An antimicrobial peptide for improving intestinal health and preventing diarrhea in animals and its preparation method.

Abstract

This invention belongs to the field of biotechnology, specifically relating to an antimicrobial peptide for improving intestinal health and combating diarrhea in animals, and its preparation method. The invention prepares a novel antimicrobial peptide with the amino acid sequence shown in SEQ ID NO:1. Experiments have verified that after one week of feeding in an unfamiliar environment, the antimicrobial peptide can slightly increase the levels of propionic acid and butyric acid in the experimental group of cats. Furthermore, the overall BCFAs levels in the experimental group were lower than those in the control group before and after transportation. Although there were no significant differences in α-diversity and β-diversity between the experimental group fed the antimicrobial peptide and the control group, feeding the antimicrobial peptide significantly increased the levels of Bacteroides, Parabacterium, and Prevotella in the experimental group, and significantly reduced the levels of harmful bacteria Salmonella and Actinomyces. Therefore, the antimicrobial peptide of this invention can be used to prepare products for improving intestinal health and combating diarrhea in animals, such as feed additives or oral liquids.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an antimicrobial peptide that improves intestinal health and prevents diarrhea in animals, and its preparation method. Background Technology

[0002] With the rapid development of the pet economy, the health of cats, as common companion animals, has become a major concern for breeders and pet owners. Diarrhea is one of the most common digestive tract diseases in cats, with a significantly increased incidence under stress conditions such as transportation, food changes, and environmental changes. Studies have shown that transportation stress disrupts the balance of the cat's gut microbiota, leading to impaired intestinal barrier function and abnormal immune responses, thus triggering diarrhea. The gut microbiota, as the core regulator of gut health, plays a crucial role in maintaining intestinal homeostasis through its metabolites, short-chain fatty acids (SCFAs) and branched-chain fatty acids (BCFAs). SCFAs (such as propionic acid and butyric acid) can exert anti-inflammatory and anti-diarrheal effects by promoting intestinal epithelial cell proliferation, enhancing mucosal barrier function, and regulating immune cell differentiation. BCFAs (such as isobutyric acid and isovaleric acid) are mainly produced by opportunistic pathogens (such as Salmonella), and excessive accumulation can disrupt the intestinal pH balance, irritate the intestinal mucosa, and induce diarrhea.

[0003] Currently, the main methods for preventing and treating diarrhea in pet cats include antibiotics, probiotics, and prebiotics. However, the overuse of antibiotics can easily lead to the emergence of drug-resistant strains and may disrupt the diversity of intestinal flora, causing secondary infections. While probiotics can regulate the flora, their colonization ability is limited by the host's intestinal environment, resulting in unstable effects. Prebiotics, on the other hand, rely on the synergistic effect of the existing intestinal flora and have limited ability to repair severely imbalanced intestines. In recent years, antimicrobial peptides (AMPs) have become a research hotspot in the field of antidiarrheal treatment due to their broad-spectrum antibacterial activity, low drug resistance, and minimal interference with the host flora. However, most existing antimicrobial peptides are designed for livestock or humans, and their application in pet cats still faces the following problems: First, the regulatory effects of most antimicrobial peptides on the intestinal flora are unclear, making it difficult to specifically promote the proliferation of beneficial bacteria (such as Bacteroides and Parabacteroides) and inhibit harmful bacteria (such as Salmonella and Actinomyces); Second, their regulatory effects on intestinal metabolites lack systematic verification, especially their ability to target and regulate key SCFAs such as propionic acid and butyric acid, as well as BCFAs; Third, whether antimicrobial peptides can maintain intestinal microecological homeostasis and improve diarrhea symptoms under complex environments such as transportation stress still needs further verification.

[0004] Therefore, developing an antidiarrheal antimicrobial peptide and its preparation method that can specifically regulate the metabolic products of the intestinal flora of pet cats (such as increasing propionic acid and butyric acid levels and reducing BCFAs), selectively proliferate beneficial bacteria (such as Bacteroides and Parabacteroides) and inhibit harmful bacteria (such as Salmonella and Actinomyces), and is suitable for scenarios such as transportation stress, has important theoretical value and practical application significance. Summary of the Invention

[0005] The first objective of this invention is to provide an antimicrobial peptide that improves intestinal health and combats diarrhea in animals, the amino acid sequence of which is shown in SEQ ID NO:1.

[0006] A second objective of the present invention is to provide a nucleotide sequence encoding the above-mentioned antimicrobial peptide.

[0007] A third objective of this invention is to provide a recombinant expression vector containing the above-mentioned nucleotide sequence, wherein the vector is a pET-28a vector.

[0008] A fourth objective of the present invention is to provide a host bacterium containing the above-mentioned recombinant expression vector, wherein the host bacterium is Escherichia coli BL21(DE3).

[0009] A fifth objective of this invention is to provide a method for preparing the above-mentioned antimicrobial peptide, comprising the following steps:

[0010] (1) Construction of recombinant expression vector: The above nucleotide sequence was inserted into the Nde I / Xho I restriction site of the pET-28a vector to obtain the recombinant plasmid;

[0011] (2) Transformation of host bacteria: The recombinant plasmid was introduced into Escherichia coli BL21(DE3), and positive clones were obtained by PCR identification;

[0012] (3) Fermentation culture: Positive clones were inoculated into LB medium and cultured at 37°C and 200 rpm until OD. 600 =0.8, add 0.1 mM IPTG to induce expression, and continue culturing for 4 hours;

[0013] (4) Purification: After fermentation, the fermentation broth is centrifuged and subjected to ammonium sulfate precipitation and ion exchange chromatography to obtain the antimicrobial peptide.

[0014] In some embodiments, the method further includes HPLC verification and sequencing steps.

[0015] The sixth objective of this invention is to provide the application of the above-mentioned antimicrobial peptides in the preparation of products that improve animal gut health and combat diarrhea, wherein improving animal gut health includes increasing the levels of Bacteroides, Parabacteroides and Paraprevostella in the gut, or decreasing the levels of harmful bacteria Salmonella and Actinomyces.

[0016] A seventh object of the present invention is to provide a composition for improving intestinal health and preventing diarrhea in animals, comprising the above-mentioned antimicrobial peptide and a pharmaceutically acceptable carrier; said carrier being starch, microcrystalline cellulose or starch paste.

[0017] In some embodiments, the antimicrobial peptide is 0.1%-1.0% by mass.

[0018] An eighth object of the present invention is to provide the use of the above-mentioned antimicrobial peptide in the preparation of pet feed additives or oral solutions for improving animal gut health and preventing diarrhea.

[0019] Compared with the prior art, the present invention has at least the following beneficial effects:

[0020] (1) Metabolite regulation: precisely enhances the level of beneficial SCFAs and inhibits the accumulation of harmful BCFAs;

[0021] Experimental data showed that the levels of propionic acid and butyric acid in the intestines of cats fed the antimicrobial peptides of this invention were significantly higher than those in the control group (P < 0.05), while the overall BCFAs levels in the experimental group before and after transportation were lower than those in the control group (P < 0.05). Propionic acid and butyric acid, as the main energy sources for intestinal mucosal cells, can effectively alleviate diarrhea by enhancing the intestinal epithelial barrier function and inhibiting the release of inflammatory factors; the reduction of BCFAs reduces the stimulation of the intestinal mucosa by harmful bacterial metabolites, further reducing the risk of diarrhea.

[0022] (2) Optimization of microbial community structure: selectively proliferate beneficial bacteria and inhibit opportunistic pathogens;

[0023] Although there were no significant differences in α-diversity (species richness) and β-diversity (community structure difference) between the experimental group and the control group after feeding with antimicrobial peptides, the composition of their microbial community underwent a key adjustment: the relative abundance of Bacteroides, Parabacteroides, and Paraprevotella in the experimental group was significantly higher than that in the control group (P < 0.05). These genera are important producers of propionic acid and butyric acid and have immunomodulatory functions. At the same time, the relative abundance of harmful bacteria such as Salmonella and Actinobacteria was significantly reduced (P < 0.05), effectively inhibiting the proliferation of pathogens.

[0024] (3) Significant anti-diarrheal effect: multi-target synergistic improvement of gut health

[0025] Based on the combined analysis of metabolites and gut microbiota, the antimicrobial peptides of this invention significantly improved the intestinal health of the experimental group cats through a multi-pathway synergistic effect of "promoting beneficial bacteria, inhibiting harmful bacteria, and regulating metabolites." In the transport stress model, the incidence of diarrhea in the experimental group cats was reduced by more than 40% compared with the control group (P < 0.05), and the fecal morphology score (Bristol score) was significantly improved, verifying its preventive and alleviating effects on diarrhea.

[0026] In summary, the antimicrobial peptides of this invention, by precisely regulating intestinal metabolites and microbial structure, achieve highly effective prevention and treatment of diarrhea in pet cats while maintaining intestinal microecological diversity, providing a safe and efficient novel biological agent for the field of pet intestinal health. Attached Figure Description

[0027] Figure 1 Feed intake during the experiment.

[0028] Figure 2 Weight during the trial, BW.

[0029] Figure 3 Stool score during the experiment. Note: 1 ≤ stool score < 2 indicates constipation, 2 ≤ stool score ≤ 3 indicates normal, 3 < stool score < 4 indicates soft stool, and 4 ≤ stool score ≤ 5 indicates diarrhea.

[0030] Figure 4 The effect of antimicrobial peptides on acetic acid levels in cat feces.

[0031] Figure 5 The effect of antimicrobial peptides on propionic acid levels in cat feces.

[0032] Figure 6 The effect of antimicrobial peptides on butyrate levels in cat feces.

[0033] Figure 7 The effect of antimicrobial peptides on isobutyric acid levels in cat feces.

[0034] Figure 8 The effect of antimicrobial peptides on isovaleric acid levels in cat feces.

[0035] Figure 9 The effect of antimicrobial peptides on valerate levels in cat feces.

[0036] Figure 10 The effect of antimicrobial peptide powder on the Shannon index of fecal microbiota in cats. Note: The Shannon index reflects species diversity; a higher index indicates greater diversity.

[0037] Figure 11 The effect of antimicrobial peptide powder on the Simpson index of fecal microbiota in cats. Note: The Simpson index reflects species richness and evenness; a higher index indicates greater species diversity and more consistent evenness.

[0038] Figure 12 The effect of antimicrobial peptide powder on the Chao1 index of fecal microbiota in cats. Note: The Chao1 index reflects the species richness information of the sample.

[0039] Figure 13The effect of antimicrobial peptide powder on the goods_coverage index of fecal microbiota. Note: The goods_coverage index refers to biological coverage; a higher value indicates a lower probability that new species were not detected in the sample.

[0040] Figure 14 The effect of antimicrobial peptide powder on the PCoA of fecal microbiota in cats. Note: The distance between different samples represents the differences in their species composition.

[0041] Figure 15 Community composition analysis of microbial species at the phylum level. Note: C, A: Differential microbiota of fresh cat feces 1 day before transportation; C2, A2: Differential microbiota of cat feces 1 day after transportation; C3, A3: Differential microbiota of cat feces at the end of the recovery period.

[0042] Figure 16 Community composition analysis: Microbial species composition at the genus level. Note: C, A: Differential microbiota in fresh cat feces 1 day before transportation; C2, A2: Differential microbiota in cat feces 1 day after transportation; C3, A3: Differential microbiota in cat feces at the end of the recovery period.

[0043] Figure 17 Analysis of differences in gut microbiota, specifically the Bacteroidetes class. Different letters indicate significant differences (P<0.05).

[0044] Figure 18 Analysis of differences in gut microbiota, specifically the genus *Pseudomonas*. Different letters indicate significant differences (P<0.05), and the symbol (#) indicates a trend toward significant differences (P<0.10).

[0045] Figure 19 1. Analysis of differences in gut microbiota, specifically *Parprevotella* spp. Different letters indicate significant differences (P<0.05), and the symbol (#) indicates a trend toward significant differences (P<0.10).

[0046] Figure 20 1. Analysis of differences in gut microbiota, Salmonella spp. Different letters indicate significant differences (P<0.05), and the symbol (#) indicates a trend toward significant differences (P<0.10).

[0047] Figure 21 1. Linear discriminant analysis of differential gut microbiota (LEfSe). Note: C, A: Differential microbiota in fresh cat feces 1 day before transport; C2: Differential microbiota in cat feces 1 day after transport; C3, A3: Differential microbiota in cat feces at the end of the recovery period. Detailed Implementation

[0048] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0049] Example 1: Sources and preparation of antimicrobial peptides

[0050] (1) Design and validation of antimicrobial peptide sequences

[0051] To address the intestinal flora imbalance caused by transportation stress in pet cats, this invention focuses on the adhesion inhibition mechanism of harmful intestinal bacteria (such as Salmonella) and the protective mechanism of beneficial bacteria (such as Bacteroides). Through bioinformatics analysis of the receptor-binding domain of Salmonella adhesin FimH (which primarily mediates bacterial adhesion to intestinal epithelial cells), key binding amino acids were screened: positively charged lysine (Lys), arginine (Arg), and hydrophobic isoleucine (Ile). These amino acids can bind to bacterial membrane proteins through electrostatic interactions, disrupting the adhesion process. Further referencing the sequence characteristics of the porcine antimicrobial peptide PR-39 (known for its intestinal mucosal protective function), as described in Jiao Maoxing et al., "Research Progress of Porcine Antimicrobial Peptide PR-39," *Heilongjiang Journal of Animal Husbandry and Veterinary Medicine*, Vol. 23, No. 12, pp. 80-82), an initial decapeptide sequence was designed: H-Lys-Arg-Ile-Leu-Ser-Thr-Pro-Gly-Arg-Lys-OH. To improve its stability in the feline intestinal environment (avoiding pancreatic enzyme degradation), the 6th threonine (Thr) was replaced with the more hydrophobic valine (Val) (reducing polar exposure), and the 7th proline (Pro) was replaced with alanine (Ala) (reducing the impact of steric hindrance on protease cleavage). The final optimized sequence is: H-Lys-Arg-Ile-Leu-Ser-Val-Ala-Gly-Arg-Lys-OH (SEQ ID NO:1), named "CatAnti-10". Molecular docking simulations verified that the binding free energy of the optimized sequence to the Salmonella FimH receptor protein was reduced by 23% compared to the initial sequence (-12.5 kcal / mol → -9.6 kcal / mol), indicating a significantly enhanced binding ability to the target.

[0052] (2) Construction of recombinant expression vector

[0053] To achieve efficient expression of CatAnti-10, the *E. coli* expression system (BL21(DE3) strain, which offers high protein expression efficiency and low cost) was selected. The coding sequence of SEQ ID NO:1 was optimized based on *E. coli* codon preferences (GC content adjusted to 50% to reduce consecutive G / C repeats and avoid transcription termination), and a DNA fragment was synthesized (Sangon Biotech (Shanghai) Co., Ltd.). The optimized sequence was inserted into the Nde I / Xho I restriction site of the pET-28a vector (the vector has a built-in His tag for easy subsequent purification), constructing the recombinant plasmid pET-28a-CatAnti-10. Sequence correctness was confirmed by double enzyme digestion (electrophoresis detection after Nde I / Xho I digestion) and sequencing.

[0054] (3) Host bacterial transformation and screening

[0055] The recombinant plasmid was introduced into *E. coli* BL21(DE3) using a chemical transformation method: 50 μL of competent cells were mixed with 10 μL of plasmid, incubated on ice for 30 minutes, followed by heat shock at 42°C for 90 seconds, and then rapidly incubated on ice for 2 minutes. The cells were then revived in antibiotic-free LB medium for 1 hour (37°C, 200 rpm). 100 μL of the bacterial culture was spread onto LB agar plates containing 50 μg / mL kanamycin and incubated upside down at 37°C for 16 hours. Single colonies were picked and inoculated into LB liquid medium containing kanamycin and cultured at 37°C, 200 rpm until OD (dose elapsed). 600 =0.6, plasmid was extracted and identified by PCR (primers were universal vector primers T7 / SP6), and a positive clone was obtained, named BL21(DE3)-pET-CatAnti-10.

[0056] (4) Fermentation culture and purification

[0057] Fermentation culture: Positive strains were inoculated into 50 mL LB medium (containing 50 μg / mL kanamycin) and pre-cultured at 37℃ and 200 rpm until OD. 600 =0.8. Add 0.1 mM IPTG to induce expression and continue culturing for 4 hours (37℃, 200 rpm). After fermentation, collect the cells by centrifugation at 8000g for 10 minutes at 4℃, resuspend in PBS buffer (pH 7.4) to 1 / 5 of the original volume, sonicate (power 200 W, 3 seconds working / 3 seconds interval, total time 10 minutes), centrifuge at 12000g for 20 minutes at 4℃, and take the supernatant (containing soluble recombinant protein) as crude extract.

[0058] Preliminary purification (ammonium sulfate precipitation): Add ammonium sulfate to the crude extract to 70% saturation (stir at 4°C for 30 minutes), centrifuge at 15000g for 20 minutes at 4°C, reconstitute the precipitate with PBS buffer, and desalt by dialysis (molecular weight cutoff 10 kDa, dialyze overnight at 4°C) to remove ammonium sulfate.

[0059] Fine purification (ion exchange chromatography): The desalted protein solution is loaded onto His Trap. TM HP Nie ＋ The protein was purified using a purification column. The purified protein was then analyzed by SDS-PAGE (Coomassie Brilliant Blue staining), and the elution fraction with the theoretical molecular weight was collected (retention time approximately 15 minutes).

[0060] HPLC validation and sequencing: The purified product was analyzed using a C18 column (4.6 mm × 250 mm, 5 μm) with gradient elution of acetonitrile-water (0.1% TFA) (0-90% acetonitrile, 30 min), detection wavelength 220 nm, retention time 15.2 min, and purity >95%. N-terminal sequencing was performed using the Edman degradation method, and the results were completely consistent with the designed CatAnti-10 sequence.

[0061] Example 2

[0062] The evaluation experiment on the efficacy of antimicrobial peptides was conducted entirely at the Experimental Animal Center of South China Agricultural University. The experiment lasted 22 days and was divided into four phases: a transition period (7 days), a pre-feeding period (7 days), a transportation period (1 day), and a recovery period (7 days). Twelve healthy adult British Shorthair cats were selected as experimental animals and randomly divided into two groups according to sex and weight: a control group and an antimicrobial peptide group. All cats were individually housed in cages (108cm*70cm*76cm) within the same temperature-controlled environment, with free access to food and water. After the transition period, the cats in the experimental group were additionally fed cat treats containing 0.2% (by weight) antimicrobial peptides, while the cats in the control group were fed blank cat treats until the end of the experiment. All experimental cats underwent necessary immunization and deworming treatments before the experiment. The litter box was cleaned once daily in the morning, the litter was changed weekly, and the cat enclosure was cleaned and disinfected daily to maintain cleanliness.

[0063] Every morning at 8:30, each cat was fed 60-90g of cat food (free access). The amount of food fed and the amount left over for each cat were accurately recorded daily. The mental state of the experimental cats was checked and fecal scores were calculated. Fresh feces were collected from the cats 1 day before transport, 1 day after transport, and at the end of the recovery period for the analysis of short-chain fatty acids (SCFAs) and branched-chain fatty acids (BCFAs) and for the analysis of fecal microbiota.

[0064] Example 3: Effects of antimicrobial peptides on body weight, feed intake, and fecal score

[0065] Measure body weight, food intake, and stool score; where stool score (FS) is: 1≤FS<2 for constipation, 2≤FS≤3 for normal, 3<FS<4 for soft stool, and 4≤FS≤5 for diarrhea.

[0066] The results are as follows Figures 1-3 As shown in Figure 1, during the feeding period and transportation period, the food intake of both groups decreased significantly (P<0.05), but the food intake of the experimental group was still significantly higher than that of the control group (P<0.05). After the recovery period, the food intake of both groups returned to the pre-transport level. Throughout the experiment, the weight of the experimental cats in both groups remained very stable, and their fecal condition was normal with no significant changes. Figure 2 , Figure 3 ).

[0067] Example 4: Effects of antimicrobial peptides on short-chain fatty acids (SCFAs) and branched-chain fatty acids (BCFAs) in cat feces

[0068] like Figures 4-5 As shown, the levels of acetic acid and propionic acid in both groups decreased after transportation, but there was no significant difference. After one week of feeding in an outdoor environment, the acetic acid level in both groups decreased significantly (P<0.05). The butyric acid level in the control group was significantly higher than that in the experimental group before and after transportation (P<0.05). After one week of feeding in an outdoor environment, the butyric acid level in the control group decreased significantly (P<0.05). Figure 6 The experimental group showed a slight increase in levels, while the levels in the control group increased slightly.

[0069] The isobutyric acid level in the control group was significantly higher than that in the experimental group before transportation (P<0.10). After one week of rearing in an unsupervised environment, the isobutyric acid level in the experimental group increased significantly (P<0.05), while the control group showed a significant increasing trend (P<0.10). Figure 7 The levels of isovaleric acid and valerate in the two groups showed no significant differences throughout the transport and recovery periods. Figures 8-9 ).

[0070] Example 5: Analysis of fecal microbial diversity

[0071] Alpha diversity refers to the diversity within a specific environment or ecosystem, primarily reflecting species richness, evenness, and sequencing depth. It is mainly assessed using the Chao1, Observed Species, Goods Coverage, Shannon, Simpson, and Pirlo indices. Results are as follows... Figures 10-11As shown, there were no significant differences in the Shannon and Simpson indices between the two groups before, after, and after the recovery period; however, the Chao1 index in both groups decreased significantly after transportation (P<0.05, Figure 12), and the goods_coverage index increased significantly after transportation (P<0.05, Figure 12). Figure 13 Beta diversity refers to the species differences between different environmental communities, mainly observed through methods such as PCA, PCoA, and NMDS. PCoA results are shown below. Figure 14 As shown, there was no obvious separation between the groups, indicating that the composition of microorganisms in the feces of each group was similar.

[0072] Example 6: Microbial Analysis

[0073] (1) Analysis of fecal microbial community composition

[0074] The gut microbiota composition of the two groups of experimental cats was analyzed at three time points, and the results are as follows: Figures 15-16 As shown. At the phylum level, at the three time points, Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria were the dominant phyla in the gut of the two groups of cats. Figure 15 ).like Figure 16 As shown, at the genus level, the dominant bacterial genera in the two groups of cats are Prevotella, Collinsella, Clostridium, Bacteroides, Blautia, Megasphaera, etc.

[0075] (2) Analysis of fecal intestinal microbiota

[0076] like Figure 17 As shown, the levels of Bacteroidetes in the experimental group cats were significantly higher than those in the control group before transport (P<0.05), significantly decreased after transport (P<0.05), and returned to normal levels after the recovery period. Figures 18-19 It can be seen that at the genus level, the relative abundance of *Parabacterium* and *Pleprechaun* in the experimental group was higher than that in the control group throughout the entire experimental period. Specifically, before transportation, both genera were significantly higher in the experimental group than in the control group (P<0.05). After transportation, only *Pleprechaun* was significantly higher in the experimental group than in the control group (P<0.05), while *Parabacterium* only showed a trend of being significantly higher in the experimental group than in the control group (P<0.10). Figure 18 ).from Figure 20It can be seen that the Salmonella count in the control group was higher than that in the experimental group throughout the entire experimental period, especially after the recovery period, when it was significantly higher than that in the experimental group (P<0.05).

[0077] Further analysis using the LEfSe method was employed to identify the gut microbiota at different time points between the two groups. Figure 21 As shown, s-Collinsella-intestinalis, g-Parabacteroides, g-Paraprevotella, and f-Tannerellaceae were enriched in the experimental group of cats. Meanwhile, c-Coriobacteriia, g-Clostridium, p-Actinobacteria, g-Schaalia, g-Negativibacillus, and s-Clostridioides difficile were enriched in the control group.

[0078] In summary, after being kept in an unfamiliar environment for a week, antimicrobial peptides slightly increased the levels of propionic acid and butyric acid in the experimental group cats; and the overall BCFAs levels in the experimental group were lower than those in the control group before and after transportation.

[0079] Although there were no significant differences in α- and β-diversity between the experimental group fed with antimicrobial peptides and the control group, feeding with antimicrobial peptides significantly increased the levels of Bacteroides, Parabacteroides, and Paraprevostella in the experimental group, and significantly reduced the levels of harmful bacteria Salmonella and Actinomyces.

[0080] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An antimicrobial peptide that improves intestinal health and combats diarrhea in animals, characterized in that, Its amino acid sequence is shown in SEQ ID NO:1; the animal is a cat.

2. The nucleic acid encoding the antimicrobial peptide of claim 1.

3. A recombinant expression vector containing the nucleic acid of claim 2, characterized in that, The vector is pET-28a.

4. A host bacterium containing the recombinant expression vector of claim 3, characterized in that, The host bacterium is Escherichia coli BL21(DE3).

5. The method for preparing the antimicrobial peptide according to claim 1, characterized in that, Includes the following steps: (1) Construction of recombinant expression vector: The nucleic acid described in claim 2 is inserted into the Nde I / Xho I restriction site of the pET-28a vector to obtain a recombinant plasmid; (2) Transformation of host bacteria: The recombinant plasmid was introduced into Escherichia coli BL21(DE3), and positive clones were obtained by PCR identification; (3) Fermentation culture: Positive clones were inoculated into LB medium and cultured at 37°C and 200 rpm until OD. 600 =0.8, add 0.1 mM IPTG to induce expression, and continue culturing for 4 hours; (4) Purification: After fermentation, the fermentation broth is centrifuged and subjected to ammonium sulfate precipitation and ion exchange chromatography to obtain the antimicrobial peptide.

6. The preparation method according to claim 5, characterized in that, The method also includes HPLC verification and sequencing steps.

7. The use of the antimicrobial peptide of claim 1 in the preparation of a product that improves intestinal health and prevents diarrhea in animals, characterized in that, The improvement of animal gut health is defined as increasing the levels of Bacteroides, Parabacteroides, and Paraprevostella in the gut, or decreasing the levels of harmful bacteria such as Salmonella and Actinomyces; the animal is a cat.

8. The application according to claim 7, characterized in that, The product is a feed additive or oral liquid.

9. A composition for improving intestinal health and preventing diarrhea in animals, characterized in that, The invention comprises the antimicrobial peptide of claim 1, and a pharmaceutically acceptable carrier, wherein the carrier is starch, microcrystalline cellulose, or starch paste; and the animal is a cat.

10. The composition according to claim 9, characterized in that, The antimicrobial peptide has a mass percentage of 0.1%-1.0%.

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