-1 and its applications.

[0008] To achieve the above objectives, the specific technical solution of the present invention is as follows: In a first aspect, the present invention provides a strain of *Lactobacillus reuteri*. (Limosilactobacillus) reuteri) YZ-1, the Lactobacillus reuteri YZ-1, is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 34974, and the Lactobacillus reuteri YZ-1 can alleviate liver and intestinal damage induced by high iron feed in fish.

[0009] Furthermore, the *Lactobacillus reuteri* YZ-1 was isolated from the intestines of zebrafish.

[0010] Furthermore, the method of mitigating hepatobiliary damage induced by high-iron feed includes reducing the total iron content and / or ferrous iron content in liver tissue.

[0011] Furthermore, the fermentation supernatant of *Lactobacillus reuteri* YZ-1 can inhibit *Streptococcus pyogenes*. (Staphylococcus dysgalactiae) The bacteria of *Lactobacillus reuteri* YZ-1 have no inhibitory effect on *Streptococcus dysgalactiae*.

[0012] Secondly, the present invention provides a microbial agent comprising the aforementioned *Lactobacillus reuteri* YZ-1.

[0013] Thirdly, the present invention provides the application of the above-mentioned microbial agent in the preparation of functional fish feed, wherein the functional fish feed is used to alleviate liver and intestinal damage in fish induced by high-iron feed.

[0014] Furthermore, the functional fish feed is also used to improve fish resistance to Edwardsiella tarda. (Edwardsiella) tarda) Resistance to infection.

[0015] Furthermore, the functional fish feed is also used to improve fish intestinal inflammation induced by sodium dextran sulfate, and the improvement includes inhibiting the expression of inflammatory factor-related genes TNF-α, IL-1β, IL-6, and IL-8.

[0016] Fourthly, the present invention provides a functional fish feed comprising the above-mentioned microbial agent and a basic feed, wherein the functional fish feed is used to alleviate liver and intestinal damage in fish induced by high-iron feed.

[0017] Furthermore, the number of viable Lactobacillus reuteri YZ-1 bacteria per gram of the aforementioned functional fish feed is not less than 1 × 10⁻⁶. 9 CFU.

[0018] Fifthly, the present invention provides a method for using the above-mentioned functional fish feed, wherein the fish are fed the functional fish feed to their satiated state 2 to 3 times a day for more than 2 weeks.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The *Lactobacillus reuteri* YZ-1 strain of this invention can alleviate hepatointestinal damage induced by high-iron feed in fish. Experiments have shown that this strain can significantly reduce the total iron and ferrous iron content in the liver tissue of fish fed high-iron feed, alleviating liver tissue damage caused by high iron intake, while improving intestinal structure and increasing intestinal villus height. This function is of great significance for solving the problem of iron overload caused by protein source substitution and micronutrient imbalance in aquaculture.

[0020] 2. The fermentation supernatant of *Lactobacillus reuteri* YZ-1 of this invention can significantly inhibit *Streptococcus pyogenes*. (Staphylococcus dysgalactiae) The bacteria themselves have no inhibitory effect on the pathogen, suggesting that this strain can exert its antibacterial function by secreting specific metabolites. This unique antibacterial mechanism differs from the competitive inhibition of conventional probiotics, providing a new approach for developing novel antibacterial agents for aquaculture. 3. The *Lactobacillus reuteri* YZ-1 strain of this invention can significantly enhance the host's resistance to pathogens. This strain can improve the resistance of *Edwards tarda*. (Edwardsiella tarda) The survival rate of infected zebrafish fry and adults showed a good anti-infection protective effect; 4. The Lactobacillus reuteri YZ-1 of this invention can inhibit the expression of inflammatory factor-related genes tnf-α, il-1β, il-6, and il-8, improve intestinal inflammation induced by sodium dextran sulfate in zebrafish larvae, and increase intestinal villus height.

[0021] Instructions for the Preservation of Biological Materials YZ-1, referred to in this invention as *Lactobacillus reuteri* YZ-1, is classified as *Lactobacillus reuteri*. Limosilactobacillus reuteri Its Latin name is: Limosilactobacillus reuteri It was deposited on June 23, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34974. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The results of the isolation and identification of *Lactobacillus reuteri* YZ-1 in this invention; Figure 1 In the image, A represents the morphology of *Lactobacillus reuteri* YZ-1 under a scanning electron microscope. Figure 1 In the figure, B represents the growth curve of *Lactobacillus reuteri* YZ-1. Figure 1 In the figure, C represents the antibiotic susceptibility result of Lactobacillus reuteri YZ-1.

[0024] Figure 2 This invention demonstrates the antibacterial effect of *Lactobacillus reuteri* YZ-1. Figure 2 In the figure, A represents the inhibitory effect of Lactobacillus reuteri YZ-1 PBS resuspension on the pathogen. Figure 2 In the figure, B represents the inhibitory effect of the fermentation supernatant of Lactobacillus reuteri YZ-1 on pathogens.

[0025] Figure 3 This presents the results of the effect of Lactobacillus reuteri YZ-1 on the resistance of zebrafish to Edwardsiella tarda infection in this invention; Figure 3 In the figure, A represents the survival rate statistics of zebrafish fry. Figure 3 In the figure, B represents the survival rate statistics of adult zebrafish; * indicates p <0.05, **** indicates p <0.0001.

[0026] Figure 4 This invention relates to the effect of Lactobacillus reuteri YZ-1 on intestinal inflammation induced by sodium dextran sulfate in zebrafish larvae. Figure 4 In the figure, A represents the survival rate statistics for each group. Figure 4 B in the text represents genes related to inflammatory factors. tnf -α , il - 1β , il - 6、il - 8 The relative expression level. Figure 4 In the image, C represents the HE staining results of whole sections of juvenile fish. The images in row b are magnified views of the area circled in black in row a. The scale bar of row a is 50 μm, and the scale bar of row b is 20 μm. Figure 4 D in the figure represents the statistical result of the intestinal villi height of the larvae. * indicates p <0.05, ** indicates p <0.01, *** indicates p <0.001, **** indicates p <0.0001.

[0027] Figure 5 This invention relates to the effect of Lactobacillus reuteri YZ-1 on hepatointestinal damage induced by high-iron diet in adult zebrafish. Figure 5 In the figure, A represents the observation of HE staining of liver sections. From left to right, the HE staining results are for the Ctr1 group, HID group, and HID+LR group. Scale bar: 50μm. Figure 5 B in the graph represents the total iron content of liver tissue. Figure 5 The figure C represents the statistical graph of ferrous iron content in liver tissue. Figure 5 D in the text represents a gene related to intestinal inflammatory factors. tnf - α , il - 1β , il - 6 The relative expression level. Figure 5 In the image, E represents the observation of intestinal sections stained with HE. The image in row b is a magnified view of the area circled in black in the image in row a. The scale bar of the image in row a is 100 μm, and the scale bar of the image in row b is 50 μm. Figure 5 F in the figure represents the statistical height of zebrafish intestinal villi. ** indicates p <0.01, **** indicates p <0.0001. Detailed Implementation

[0028] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific 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. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0029] Edwardsiella tarda in this invention ( Edwardsiella tarda ) and Streptococcus lactis ( Staphylococcus dysgalactiae The Vibrio anguillarum (a type of bacteria) was donated by the team led by Chi Heng at Ocean University of China. Vibrio anguillarum ), Citrobacter ( Citrobacter portucalensis ), Enterobacteriaceae ( Enterobacter asburiae ) and Staphylococcus aureus ( Staphylococcus aureus The strains were donated by the team of Chen Shiyong from Qingdao Agricultural University. All of the above strains could be obtained through conventional means from public culture collection centers or relevant research institutions, or isolated using methods known in the field.

[0030] The high-iron feed in this invention is made by adding ferrous sulfate heptahydrate to conventional fish feed. The composition of conventional fish feed and high-iron feed is shown in Table 1, and the corresponding nutrient ratio is shown in Table 2.

[0031] Table 1 Feed composition

[0032] Note: Each gram of vitamin premix contains the following components: 5 mg thiamine hydrochloride, 10 mg riboflavin, 10 mg calcium pantothenate, 0.6 mg D-biotin, 4 mg pyridoxine hydrochloride, 1.5 mg folic acid, 200 mg inositol, 60 mg L-vitamin C-2-phosphate magnesium, 6.05 mg niacin, 50 mg α-vitamin E acetate, 4 mg vitamin K, 2000 IU retinyl acetate, 400 IU vitamin D3, and is supplemented to 1 g with microcrystalline cellulose. Each gram of mineral salt premix contains the following components: 135.8 mg calcium dihydrogen phosphate, 327 mg calcium lactate, 137 mg magnesium sulfate, 87.2 mg sodium dihydrogen phosphate, 43.5 mg sodium chloride, 0.15 mg aluminum chloride, 0.125 mg potassium iodate, 75 mg potassium chloride, 0.1 mg copper chloride, 0.80 mg manganese sulfate, 1 mg cobalt chloride, 3 mg zinc sulfate, and is made up to 1 g with microcrystalline cellulose.

[0033] Table 2 Nutritional composition of feed

[0034] With the development of aquaculture, health problems in aquatic animals are becoming increasingly prominent. Frequent bacterial and viral infections, coupled with the substitution of dietary protein sources leading to impaired intestinal function, excessive inflammation, and weakened immunity, result in slow growth. Furthermore, iron overload can easily damage organs such as the liver and intestines. Probiotics, as beneficial live microorganisms, can significantly improve fish growth and immune performance. However, most current aquatic probiotics are derived from humans or livestock, and due to species differences, their application effects are often unsatisfactory or even produce negative effects. Therefore, there is an urgent need to develop native aquatic probiotics, explore their beneficial functions, and promote their application and industrialization.

[0035] This invention provides a strain of *Lactobacillus reuteri* YZ-1 and its applications. This invention isolates and screens a strain of *Lactobacillus reuteri* YZ-1 from the intestines of aquatic animals, and classifies it as *Lactobacillus reuteri*. Limosilactobacillus reuteri This strain, *Lactobacillus reuteri* YZ-1, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34974. It exhibits significant antibacterial effects against a variety of common aquatic pathogens. This strain can effectively improve the survival rate of aquatic animals under *Edwards tarda* infection, alleviate intestinal inflammation in zebrafish larvae and juveniles induced by sodium dextran sulfate and improve intestinal morphology. It can also reduce liver and intestinal damage in fish caused by high-iron feed, demonstrating good probiotic potential and application value.

[0036] Example 1: Isolation and identification of Lactobacillus reuteri YZ-1 I. Strains Isolation Intestinal samples were collected from healthy zebrafish in the zebrafish breeding facility at the Yushan Campus of Ocean University of China. The intestinal samples were homogenized under aseptic conditions and spread on LBS agar plates. After incubation at 37°C for 24 hours, single colonies were randomly picked and purified by streak culturing twice to obtain a strain YZ-1.

[0037] II. Identification and biological characteristics analysis of *Lactobacillus reuteri* YZ-1 1. Morphological observation Morphological observation of strain YZ-1 using scanning electron microscopy yielded the following results: Figure 1 As shown in A, Lactobacillus reuteri YZ-1 presents as a long rod-shaped organism.

[0038] 2. Growth status measurement The YZ-1 strain was inoculated into MRS medium and cultured at 37℃. The growth of the YZ-1 strain was observed. Results are shown below. Figure 1 In strain B, it was found that the exponential growth phase of the strain was from 3 to 9 hours of culture, and it tended to stabilize after 12 hours of culture.

[0039] 3. Physiological and biochemical identification The physiological and biochemical characteristics of strain YZ-1 were identified using a bacterial physiological and biochemical identification kit. The physiological and biochemical indicators included: mannose, galactose, glucose, fructose, sucrose, maltose, trehalose, dextrin, mannitol, starch, arabinose, amygdalin, raffinose, rhamnose, lactose, cellobiose, xylose, salicin, sorbitol, and gluconate. Each indicator was tested in triplicate, with a blank culture medium as a blank control. The results were observed and recorded after overnight incubation at 37°C.

[0040] Table 3. Physiological and biochemical assay results of strain YZ-1

[0041] Note: "+" represents positive, and "-" represents negative.

[0042] The results are shown in Table 3. The YZ-1 strain can utilize mannose, galactose, glucose, fructose, sucrose, maltose, dextrin, mannitol, starch, arabinose, amygdalin, raffinose, rhamnose, lactose, cellobiose, xylose, salicin and sorbitol, but cannot utilize gluconate.

[0043] 4. Antibiotic susceptibility analysis The antibiotic susceptibility of *Lactobacillus reuteri* YZ-1 was assessed using the disk diffusion method. The bacterial culture was evenly spread onto LBS agar plates, and antibiotic disks (purchased from Hangzhou Binhe Microbial Reagent Co., Ltd.) were attached. After incubation at 37°C overnight, the diameter of the inhibition zone was observed and recorded. The antibiotics used included: kanamycin, chloramphenicol, neomycin, gentamicin, tetracycline, polymyxin B, penicillin G, vancomycin, clarithromycin, and ampicillin.

[0044] Experimental results are as follows Figure 1 As shown in C, *Lactobacillus reuteri* YZ-1 exhibits resistance to kanamycin, gentamicin, polymyxin B, and vancomycin, but sensitivity to chloramphenicol, neomycin, tetracycline, penicillin G, clarithromycin, and ampicillin.

[0045] 5. Molecular biological identification The YZ-1 strain was identified by 16S rRNA sequencing. Using the commonly used upstream primer 27F and downstream primer 1492R for 16S rRNA sequencing, PCR amplification was performed on the YZ-1 bacterial culture according to the following PCR reaction system and procedure to obtain the PCR amplification products. The nucleotide sequence of 27F is 5'-AGAGTTTGATCCTGGCTCAG-3'; and the nucleotide sequence of 1492R is 5'-CTACGGCTACCTTGTTACGA-3'.

[0046] PCR reaction system: 1 μL bacterial template, 12.5 μL 2×Taq, 1 μL 27F, 1 L 1492R, and DEPC water to a final volume of 25 μL.

[0047] Reaction program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 51℃ annealing for 30 s, 72℃ extension for 1.5 min, 35 cycles; 72℃ extension for 5 min.

[0048] After passing agarose gel electrophoresis, the PCR amplification products, with a fragment size of approximately 1500 bp, were sent to a biotechnology company for sequencing to obtain the 16S rRNA sequence of strain YZ-1.

[0049] The sequencing results were compared with BLAST in NCBI, and the YZ-1 strain was identified as belonging to Lactobacillus reuteri, and named Lactobacillus reuteri YZ-1.

[0050] 6. Whole genome sequencing The library was constructed using the Illumina TruSeq™ Nano DNA Sample Prep Kit method, and the specific steps are as follows: Starting amount: Library construction was initiated with 1 μg of Lactobacillus reuteri YZ-1 genomic DNA; DNA fragmentation: Genomic DNA was broken down into 300-500 bp fragments using a Covaris M220 sonicator; Library Construction: Fill in the ends of DNA fragments Add an "A" base to the 3' end Connect the index adapter (TruSeq™ Nano DNA Sample Prep Kit) Library enrichment: Perform PCR amplification, with 8 amplification cycles; Fragment screening: The target band (Certified Low Range UltraAgarose) was recovered by 2% agarose gel electrophoresis. Library quantification: The library was quantified using a TBS380 (Picogreen), and the data were mixed according to the specified proportions before being fed into the machine. Cluster generation: Bridged PCR amplification was performed on the cBot solid-phase support to generate clusters; Sequencing: Paired-end sequencing was performed on the Illumina Hiseq sequencing platform, with a read length of 2×150bp.

[0051] The raw sequencing data was subjected to quality trimming and filtering to obtain high-quality sequencing data, which was then used for genome assembly. The assembly results showed that the genome of *Lactobacillus reuteri* YZ-1 had been completely assembled.

[0052] As shown in SEQ ID NO.1: Genome size 2056191 bp, complete circular genome. GC content 40.56%. Consistent with Lactobacillus reuteri characteristics.

[0053] The genome was screened for virulence and resistance genes, and the results showed: Virulence genes: No known bacterial virulence genes were detected. Antibiotic resistance genes: No known antibiotic resistance genes were detected. Integrase gene: Not detected Repressor protein gene: Not detected The above results indicate that Lactobacillus reuteri YZ-1 has good biocompatibility, does not carry transferable drug resistance genes and virulence factors, and is suitable as a probiotic for aquaculture.

[0054] Example 2: Antibacterial activity of Lactobacillus reuteri YZ-1 I. Antibacterial effect of YZ-1 Using Edwardsiella tarda, Streptococcus pyogenes, Vibrio anguillarum, Citrobacter, Enterobacter, and Staphylococcus aureus as pathogens, a pathogenic fermentation broth was prepared. The Oxford cup method was used to prepare LB agar plates. The aforementioned pathogens were added to cooled LB agar plates to achieve a final pathogen concentration of 10-1 in the resulting LB plates. 8 CFU / mL.

[0055] Preparation of fermentation supernatant of Lactobacillus reuteri YZ-1: Centrifuge 8000g of Lactobacillus reuteri YZ-1 bacterial culture for 5min and the obtained centrifugation supernatant is the fermentation supernatant of Lactobacillus reuteri YZ-1.

[0056] Preparation of *Lactobacillus reuteri* YZ-1 PBS resuspension: Centrifuge *Lactobacillus reuteri* YZ-1 bacterial suspension at 8000g for 5 min, collect the bacterial pellet, and dilute the bacterial cells with PBS to a viable count of 102. 8 CFU / mL was used to obtain a PBS resuspension of Lactobacillus reuteri YZ-1.

[0057] Using a pipette, 100 μL of Lactobacillus reuteri YZ-1 fermentation supernatant and Lactobacillus reuteri YZ-1 PBS resuspension were respectively inoculated into LB agar plates containing pathogens. After overnight incubation at 37°C, the diameter of the inhibition zone was accurately measured using calipers.

[0058] The results of the inhibition test of Lactobacillus reuteri YZ-1 against the pathogen are shown in the figure. Figure 2 And Table 4.

[0059] Table 4. Antibacterial effect of Lactobacillus reuteri YZ-1 against pathogens.

[0060] Note: "+" indicates antibacterial effect, and "-" indicates no antibacterial effect.

[0061] from Figure 2As shown in Table 4, except for the PBS resuspension of *Lactobacillus reuteri* YZ-1 which had no inhibitory effect on *Streptococcus dysgalactiae*, the PBS resuspension and supernatant of *Lactobacillus reuteri* YZ-1 showed inhibitory effects on other tested pathogens, with the strongest inhibitory effect on *Vibrio anguillarum*.

[0062] II. Protective effect of YZ-1 on challenge with Edwardsiella tarda in zebrafish 1. Protective effect on zebrafish fry Three days after zebrafish fertilization, zebrafish fry were evenly placed into two separate culture media containing 4 mL of normal embryo culture medium and 10 mL of live bacteria culture medium. 7 In a six-well plate containing embryonic culture medium of *Lactobacillus reuteri* YZ-1 at CFU / mL, after incubation for three days, use a medium containing 10 CFU / mL of viable bacteria. 8 Embryo culture medium containing CFU / mL Edwardsiella tarda was used for challenge experiments, and the survival rate was calculated.

[0063] The results are as follows Figure 3 As shown in A, using 10 7 Incubation with CFU / mL of *Lactobacillus reuteri* significantly improved the resistance of zebrafish larvae to infection by *Edwards tarda*.

[0064] 2. Protective effect on adult zebrafish Preparation of feed containing *Lactobacillus reuteri* YZ-1: *Lactobacillus reuteri* YZ-1 bacterial solution was evenly sprayed onto conventional fish feed, ensuring that the viable count of *Lactobacillus reuteri* YZ-1 per gram of feed was 10-1. 9 CFU.

[0065] Three-month-old adult zebrafish were evenly placed in 3L tanks, with 10 fish per tank. They were fed either regular fish food or a diet containing *Lactobacillus reuteri* YZ-1. The fish were fed twice daily, at 9:00 AM and 6:00 PM, with water changes after each feeding, for two weeks. After the feeding experiment, each treatment group of zebrafish was intraperitoneally injected with 10... 8 CFU-treated Edwardsiella tarda was used in challenge experiments to determine the survival rate.

[0066] Experimental results are as follows Figure 3 As shown in B, the diet containing Lactobacillus reuteri YZ-1 significantly improved the infection resistance of adult zebrafish, indicating that Lactobacillus reuteri YZ-1 can significantly improve the infection resistance of adult zebrafish against pathogens.

[0067] Example 3: Anti-inflammatory ability of Lactobacillus reuteri YZ-1 The experimental groups were as follows: Control group: Zebrafish larvae were incubated in 4 mL of normal embryo culture medium 3 days after fertilization; DSS group: Zebrafish larvae were incubated in 0.5 w / v% sodium dextran sulfate solution 3 days after fertilization to induce colitis; LR group: Lactobacillus reuteri YZ-1 bacterial suspension was added to 0.5 w / v% sodium dextran sulfate solution to obtain a mixed solution with 10 viable YZ-1 bacteria. 7 Three days after zebrafish fertilization, zebrafish fry were placed in the above-mentioned mixed solution for incubation. Each group was incubated for three days, with the solution changed daily and survival rates recorded. After three days of incubation, the fish were washed three times with PBS. Five fish were collected per sample and placed in 1.5 mL centrifuge tubes. The entire fish were homogenized for the determination of relative gene expression levels.

[0068] RNA was extracted from collected samples and reverse transcribed to obtain cDNA. Using cDNA as a template, real-time quantitative PCR was used to analyze the inflammatory factor-related genes in zebrafish cDNA. tnf - α , il - 1β , il - 6、il - 8 Expression levels, detection tnf - α , il - 1β , il - 6、il - 8 The upstream and downstream primer sequence information is as follows.

[0069] tnf - α Upstream primer: 5'-GCGCTTTTCTGAATCCTACG-3', SEQ ID NO.4; tnf - α Downstream primer: 5'-TGCCCAGTCTGTCTCCTTCT-3', SEQ ID NO.5; il - 1β Upstream primer: 5'-TGGACTTCGCAGCACAAAATG-3', SEQ ID NO.6; il - 1β Downstream primer: 5'-GTTCACTTCACGCTCTTGGATG-3', SEQ ID NO.7; il - 6Upstream primer: 5'-CACGGAAAGATGTCTAACGCGAAT-3', SEQ ID NO.8; il - 6 Downstream primer: 5'-TTTATGGCCTCCAGCAGTCGTTT-3', SEQ ID NO.9; il - 8 Upstream primer: 5'-CACGGAAAGATGTCTAACGCGAAT-3', SEQ ID NO.10; il - 8 Downstream primer: 5'-CTTAACCCATGGAGCAGAGG-3', SEQ ID NO.11.

[0070] PCR reaction system: 2 μL cDNA, 7.5 μL 2×ChamQ SYBR qPCR Master, 0.3 μL upstream primer, 0.3 L downstream primer, and DEPC water to a final volume of 25 μL.

[0071] The reaction program was as follows: pre-denaturation at 95℃ for 2 min; denaturation at 95℃ for 10 s, annealing at 58℃ for 10 s, extension at 72℃ for 20 s, for 40 cycles.

[0072] Meanwhile, another 1.5 mL centrifuge tube was used to collect larvae, which were fixed with 4% paraformaldehyde for 24 h, then dehydrated in a gradient manner, embedded in paraffin, and 7 mm paraffin sections were prepared using a microtome. The zebrafish larvae were stained with HE using the HE staining kit from Beyotime Biotechnology Co., Ltd., according to the instructions, to observe the intestinal morphology.

[0073] GraphPad Prism 8.0 was used for statistical analysis of the experimental data. One-way ANOVA was used to process the data, and Dunnett's test was used for multiple comparisons. p <0.05 indicates a significant difference between treatment groups. All experimental data are expressed as mean ± standard error.

[0074] Experimental results are as follows Figure 4 As shown, *Lactobacillus reuteri* YZ-1 significantly improved the survival rate of zebrafish larvae under DSS treatment and significantly reduced related inflammatory factors. tnf - α , il - 1β , il - 6、il - 8 Gene expression significantly improved intestinal damage in zebrafish larvae induced by sodium dextran sulfate and increased intestinal villus height.

[0075] Example 4: The ability of *Lactobacillus reuteri* YZ-1 to alleviate iron overload Experimental Design: Three-month-old wild-type male zebrafish were selected as experimental subjects and randomly assigned to three experimental tanks. Each experimental group had three parallel groups, with 10 zebrafish in each tank. The experimental group fed with conventional fish food was designated as the Ctr1 group; the experimental group fed with high-iron feed was designated as the HID group; and the experimental group fed with high-iron feed containing *Lactobacillus reuteri* YZ-1 was designated as the HID+LR group. The viable count of *Lactobacillus reuteri* YZ-1 in this feed was 10-1. 9 CFU / g. First, all zebrafish were fed a normal diet for one week to acclimatize to the experimental environment. They were fed to satiety at 9:00 AM and 6:00 PM daily, with the tank water changed after each feeding, for a period of two weeks. After the feeding experiment, the zebrafish in each group were fasted for 24 hours before sampling. The zebrafish were anesthetized with a 0.1% tricaine solution, and after they lost mobility, they were dissected. Intestinal and liver tissue samples were collected, placed in centrifuge tubes, frozen in liquid nitrogen, and then stored at -80°C for subsequent analysis.

[0076] The ferrous ion content and total iron content in the liver were detected according to the instructions of the ferrous ion detection kit from Beijing Solarbio Science & Technology Co., Ltd. and the tissue iron assay kit from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., respectively. The intestines and liver were stained with hematoxylin and eosin (HE) to observe their morphology.

[0077] The results are as follows Figure 5 As shown, *Lactobacillus reuteri* YZ-1 can significantly alleviate liver damage induced by high-iron diets and reduce the accumulation of ferrous iron and total iron in the liver caused by high-iron diets. It can also significantly reduce the gene expression of intestinal inflammatory factors induced by high-iron diets. tnf - α , il - 1β , il - 6 It can upregulate and significantly improve intestinal structure and increase intestinal villus height.

[0078] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

[0079] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A strain of *Lactobacillus reuteri* (Limosilactobacillus reuteri) YZ-1, characterized in that, The *Lactobacillus reuteri* YZ-1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34974. *Lactobacillus reuteri* YZ-1 was isolated from the intestine of zebrafish, and it can alleviate liver and intestinal damage induced by high-iron feed in fish.

2. The *Lactobacillus reuteri* YZ-1 according to claim 1 or 2, characterized in that, The method for mitigating hepatobiliary damage induced by high-iron feed includes reducing the total iron content and / or ferrous iron content in liver tissue.

3. The *Lactobacillus reuteri* YZ-1 according to claim 1, characterized in that, The fermentation supernatant of *Lactobacillus reuteri* YZ-1 can inhibit *Streptococcus galactiae*. (Staphylococcus dysgalactiae) .

4. A microbial agent, characterized in that, It contains Lactobacillus reuteri YZ-1 as described in claim 1.

5. The application of the microbial agent according to claim 4 in the preparation of functional fish feed, characterized in that, The functional fish feed is used to alleviate liver and intestinal damage in fish induced by high-iron feed.

6. The application according to claim 5, characterized in that, The functional fish feed is also used to improve fish resistance to Edwardsiella tarda. (Edwardsiella tarda) Resistance to infection.

7. The application according to claim 5, characterized in that, The functional fish feed is also used to improve intestinal inflammation in fish induced by sodium dextran sulfate, and the improvement includes inhibiting the expression of inflammatory factor-related genes TNF-α, IL-1β, IL-6, and IL-8.

8. A functional fish feed, characterized in that, The product comprises the microbial agent as described in claim 4 and a basic feed, wherein the functional fish feed is used to alleviate liver and intestinal damage in fish induced by high-iron feed.

9. The functional fish feed according to claim 8, characterized in that, The number of viable Lactobacillus reuteri YZ-1 per gram of the functional fish feed shall not be less than 1 × 10⁻⁶. 9 CFU.

10. A method of using the functional fish feed as described in claim 9, characterized in that, Feed the fish with the functional fish feed described above 2 to 3 times a day for at least 2 weeks.

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