An antimicrobial peptide derived from kitchen waste liquid and its preparation method

Abstract

This invention relates to an antimicrobial peptide derived from kitchen waste liquid and its preparation method, belonging to the field of biotechnology. Using kitchen waste liquid as raw material, this invention employs processes such as ultrasonic enzymatic hydrolysis, microbial fermentation, and purification to obtain peptides with antimicrobial activity. The antimicrobial peptides prepared by this invention exhibit broad-spectrum antimicrobial activity, inhibiting and killing various pathogens such as bacteria and fungi. Furthermore, the antimicrobial peptides show low hemolytic activity against erythrocytes, have short synthetic sequences, small molecular weights, and are easy to synthesize chemically. They can specifically kill pathogens in organisms while significantly reducing the cost of large-scale production. They can be used as peptide-based microbial inhibitors in fields such as feed, food, skincare products, pharmaceuticals, medical devices, animal husbandry, and agriculture.

Description

Technical Field

[0001] This invention relates to an antimicrobial peptide derived from kitchen waste liquid and its preparation method, belonging to the field of biotechnology. Background Technology

[0002] In recent years, with the overuse and long-term use of antibiotics, drug resistance has become increasingly serious, and the efficacy of traditional antibiotics has gradually declined. Furthermore, food poisoning and systemic infections caused by foodborne pathogens such as Staphylococcus aureus and Bacillus cereus remain significant threats to public health. Therefore, developing novel antimicrobial drugs to replace chemical preservatives and traditional antibiotics has become an urgent need to address current health and food safety issues.

[0003] Antimicrobial peptides are a class of polypeptides widely found in nature, possessing significant antimicrobial activity. Compared to traditional antibiotics, antimicrobial peptides offer numerous advantages: they have a simple structure, typically composed of short-chain amino acids, resulting in lower drug resistance. They act on microorganisms through various mechanisms, such as cell membrane damage, interference with cell wall synthesis, and inhibition of nucleic acid or protein synthesis, effectively resisting pathogen invasion and serving as crucial components for immune function. Furthermore, antimicrobial peptides exhibit broad-spectrum antimicrobial activity, inhibiting and killing various pathogens, including Gram-positive and Gram-negative bacteria, fungi, and viruses, without damaging normal cells in animals. This allows for wide applications in medicine, livestock farming, aquaculture, and the food industry.

[0004] Although over 3,000 antimicrobial peptides have been developed, only a small number are currently used clinically as antibiotic alternatives, such as nisin, bacitracin, and polymyxin. Currently, most antimicrobial peptides suffer from high production costs, low bioactivity, and a tendency to cause hemolysis, failing to meet clinical treatment needs. Therefore, developing peptides with simple structures, high bioactivity, and no significant toxic side effects is an urgent problem to be solved.

[0005] With the continuous advancement of waste sorting policies in my country, the amount of kitchen waste is showing an increasing trend. As a major component of kitchen waste, food waste liquid mainly consists of carbohydrates, proteins, cellulose, and a small amount of lipids. As a rich natural resource, food waste liquid contains a large number of bioactive substances, including antimicrobial peptides, and has high resource recovery and utilization value. Given the current situation of energy and resource scarcity, the recycling and utilization of kitchen waste can effectively alleviate these problems and achieve good economic benefits. Therefore, effectively extracting and purifying antimicrobial peptides from food waste liquid and conducting in-depth research on their sequences and functions provides new ideas and possibilities for the development of novel antimicrobial drugs. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides an antimicrobial peptide derived from kitchen waste liquid and its preparation method. The antimicrobial peptide MKLPAKGYALVSP provided by this invention exhibits broad-spectrum antibacterial and antifungal activity, and demonstrates low hemolytic activity against erythrocytes, showing great application potential in feed, food, skincare products, pharmaceuticals, medical devices, animal husbandry, and agriculture.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] The present invention provides an antimicrobial peptide, the amino acid sequence of which is shown in SEQ ID NO:1.

[0009] Furthermore, the antimicrobial peptide is derived from kitchen waste liquid or is artificially synthesized.

[0010] The present invention also provides a medicament for the prevention or treatment of bacterial and / or fungal infectious diseases, the medicament containing the aforementioned antimicrobial peptide.

[0011] The present invention also provides the use of antimicrobial peptides in the preparation of medicaments for the prevention or treatment of bacterial and / or fungal infectious diseases.

[0012] Furthermore, the bacteria are selected from Escherichia coli, Staphylococcus aureus, Salmonella, or Bacillus thuringiensis.

[0013] Furthermore, the bacteria are selected from Escherichia coli GDMCC NO.801303, Staphylococcus aureus GDMCC NO.1.160, Salmonella GDMCC NO.1.5271, or Bacillus thuringiensis GDMCC NO.1.24.

[0014] Furthermore, the fungus is selected from Candida albicans, rice sheath blight pathogen, pepper phytosis pathogen, or Fusarium oxysporum.

[0015] Furthermore, the fungus is selected from Candida albicans GDMCC NO.2.194, rice sheath blight fungus GDMCC NO.3.724, pepper phytophthora GDMCC NO.3.452, or Fusarium oxysporum GDMCC NO.3.558.

[0016] The present invention also provides a method for preparing the antimicrobial peptide, characterized by comprising the following steps:

[0017] S1. Pretreatment of kitchen waste liquid: Collect kitchen waste liquid and use a high-speed pulverizer to crush large pieces of food residue to control the particle size of the material to 1-3mm to obtain pretreated liquid;

[0018] S2. Ultrasonic-assisted enzymatic hydrolysis: The pH of the pretreatment solution was adjusted to 4, pepsin was added, and the mixture was ultrasonically reacted and then heated to a warm temperature to obtain the first enzymatic hydrolysate; the pH of the first enzymatic hydrolysate was adjusted to 7.5, flavor protease was added, and the mixture was ultrasonically reacted and then heated to a warm temperature to obtain the second enzymatic hydrolysate;

[0019] S3. Microbial fermentation: Add Lactobacillus acidophilus to the second enzymatic hydrolysate for fermentation. After the fermentation is completed, add Bacillus subtilis for secondary fermentation. After the fermentation is completed, heat the fermentation mixture to inactivate it and obtain the fermentation broth.

[0020] S4. Crude extraction of antimicrobial peptides: After the microbial fermentation is completed and the fermentation broth has cooled completely, the supernatant of the fermentation broth is collected by centrifugation; ammonium sulfate powder is slowly added to the supernatant while stirring, so that the saturation of ammonium sulfate reaches 60%; after standing, the supernatant is discarded and the protein precipitate at the bottom is collected; the protein precipitate is dissolved in phosphate buffer to obtain a protein solution, which is placed in a dialysis bag and dialyzed in phosphate buffer to obtain the crude extract of antimicrobial peptides;

[0021] S5. Column chromatography purification: The crude antimicrobial peptide extract was initially purified by Superdex-20010 / 300GL gel column chromatography. The mobile phase was 20mM citrate-phosphate buffer at pH=4, the flow rate was 2mL / min, 5mL was collected each time by an automatic collector, the detection wavelength was 280nm, and each elution peak was collected for antibacterial test.

[0022] S6. Reverse-phase high-performance liquid chromatography (RP-HPLC) purification: The peak exhibiting anti-E. coli activity after the previous column chromatography purification was finally purified using reverse-phase HPLC. First, the sample was filtered through a 0.22 μm filter membrane, followed by fine purification using a preparative C18 RP-HPLC column. The column used was an Agilent Shim-pack Arata C18. The injection volume was 0.6 mL. Mobile phase A was an aqueous solution containing 0.1% trifluoroacetic acid, and mobile phase B was an acetonitrile solution containing 0.1% trifluoroacetic acid. The flow rate was 3 mL / min, and the detection wavelength was 220 nm. The gradient elution program was: 0-8 min, 10-40% B; 8-15 min, 40-80% B; 15-25 min, 80-93% B; 25-30 min, 93-10% B.

[0023] S7. Structural identification of antimicrobial peptides: An Agilent 1200 rapid separation liquid chromatography system and an Agilent 6520 Q-TOF mass spectrometer were used. Liquid chromatography conditions: column: Agilent Eclipse Plus C18; binary linear gradient elution: mobile phase A was 0.1% formic acid aqueous solution; B was acetonitrile; mobile phase gradient: 0–30 min, 5%–30% B; column temperature 35℃, flow rate 0.4 mL / min, injection volume 5 μL.

[0024] Further, in step S2, ultrasound-assisted enzymatic hydrolysis, pepsin was added at a dosage of 20,000 U / g, and the mixture was ultrasonically reacted at 37°C for 2 hours. After hydrolysis, the mixture was heated to 90°C and kept at that temperature for 15 minutes. Then, flavor protease was added at a dosage of 40,000 U / g, and the mixture was ultrasonically reacted at 40°C for 2 hours. After hydrolysis, the mixture was heated to 90°C and kept at that temperature for 15 minutes to obtain the second enzymatic hydrolysate.

[0025] Further, in step S3, during microbial fermentation, 2% by volume of Lactobacillus acidophilus ATCC4356 was added to the second enzymatic hydrolysate, and fermentation was carried out at a constant temperature of 37°C for 12 hours. After the fermentation was completed, 4% by volume of Bacillus subtilis ATCC6633 was added for secondary fermentation, and fermentation was carried out at 37°C for 24 hours. After the fermentation was completed, the fermentation mixture was subjected to heat inactivation treatment to obtain the fermentation broth.

[0026] The present invention also provides the use of the antimicrobial peptide in the preparation of compositions having antibacterial and / or antifungal effects.

[0027] Furthermore, the composition is a skin care composition, a pharmaceutical composition, a feed additive, or a biological preservative.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) This invention uses kitchen waste liquid as raw material and employs processes such as ultrasonic enzymatic hydrolysis, microbial fermentation, and purification to obtain peptides with antibacterial activity. This avoids the drug resistance problem caused by long-term use of traditional antibiotics and provides a new approach to solving bacterial drug resistance. The obtained antimicrobial peptides have the characteristics of simple structure and well-defined amino acid sequence. Sequence analysis shows that its amino acid sequence is MKLPAKGYALVSP, which can effectively disrupt the permeability and barrier function of bacterial cell membranes and achieve bactericidal effect.

[0030] (2) The antimicrobial peptides prepared by this invention have broad-spectrum antimicrobial activity and can inhibit and kill a variety of pathogens such as bacteria and fungi, especially Escherichia coli, Staphylococcus aureus, Salmonella, Bacillus thuringiensis, Candida albicans, rice sheath blight, Phytophthora capsici, Fusarium oxysporum, etc., providing a multi-target, broad-spectrum antimicrobial strategy and providing a new option for the widespread application of antimicrobial peptides.

[0031] (3) The antimicrobial peptides described in this invention have low hemolytic activity, short synthetic sequences, small molecular weights, and are easy to synthesize chemically. They can specifically kill pathogens in organisms while saving large-scale production costs. They can be used as polypeptide microbial inhibitors in feed, food, skin care products, pharmaceuticals, medical devices, animal husbandry and agriculture. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of this application 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 this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 This is the reverse high performance liquid chromatogram of Example 1 of the present invention;

[0034] Figure 2 This is the liquid chromatogram of the antimicrobial peptide in Example 2 of the present invention;

[0035] Figure 3 This is a schematic diagram of the predicted three-dimensional structure of the antimicrobial peptide of the present invention;

[0036] Figure 4 This is a graph showing the antibacterial effect of the antimicrobial peptide in Example 3 of the present invention against Escherichia coli at a concentration of 128 μg / mL;

[0037] Figure 5 This is a graph showing the antimicrobial effect of the antimicrobial peptide in Example 3 of the present invention against Staphylococcus aureus at a concentration of 128 μg / mL;

[0038] Figure 6 This is a graph showing the antibacterial effect of the antimicrobial peptide in Example 3 of the present invention against Bacillus thuringiensis at a concentration of 128 μg / mL;

[0039] Figure 7 This is a graph showing the antimicrobial effect of the antimicrobial peptide in Example 3 of the present invention against Candida albicans at a concentration of 128 μg / mL;

[0040] Figure 8 This is a graph showing the antimicrobial effect of the antimicrobial peptide in Example 3 of the present invention against rice sheath blight pathogen at a concentration of 100 μg / mL (the left side is the blank group).

[0041] Figure 9 This is a graph showing the antimicrobial effect of the antimicrobial peptide in Example 3 of the present invention against Phytophthora capsici at a concentration of 100 μg / mL (the left side is the blank group).

[0042] Figure 10 This is a graph showing the antibacterial effect of the antimicrobial peptide in Example 3 of the present invention against Fusarium oxysporum at a concentration of 100 μg / mL (the left side is the blank group). Detailed Implementation

[0043] The present invention will be illustrated below with examples to make the technical solution of the present invention easier to understand and master, but the present invention is not limited thereto. Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the medicinal materials and reagents can be obtained commercially; and the performance of products from different sources does not have a significant impact.

[0044] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0045] Example 1

[0046] This embodiment provides a method for extracting antimicrobial peptides from kitchen waste liquid, the specific steps of which are as follows:

[0047] S1. Pretreatment of kitchen waste liquid:

[0048] Kitchen waste liquid (taken from the kitchen of Guangdong Hainabaichuan Environmental Protection Technology Co., Ltd., mainly composed of rice, meat, vegetable scraps and oil mixture) was collected. Large pieces of food scraps were crushed using a high-speed pulverizer (model: DFT-200C). The pulverizing speed was set to 5000r / min and the pulverizing time was 5min to control the particle size of the material to 1-3mm, thus obtaining pretreated liquid.

[0049] S2. Ultrasound-assisted enzymatic hydrolysis:

[0050] The pH of the pretreatment solution was adjusted to 4, and pepsin (purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number S10027-100g) was added at a dosage of 20,000 U / g. The solution was ultrasonically reacted at 37℃ for 2 hours, followed by hydrolysis and heating to 90℃ for 15 minutes to obtain the first enzymatic hydrolysate. The pH of the first enzymatic hydrolysate was then adjusted to 7.5, and flavor protease (purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number S10153-100g) was added at a dosage of 40,000 U / g. The solution was ultrasonically reacted at 40℃ for 2 hours, followed by hydrolysis and heating to 90℃ for 15 minutes to obtain the second enzymatic hydrolysate.

[0051] During the two enzymatic hydrolysis processes described above, the ultrasonic power was set to 300W and the ultrasonic frequency to 25kHz.

[0052] S3. Microbial fermentation:

[0053] Add 2% by volume of Lactobacillus acidophilus ATCC4356 (purchased from Shanghai Baolu Biotechnology Co., Ltd., catalog number 0243P) to the second enzymatic hydrolysate and ferment at 37℃ for 12 hours. After fermentation, add 4% by volume of Bacillus subtilis ATCC6633 (purchased from Shanghai Baolu Biotechnology Co., Ltd., catalog number 0486X) for a second fermentation at 37℃ for 24 hours. After fermentation, heat the fermentation mixture at 110℃ for 15 minutes to inactivate the bacteria and obtain the fermentation broth.

[0054] S4. Crude extraction of antimicrobial peptides:

[0055] After microbial fermentation is complete and the fermentation broth has cooled completely, centrifuge at 10000 rpm for 20 minutes and collect the supernatant. Slowly add ammonium sulfate powder to the supernatant while stirring until the ammonium sulfate saturation reaches 60%, and let it stand at 25°C for 30 minutes. Then centrifuge at 6000 rpm for 20 minutes, discard the supernatant, and collect the protein precipitate at the bottom.

[0056] The protein precipitate was dissolved in 0.02M phosphate buffer (pH 7.2) to obtain a protein solution. The solution was then placed in a dialysis bag and immersed in 10 times the volume of 0.02M phosphate buffer (pH 7.2). Dialysis was performed at 4°C for 24 hours, with the buffer solution being changed 3 times during the process, to obtain a crude extract of the antimicrobial peptide.

[0057] S5. Column chromatography purification:

[0058] The crude antimicrobial peptide extract was initially purified by Superdex-20010 / 300GL gel column chromatography. The mobile phase was 20 mM citrate-phosphate buffer (pH 4), the flow rate was 2 mL / min, and 5 mL was collected each time using an automated collector. The detection wavelength was 280 nm, and each elution peak was collected for antibacterial testing. The fraction from tube 16, which showed the strongest antibacterial activity, was combined and collected for further purification.

[0059] S6. Reverse high performance liquid chromatography purification:

[0060] The peak exhibiting anti-E. coli activity, purified by the previous column chromatography step, was finally purified using reversed-phase high-performance liquid chromatography (RP-HPLC). First, the sample was filtered through a 0.22 μm filter membrane, followed by fine purification using a preparative C18 RP-HPLC column. The column used was an Agilent Shim-pack Arata C18; the injection volume was 0.6 mL; mobile phase A was an aqueous solution containing 0.1% trifluoroacetic acid, and mobile phase B was an acetonitrile solution containing 0.1% trifluoroacetic acid; the flow rate was 3 mL / min; the detection wavelength was 220 nm; and the gradient elution program was: 0-8 min, 10-40% B; 8-15 min, 40-80% B; 15-25 min, 80-93% B; 25-30 min, 93-10% B. Figure 1 The reversed-phase high-performance liquid chromatography (RP-HPLC) separation chromatogram obtained under these conditions shows that the absorption peak intensity is highest at 13.28 min, and the eluent of this component is collected.

[0061] S7. Structural identification of antimicrobial peptides:

[0062] An Agilent 1200 rapid separation liquid chromatography system and an Agilent 6520 Q-TOF mass spectrometer were used. HPLC conditions: Column: Agilent Eclipse Plus C18 (2.1 × 150 mm, 3.5 μm); Binary linear gradient elution; Mobile phase: (A) 0.1% (w / w) formic acid aqueous solution; (B) acetonitrile; Mobile phase gradient: 0–30 min, 5%–30% B; Column temperature: 35℃; Flow rate: 0.4 mL / min; Injection volume: 5 μL.

[0063] Mass spectrometry conditions: Electrospray ionization (ESI+) mode was used; mass scan range m / z 100–2000; drying gas flow rate (N2) was 9 L / min; desiccator temperature was 300 °C; nebulization voltage was 35 psig; capillary voltage was 3.5 kV; fragmentation voltage was 175 V; cone voltage was 65 V; octapole RF voltage was 250 V; and secondary mass spectrometry collision voltage was 12–15 eV. Mass spectrometry output data were analyzed using Peaks 7.5 software (Bioinformatics Solutions Inc.). Results showed that the molecular weight of the antimicrobial peptide was 1.37 kDa (1374.69 Da), the amino acid sequence SEQ ID NO:1 was MKLPAKGYALVSP, and the theoretical isoelectric point PI was 10.33.

[0064] Example 2

[0065] Based on the amino acid sequence of the antimicrobial peptide in Example 1, Nanjing Genscript Biotech Co., Ltd. was commissioned to synthesize the antimicrobial peptide using a solid-phase chemical synthesis method.

[0066] (1) According to the first amino acid Pro at the C-terminus of the antimicrobial peptide sequence in Example 1, 0.5 mmol of the corresponding Fmoc protected amino acid - WangResin was selected and added to the solid-phase reactor. After adding DCM swelling resin for 30 min, the mixture was dried and washed three times with DMF. Then, a 20% hexahydropyridine DMF solution was added and reacted for 5 min. Another 20% hexahydropyridine DMF solution was added and reacted for 10 min. DMF was washed once in the middle. After the reaction was completed, the mixture was dried and washed three times with DMF.

[0067] (2) Following the sequence of the peptide from the C-terminus to the N-terminus, the condensation reaction and Fmoc removal reaction were carried out alternately with an amino acid reaction amount of 1.5 mmol. According to the amino acid sequence of the antimicrobial peptide (from the C-terminus), all amino acids were condensed onto the resin. After the last amino acid Met was condensed, a 20% hexahydropyridine DMF solution was added and reacted for 5 min. Then, another 20% hexahydropyridine DMF solution was added and reacted for 10 min. DMF was washed once in the middle. After the reaction was completed, the solution was dried and washed with DMF 3 times.

[0068] (3) Add 1 mmol of FITC and N-methylmorpholine to the reactor and react for 5-10 minutes. Use ninhydrin to check whether the reaction is complete. After the reaction is complete, wash three times with DMF and DMC alternately, and then wash with methanol to shrink and obtain dry MKLPAKGYALVSP-resin.

[0069] (4) Place the MKLPAKGYALVSP-resin into a round-bottom flask and slowly add the prepared lysis buffer at 0°C (the volume ratio of the reagent formula used in the lysis buffer is TFA: benzyl sulfide: phenol: triisopropylsilane: water = 82.5: 7.5: 5: 3: 2). Stir slowly and react at low temperature for 0.5 hours, then at room temperature for 2 hours. Filter to obtain the lysis buffer. Slowly add the lysis buffer to anhydrous ice-cold ether and stir. Filter to separate the crude antimicrobial peptide. Wash with ice-cold ether 3 times to obtain the antimicrobial peptide MKLPAKGYALVSP.

[0070] (5) The liquid chromatography profile of the antimicrobial peptide was detected using the same elution procedure as step S6 in Example 1, as shown in the figure. Figure 2 As shown, the purity is greater than 95%. White or pale yellow powdered antimicrobial peptides are obtained using vacuum freeze-drying or spray drying processes.

[0071] Example 3 Antimicrobial test of antimicrobial peptides

[0072] Using the 2:5 dilution method, the antimicrobial peptide from Example 2 was prepared with distilled water to create antimicrobial peptide solutions with concentration gradients of 512 μg / mL, 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, and 4 μg / mL. These solutions were then stored at 4°C for later use.

[0073] E. coli ( Escherichia coli (Purchased from Guangdong Provincial Microbial Culture Collection Center, GDMCC NO. 801303), Staphylococcus aureus ( Staphylococcus aureus (Purchased from Guangdong Provincial Microbial Culture Collection Center, GDMCC NO.1.160), Salmonella ( Salmonella enterica (Purchased from Guangdong Provincial Microbial Culture Collection Center, GDMCC NO.1.5271), Bacillus thuringiensis ( Bacillus thuringiensis (Purchased from Guangdong Provincial Microbial Culture Collection Center, GDMCC NO.1.24), Candida albicans ( Candida albicans (Purchased from Guangdong Provincial Microbial Culture Collection Center, GDMCC NO.2.194) Stranded onto LB solid medium and incubated at 37℃ for 12 h. Single colonies of each strain were then picked and placed in LB liquid medium and incubated at 37℃ with shaking for 12 h. The OD of the bacterial culture was measured. 600 Value, and dilute it to 1×10 6 CFU / mL.

[0074] Determination of minimum inhibitory concentration (MIC): 50 μL of bacterial culture of each strain (Escherichia coli, Staphylococcus aureus, Salmonella, Bacillus thuringiensis, and Candida albicans) cultured to the logarithmic growth phase was added to each well of a 96-well plate. Simultaneously, 50 μL of antimicrobial peptide solutions of varying concentrations were added to each well, with positive and negative controls provided separately. After incubation at 37°C for 12 h, the OD of each well was measured. 600 The minimum inhibitory concentration (MIC) of the antimicrobial peptide was determined by taking the lowest concentration at which the growth of the strain was completely inhibited. The test results are shown in Table 1.

[0075] Inhibition zone assay: Prepare LB semi-solid medium (0.5% agar). Add 10 μL of bacterial suspension to each 20 mL plate, shake to mix, and allow to solidify before placing the plates into Oxford cups. Add 100 μL of 128 μg / mL antimicrobial peptide solution to each Oxford cup. Incubate at 37℃ for 24 h, then measure the diameter of the inhibition zone. The test results are shown in Table 1. Figure 4-7 As shown.

[0076] Table 1. Antimicrobial activity of antimicrobial peptides (X±SD, n=3)

[0077] strain Diameter of the inhibition zone (mm) MIC (μg / mL) E. coli 16.24±2.43 16 Staphylococcus aureus 43.66±4.61 4 salmonella 21.45±1.32 8 Bacillus thuringiensis 15.22±1.28 32 Candida albicans 17.86±1.54 16

[0078] Rice sheath blight pathogen ( Thanatephorus Cucumeris (Purchased from Guangdong Provincial Microbial Culture Collection Center, GDMCC NO.3.724), Phytophthora capsici ( Phytophthora capsici(Purchased from Guangdong Provincial Microbial Culture Collection Center, GDMCC NO.3.452), Fusarium oxysporum ( Fusarium oxysporum (Purchased from Guangdong Provincial Microbial Culture Collection Center, GDMCC NO.3.558) The strain was inoculated onto PDA solid medium for activation and incubated at 28℃ for 18 hours. PDA semi-solid medium (0.5% agar) was prepared. Before solidification, a certain amount of antimicrobial peptide was mixed thoroughly with the medium to achieve a concentration of 100 μg / mL. The medium was then poured evenly into petri dishes. No antimicrobial peptide was added to the control group. After solidification, the activated strain was perforated and placed in the center of the petri dish (with the mycelial side in contact with the medium). Each treatment was repeated three times. The petri dishes were incubated at 28℃. The mycelial inhibition rate was measured based on mycelial growth. The results are shown in Table 2. Figure 8-10 As shown.

[0079] Inhibition rate (%) = [(colony diameter of blank group - colony diameter of experimental group) / colony diameter of blank group] × 100%.

[0080] Table 2. Hyphae-inhibiting effect of antimicrobial peptides (X±SD, n=3)

[0081] strain Mycelial inhibition rate (%) Rice sheath blight fungus 31.51±3.84 Phytophthora capsici 24.85±3.52 Fusarium oxysporum 52.64±6.07

[0082] Example 4 Hemolysis assay of antimicrobial peptides

[0083] Take 1 mL of blood from a healthy C57BL / 6 mouse and add it to a heparin anticoagulant tube. Centrifuge at 2000 rpm for 5 min, discard the supernatant, collect the lower layer of blood cells, wash 3 times with 0.9% sodium chloride solution, add 25 mL of 0.9% sodium chloride solution to prepare a 4% blood cell suspension, and store it in a refrigerator at 4℃ for later use.

[0084] The antimicrobial peptide was prepared into antimicrobial peptide solutions with concentration gradients of 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, and 16 μg / mL using 0.9% sodium chloride solution for later use.

[0085] Take 1 mL of 4% blood cell suspension into a 2 mL centrifuge tube, centrifuge at 2000 rpm for 5 min to collect blood cells, then add 1 mL of antimicrobial peptide solution of different concentrations. The positive control is sterile ultrapure water, and the negative control is 0.9% sodium chloride solution. Incubate in a 37℃ water bath. After 1 h of incubation, centrifuge at 2000 rpm for 5 min, collect 200 μL of supernatant, and measure the absorbance of the supernatant at 545 nm. The experiment is repeated 3 times, and the average value of each result is used to calculate the hemolysis rate. The formula for calculating the hemolysis rate is:

[0086] Hemolysis rate (%) = (Ai-A0) / (Aj-A0)×100%.

[0087] In the formula: A0 is the absorbance value of the negative control supernatant, Ai is the absorbance value of the sample supernatant, and Aj is the absorbance value of the positive control supernatant.

[0088] Table 3 Hemolytic activity of antimicrobial peptides (X±SD, n=3)

[0089] Antimicrobial peptide concentration (μg / mL) Hemolysis rate (%) 16 1.45±0.24 32 3.31±0.84 64 5.42±1.17 128 8.93±1.94 256 15.74±1.65

[0090] Table 3 shows that the hemolysis rate of antimicrobial peptides increases slowly with increasing concentration, exhibiting a positive correlation. The hemolysis rate at a concentration of 128 μg / mL is consistently below 10%, indicating that antimicrobial peptides have good safety and promising applications in feed, food, skincare products, pharmaceuticals, medical devices, animal husbandry, and agriculture. Further in-depth research and development are warranted.

[0091] Based on the foregoing description, those skilled in the art will understand that this disclosure can be implemented in different specific forms without altering its technical spirit and essential features. Therefore, it should be understood that the above embodiments are not restrictive in any respect, but rather illustrative. The scope of the invention is limited by the appended claims, not by the preceding description, and thus all changes and modifications fall within the boundaries and scope of the claims.

Claims

1. An antimicrobial peptide, characterized in that, The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO:

1.

2. The antimicrobial peptide according to claim 1, characterized in that, The antimicrobial peptides are derived from kitchen waste liquid or are artificially synthesized.

3. A medicament for the prevention or treatment of bacterial and / or fungal infectious diseases, characterized in that, The drug contains the antimicrobial peptide as described in any one of claims 1-2.

4. Use of the antimicrobial peptide according to any one of claims 1-2 in the preparation of a medicament for the prevention or treatment of bacterial and / or fungal infectious diseases, wherein the bacteria and / or fungi are selected from Escherichia coli GDMCC NO. 801303, Staphylococcus aureus GDMCC NO. 1.160, Salmonella GDMCC NO. 1.5271, Bacillus thuringiensis GDMCC NO. 1.24, Candida albicans GDMCC NO. 2.194, Rice sheath blight fungus GDMCC NO. 3.724, Phytophthora capsici GDMCC NO. 3.452, and Fusarium oxysporum GDMCC NO. 3.

558.

5. The method for preparing the antimicrobial peptide according to claim 1, characterized in that, Includes the following steps: S1. Pretreatment of kitchen waste liquid: Collect kitchen waste liquid and use a high-speed pulverizer to crush large pieces of food residue to control the particle size of the material to 1-3mm to obtain pretreated liquid; S2. Ultrasonic-assisted enzymatic hydrolysis: The pH of the pretreatment solution was adjusted to 4, pepsin was added, and the mixture was ultrasonically reacted and then heated to a warm temperature to obtain the first enzymatic hydrolysate; the pH of the first enzymatic hydrolysate was adjusted to 7.5, flavor protease was added, and the mixture was ultrasonically reacted and then heated to a warm temperature to obtain the second enzymatic hydrolysate; S3. Microbial fermentation: Add Lactobacillus acidophilus to the second enzymatic hydrolysate for fermentation. After the fermentation is completed, add Bacillus subtilis for secondary fermentation. After the fermentation is completed, heat the fermentation mixture to inactivate it and obtain the fermentation broth. S4. Crude extraction of antimicrobial peptides: After the microbial fermentation is completed and the fermentation broth has cooled completely, the supernatant of the fermentation broth is collected by centrifugation; ammonium sulfate powder is slowly added to the supernatant while stirring, so that the saturation of ammonium sulfate reaches 60%; after standing, the supernatant is discarded and the protein precipitate at the bottom is collected; the protein precipitate is dissolved in phosphate buffer to obtain a protein solution, which is placed in a dialysis bag and dialyzed in phosphate buffer to obtain the crude extract of antimicrobial peptides; S5. Column chromatography purification: The crude antimicrobial peptide extract was initially purified by Superdex-20010 / 300GL gel column chromatography. The mobile phase was 20mM citrate-phosphate buffer at pH=4, the flow rate was 2mL / min, 5mL was collected each time by an automatic collector, the detection wavelength was 280nm, and each elution peak was collected for antibacterial test. S6. Reverse-phase high-performance liquid chromatography (RP-HPLC) purification: The peak exhibiting anti-E. coli activity after the previous column chromatography purification was finally purified using reverse-phase HPLC. First, the sample was filtered through a 0.22 μm filter membrane, followed by fine purification using a preparative C18 RP-HPLC column. The column used was an Agilent Shim-pack Arata C18. The injection volume was 0.6 mL. Mobile phase A was an aqueous solution containing 0.1% trifluoroacetic acid, and mobile phase B was an acetonitrile solution containing 0.1% trifluoroacetic acid. The flow rate was 3 mL / min, and the detection wavelength was 220 nm. The gradient elution program was: 0-8 min, 10-40% B; 8-15 min, 40-80% B; 15-25 min, 80-93% B; 25-30 min, 93-10% B. S7. Structural identification of antimicrobial peptides: An Agilent 1200 rapid separation liquid chromatography system and an Agilent 6520Q-TOF mass spectrometer were used. Liquid chromatography conditions: column: Agilent Eclipse Plus C18; binary linear gradient elution: mobile phase A was 0.1% formic acid aqueous solution; B was acetonitrile; mobile phase gradient: 0–30 min, 5%–30% B; column temperature 35℃, flow rate 0.4 mL / min, injection volume 5 μL.

6. The preparation method according to claim 5, characterized in that, In step S2, ultrasound-assisted enzymatic hydrolysis, pepsin was added at a dosage of 20,000 U / g, and the mixture was ultrasonically reacted at 37°C for 2 hours. After hydrolysis, the mixture was heated to 90°C and kept warm for 15 minutes. Then, flavor protease was added at a dosage of 40,000 U / g, and the mixture was ultrasonically reacted at 40°C for 2 hours. After hydrolysis, the mixture was heated to 90°C and kept warm for 15 minutes to obtain the second enzymatic hydrolysate.

7. The preparation method according to claim 5, characterized in that, In step S3, during microbial fermentation, 2% by volume of Lactobacillus acidophilus ATCC4356 was added to the second enzymatic hydrolysate, and fermentation was carried out at a constant temperature of 37°C for 12 hours. After the fermentation was completed, 4% by volume of Bacillus subtilis ATCC6633 was added for secondary fermentation, which was carried out at 37°C for 24 hours. After the fermentation was completed, the fermentation mixture was heated to inactivate it, and the fermentation broth was obtained.

8. The use of the antimicrobial peptide according to any one of claims 1-2 in the preparation of a composition having antibacterial and / or antifungal properties, wherein the bacteria and / or fungi are selected from Escherichia coli GDMCC NO. 801303, Staphylococcus aureus GDMCC NO. 1.160, Salmonella GDMCC NO. 1.5271, Bacillus thuringiensis GDMCC NO. 1.24, Candida albicans GDMCC NO. 2.194, Rice sheath blight fungus GDMCC NO. 3.724, Phytophthora capsici GDMCC NO. 3.452, and Fusarium oxysporum GDMCC NO. 3.

558.

9. The application according to claim 8, characterized in that, The composition is a skin care product composition, a pharmaceutical composition, a feed additive, or a biological preservative.

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