Porcine sperm ultra-long-acting dilution powder containing natural antibacterial components and preparation method of porcine sperm ultra-long-acting dilution powder

Abstract

The invention relates to the technical field of biological reproduction, and discloses a pig sperm ultra-long-acting dilution powder containing natural antibacterial components and a preparation method of the pig sperm ultra-long-acting dilution powder. The feed additive is prepared from the following raw materials in parts by weight: 30 to 50 parts of glucose, 1 to 2 parts of sodium ethylene diamine tetracetate, 5 to 10 parts of sodium citrate, 1 to 2 parts of sodium bicarbonate, 0.5 to 1 part of potassium chloride, 0.5 to 2 parts of composite natural antibacterial components, 0.2 to 0.8 part of novel antibacterial peptide, 0.3 to 0.8 part of polyvinyl alcohol, 0.05 to 0.2 part of cysteine and 1 to 3 parts of freeze-dried egg yolk powder. The pig sperm long-acting dilution powder prepared by the process has remarkable advantages, the sperm motility rate can be maintained to be 65% or above within 10 days at 17 DEG C, the total number of bacterial colonies is only 3.8 * 10 < 3 > CFU / mL or so within 10 days, the sperm plasma membrane integrity rate and the acrosome integrity rate reach 62% or above and 65% or above respectively after 10 days, the sperm long-acting dilution powder far exceeds traditional dilution powder, and the requirements of long-distance transportation and large-scale breeding are met.

Description

Technical Field

[0001] This invention relates to the field of biological reproduction technology, specifically to a long-lasting diluted powder of porcine semen containing natural antibacterial components and its preparation method. Background Technology

[0002] Artificial insemination in pigs is an important means of modernizing pig production, offering numerous advantages such as reducing the number of boars needed, decreasing disease transmission, increasing mating rates, and expanding the coverage of improved breeds. It is widely used globally. In the process of artificial insemination in pigs, semen preservation technology is crucial, and the quality of the semen diluent directly affects the semen preservation effect.

[0003] Currently, there are many types of boar semen diluents on the market, but they all have some problems. Most diluents have a short shelf life; for example, some only preserve semen for 3-4 days, which cannot meet the needs of long-term semen preservation in actual production. While some diluents can extend shelf life, their preparation processes are complex and costly, and some components may negatively impact sperm quality. For example, some diluents contain high concentrations of glucose, which can maintain a low pH level in sperm, inhibiting glycolysis; while EDTA can capture metal ions, it may inhibit sperm capacitation and acrosome reaction. Furthermore, semen is susceptible to bacterial contamination during storage, leading to decreased sperm motility and affecting fertilization outcomes, and some existing antibacterial components may have issues such as drug resistance. Therefore, developing a boar semen diluent powder that has a long shelf life, is easy to prepare, has low cost, and contains natural antibacterial components is of significant practical importance. Summary of the Invention

[0004] (a) Technical problems to be solved

[0005] To address the shortcomings of existing technologies, this invention provides a long-lasting diluted porcine semen powder containing natural antibacterial components and its preparation method.

[0006] (II) Technical Solution

[0007] To achieve the above objectives, the present invention provides the following technical solution: a long-lasting diluted powder of porcine semen containing natural antibacterial components, composed of the following raw materials in parts by weight:

[0008] 30-50 parts glucose, 1-2 parts sodium EDTA, 5-10 parts sodium citrate, 1-2 parts sodium bicarbonate, 0.5-1 part potassium chloride, 0.5-2 parts compound natural antibacterial ingredients, 0.2-0.8 parts novel antibacterial peptides, 0.3-0.8 parts polyvinyl alcohol, 0.05-0.2 parts cysteine, and 1-3 parts freeze-dried egg yolk powder;

[0009] The compound natural antibacterial components consist of tea polyphenols, allicin, rosemary antibacterial components in a mass ratio of 2:1:1, and chitosan in a total mass of 0.5%.

[0010] The novel antimicrobial peptide is a polyethylene glycol derivative modified with folic acid groups that has sperm-targeting properties.

[0011] The method for preparing the polyethylene glycol derivative containing folic acid groups includes the following steps:

[0012] A1. Folic acid was dissolved in anhydrous N,N-dimethylformamide and stirred under ice bath conditions. N-hydroxysuccinimide and N,N'-dicyclohexylcarbodiimide were added sequentially. The reaction was carried out at room temperature, filtered, and the filtrate was concentrated under reduced pressure. Under vigorous stirring, the concentrate was slowly added dropwise to ice-cold ether, and a precipitate was formed. The precipitate was collected by filtration, washed with cold ether, and dried under vacuum to obtain folic acid-N-hydroxysuccinimide active ester.

[0013] A2. Dissolve amino polyethylene glycol in anhydrous dimethyl sulfoxide, add triethylamine, and slowly add an anhydrous dimethyl sulfoxide solution of folic acid-N-hydroxysuccinimide active ester under stirring. React at room temperature, dilute the reaction solution with deionized water, transfer it to a dialysis bag for dialyzing, and freeze-dry the solution after dialyzing to obtain a polyethylene glycol derivative containing folic acid groups.

[0014] Furthermore, the preparation method of tea polyphenols includes the following steps:

[0015] B1. Grind green tea into 40-60 mesh, place the ground green tea in a supercritical extraction device, and extract for 1-2 hours under the conditions of pressure 20-30 MPa, temperature 35-45℃ and carbon dioxide flow rate 15-25 L / h.

[0016] B2. Add the supercritical treated green tea powder to a 60-80% ethanol solution at a material-to-liquid ratio of 1g:10-15mL. Under the conditions of 50-70℃ and ultrasonic power of 200-300W, extract by ultrasonication for 30-40min. Filter, collect the filtrate, and recover the ethanol by vacuum distillation to obtain crude tea polyphenol extract.

[0017] B3. The crude extract of tea polyphenols was purified by passing it through a macroporous adsorption resin column. AB-8 type macroporous adsorption resin was selected. The eluent was first eluted with 30-50% ethanol solution at a flow rate of 1-2 BV / h, where BV is the resin bed volume. The eluent was collected, concentrated under reduced pressure, and then freeze-dried to obtain high-purity tea polyphenols.

[0018] Furthermore, the preparation method of allicin includes the following steps:

[0019] C1. Peel the garlic and homogenize it. Add 2 to 3 times its weight of water to the garlic homogenate and enzymatically hydrolyze it at 40 to 50°C for 1 to 2 hours. Use cellulase and pectinase, with a total enzyme addition of 0.1 to 0.3% and a mass ratio of 1:1. Then, perform vacuum distillation at 60 to 70°C and a vacuum degree of -0.05 to -0.08 MPa, collect the fraction, and obtain crude allicin extract.

[0020] C2. The crude allicin extract was purified by molecular distillation. Molecular distillation was carried out at a temperature of 80–100℃, a pressure of 0.1–1 Pa, and a scraper rotation speed of 200–300 r / min. The fractions were collected to obtain purified allicin.

[0021] Furthermore, the preparation method of the antibacterial component of rosemary includes the following steps:

[0022] D1. Cut rosemary into small pieces of 0.5-1cm, add 70-90% acetone solution at a material-to-liquid ratio of 1g:8-12mL, and extract 2-3 times at a temperature of 40-50℃ and a stirring speed of 150-200r / min, each time for 2-3 hours. Combine the extracts and recover the acetone by vacuum distillation to obtain crude extract of antibacterial components of rosemary.

[0023] D2. The crude extract of antibacterial components of rosemary was purified by silica gel column chromatography with petroleum ether-ethyl acetate at a volume ratio of 3-5:1 and an elution flow rate of 1-2 mL / min. The eluent containing antibacterial components was collected, concentrated under reduced pressure, and dried to obtain the purified antibacterial components of rosemary.

[0024] Furthermore, the preparation method of the novel antimicrobial peptide includes the following steps:

[0025] E1. Screening for the natural antimicrobial peptide sequence LL-37 with potent antimicrobial activity, and modifying its encoding gene sequence to the codons preferred by E. coli using codon optimization technology, designing a fusion expression vector, linking a glutathione S-transferase tag gene upstream of the antimicrobial peptide gene, inserting an enterokinase cleavage site sequence in the middle, constructing the recombinant expression plasmid pGEX-AMP, transforming the recombinant plasmid into E. coli BL21, and obtaining a genetically engineered bacterium that efficiently expresses the antimicrobial peptide fusion protein through resistance screening and PCR verification;

[0026] E2. Inoculate the engineered bacteria into LB medium containing 100 μg / mL ampicillin and incubate at 37°C with shaking until OD. 600=0.6-0.8, add IPTG to a final concentration of 0.1-0.5mM, induce expression at 25℃ for 12-16h, collect bacterial cells, sonicate the bacterial cells, purify the fusion protein by glutathione affinity chromatography column, elute with elution buffer containing 10mM reduced glutathione, add enterokinase, digest at 25℃ for 10-16h, and then separate by ion exchange chromatography to obtain the purified antimicrobial peptide;

[0027] E3. Nanocellulose was selected as the grafting carrier and dispersed in deionized water to prepare a 1% (w / v) suspension. 0.5% by mass of 2,2,6,6-tetramethylpiperidine-1-oxy free radical and 1% sodium bromide were added. Under the condition of pH=10, 5% sodium hypochlorite solution was slowly added dropwise and reacted at room temperature for 1-2 hours to obtain a carboxylated nanocellulose suspension.

[0028] E4. The carboxylated nanocellulose suspension and the antimicrobial peptide solution were mixed at a mass ratio of 8-10:1, the pH was adjusted to 5.5, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added as crosslinking agents at a molar ratio of 1:1 and a final concentration of 5mM for both. The reaction was carried out at room temperature in the dark for 2-4 hours with continuous stirring. After the reaction was completed, unreacted small molecules were removed by centrifugation and dialysis, and the grafted modified antimicrobial peptide was obtained by freeze drying.

[0029] E5. The grafted modified antimicrobial peptide was mixed with a polyethylene glycol derivative containing folic acid groups at a mass ratio of 3 to 5:1. In a borate buffer solution at pH 8.5, 1 mM of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was added and reacted at room temperature for 6 to 8 hours. The mixture was then subjected to ultrafiltration and lyophilization to obtain a novel antimicrobial peptide with sperm targeting properties.

[0030] Furthermore, a method for preparing a long-lasting diluted porcine semen powder containing natural antibacterial components includes the following steps:

[0031] S1. Accurately weigh each ingredient according to the above weight proportions, and place glucose, sodium EDTA, sodium citrate, sodium bicarbonate, potassium chloride, compound natural antibacterial ingredients, novel antibacterial peptides, polyvinyl alcohol, cysteine, and freeze-dried egg yolk powder in a beaker.

[0032] S2. Add double-distilled water to a beaker, then heat and stir in a 37°C water bath until all ingredients are completely dissolved and equilibrate for more than 1 hour to obtain a uniform diluted solution.

[0033] S3. The above-mentioned diluent is spray-dried to obtain a long-lasting diluent powder for pig semen.

[0034] (III) Beneficial Technical Effects

[0035] The porcine semen diluent prepared using this process has significant advantages. In terms of semen preservation, it can maintain sperm viability at over 65% for 10 days at 17°C, far exceeding traditional diluents, thus meeting the needs of long-distance transportation and large-scale farming.

[0036] It exhibits excellent antibacterial properties, with a synergistic effect of complex natural antibacterial components including tea polyphenols, allicin, and rosemary, along with novel antibacterial peptides, resulting in a total bacterial count of only 3.8 × 10⁻⁶ within 10 days. 3 With a concentration of approximately CFU / mL, it is more than 50% lower than that of single antibacterial ingredients, effectively inhibiting a variety of pathogens and reducing the risk of disease transmission.

[0037] It has an outstanding effect on sperm protection. The phospholipids provided by the freeze-dried egg yolk powder bind to the novel antimicrobial peptides in a targeted manner. After 10 days, the integrity rate of sperm plasma membrane and acrosome reaches more than 62% and 65% respectively, ensuring fertilization ability.

[0038] The preparation process of this invention is scientific and efficient. Supercritical extraction and molecular distillation technologies improve the purity of antibacterial components, spray drying facilitates storage and transportation, raw material costs are controllable, and the components work synergistically to break import dependence and promote the efficient development of the pig industry. Detailed Implementation

[0039] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0040] Unless otherwise specified, all components of the porcine semen diluent formula containing natural antibacterial ingredients in this invention are commercially available.

[0041] Example 1

[0042] A long-lasting diluted porcine semen powder containing natural antibacterial ingredients, composed of the following ingredients in parts by weight:

[0043] 30 parts glucose, 1 part sodium EDTA, 5 parts sodium citrate, 1 part sodium bicarbonate, 0.5 parts potassium chloride, 0.5 parts compound natural antibacterial ingredients, 0.2 parts novel antibacterial peptides, 0.3 parts polyvinyl alcohol, 0.05 parts cysteine, and 1 part freeze-dried egg yolk powder;

[0044] The preparation method includes the following steps:

[0045] S1. Accurately weigh each ingredient according to the above weight proportions, and place glucose, sodium EDTA, sodium citrate, sodium bicarbonate, potassium chloride, compound natural antibacterial ingredients, novel antibacterial peptides, polyvinyl alcohol, cysteine, and freeze-dried egg yolk powder in a beaker.

[0046] S2. Add double-distilled water to a beaker, then heat and stir in a 37°C water bath until all ingredients are completely dissolved and equilibrate for more than 1 hour to obtain a uniform diluted solution.

[0047] S3. Spray dry the above diluent to obtain a long-lasting diluent powder for pig semen.

[0048] The compound natural antibacterial components consist of tea polyphenols, allicin, rosemary antibacterial components in a mass ratio of 2:1:1, and chitosan in a total mass of 0.5%.

[0049] The novel antimicrobial peptide is a polyethylene glycol derivative modified with folic acid groups that has sperm-targeting properties.

[0050] The preparation method of the compound natural antibacterial ingredients is as follows:

[0051] High-purity tea polyphenols, refined allicin, and purified rosemary antibacterial components were mixed in a mass ratio of 2:1:1, and 0.5% of chitosan by mass was added. The mixture was stirred evenly at 40℃ and 200r / min to obtain a composite natural antibacterial component.

[0052] The preparation method of tea polyphenols includes the following steps:

[0053] 1. Grind the green tea to 40 mesh, place the ground green tea in a supercritical extraction device, and extract for 1 hour under the conditions of pressure 20MPa, temperature 35℃ and carbon dioxide flow rate 15L / h.

[0054] 2. Green tea powder that has undergone supercritical treatment was added to a 60% ethanol solution at a material-to-liquid ratio of 1g:10mL. The mixture was ultrasonically extracted for 30min at a temperature of 50℃ and an ultrasonic power of 200W. The mixture was then filtered, the filtrate was collected, and the ethanol was recovered by vacuum distillation to obtain crude tea polyphenol extract.

[0055] 3. The crude extract of tea polyphenols was purified by passing it through a macroporous adsorption resin column. AB-8 type macroporous adsorption resin was selected. The eluent was first eluted with 30% ethanol solution at a flow rate of 1 BV / h, where BV is the resin bed volume. The eluent was collected, concentrated under reduced pressure, and then freeze-dried to obtain high-purity tea polyphenols.

[0056] The preparation method of allicin includes the following steps:

[0057] 1. Peel the garlic and grind it into a homogenate. Add twice the amount of water to the garlic homogenate and enzymatically hydrolyze it at 40℃ for 1 hour. Use cellulase and pectinase, with a total enzyme addition of 0.1% and a mass ratio of 1:1. Then, perform vacuum distillation at 60℃ and -0.05MPa, collect the fraction, and obtain crude allicin extract.

[0058] 2. The crude allicin extract was purified by molecular distillation. Molecular distillation was carried out at a temperature of 80℃, a pressure of 0.1Pa, and a scraper rotation speed of 200r / min. The fractions were collected to obtain purified allicin.

[0059] The preparation method of the antibacterial component of rosemary includes the following steps:

[0060] 1. Cut rosemary into 0.5cm pieces and add 70% acetone solution at a ratio of 1g:8mL. Extract twice at 40℃ and 150r / min for 2 hours each time. Combine the extracts and recover the acetone by vacuum distillation to obtain crude extract of antibacterial components from rosemary.

[0061] 2. The crude extract of antibacterial components of rosemary was purified by silica gel column chromatography with petroleum ether-ethyl acetate (volume ratio 3:1) as the eluent and an elution flow rate of 1 mL / min. The eluent containing antibacterial components was collected, concentrated under reduced pressure, and dried to obtain the purified antibacterial components of rosemary.

[0062] The preparation method of the novel antimicrobial peptide includes the following steps:

[0063] 1. The natural antimicrobial peptide sequence LL-37 with strong antimicrobial activity was screened. The coding gene sequence was modified to the codon preferred by Escherichia coli using codon optimization technology. A fusion expression vector was designed, and a glutathione S-transferase tag gene was linked upstream of the antimicrobial peptide gene. An enterokinase cleavage site sequence was inserted in the middle to construct the recombinant expression plasmid pGEX-AMP. The recombinant plasmid was transformed into Escherichia coli BL21. Through resistance screening and PCR verification, a genetically engineered bacterium that efficiently expresses the antimicrobial peptide fusion protein was obtained.

[0064] 2. Inoculate the engineered bacteria into LB medium containing 100 μg / mL ampicillin and incubate at 37°C with shaking until OD reaches 100%. 600 =0.6, add IPTG to a final concentration of 0.1mM, induce expression at 25℃ for 12h, collect bacterial cells, sonicate the bacterial cells, purify the fusion protein by glutathione affinity chromatography column, elute with elution buffer containing 10mM reduced glutathione, add enterokinase (1U / mg fusion protein), digest at 25℃ for 10h, and then separate by ion exchange chromatography to obtain the purified antimicrobial peptide;

[0065] 3. Nanocellulose was selected as the grafting carrier and dispersed in deionized water to prepare a 1% (w / v) suspension. 0.5% by mass of 2,2,6,6-tetramethylpiperidine-1-oxy free radical and 1% sodium bromide were added. Under the condition of pH=10, 5% sodium hypochlorite solution was slowly added dropwise and reacted at room temperature for 1 hour to obtain a carboxylated nanocellulose suspension.

[0066] 4. The carboxylated nanocellulose suspension and the antimicrobial peptide solution were mixed at a mass ratio of 8:1, the pH was adjusted to 5.5, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added as crosslinking agents at a molar ratio of 1:1 and a final concentration of 5mM for both. The reaction was carried out at room temperature in the dark for 2 hours with continuous stirring. After the reaction was completed, unreacted small molecules were removed by centrifugation and dialysis, and the grafted modified antimicrobial peptide was obtained by freeze drying.

[0067] 5. The grafted modified antimicrobial peptide was mixed with a polyethylene glycol derivative containing folic acid groups at a mass ratio of 3:1. In a borate buffer solution at pH 8.5, 1 mM of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was added and reacted at room temperature for 6 h. The resulting product was obtained by ultrafiltration and lyophilization to obtain a novel antimicrobial peptide with sperm targeting properties.

[0068] The preparation method of polyethylene glycol derivatives containing folic acid groups includes the following steps:

[0069] 1. Folic acid was dissolved in anhydrous N,N-dimethylformamide to prepare a 0.02 g / mL solution. The solution was stirred in an ice bath, and N-hydroxysuccinimide and N,N'-dicyclohexylcarbodiimide were added sequentially. The mass ratio of folic acid, N-hydroxysuccinimide, and N,N'-dicyclohexylcarbodiimide was 1:0.1:0.5. The reaction was carried out at room temperature for 12 h. The dicyclohexylurea precipitate was removed by filtration. The filtrate was concentrated under reduced pressure. The concentrate was slowly added dropwise to ice-cold diethyl ether under vigorous stirring. The precipitate was collected by filtration, washed with cold diethyl ether, and dried under vacuum to obtain folic acid-N-hydroxysuccinimide active ester.

[0070] 2. Dissolve amino polyethylene glycol in anhydrous dimethyl sulfoxide to prepare a 0.1 g / mL solution. Add triethylamine, and under stirring, slowly add anhydrous dimethyl sulfoxide solution of folic acid-N-hydroxysuccinimide active ester. The mass ratio of amino polyethylene glycol, triethylamine, and folic acid-N-hydroxysuccinimide active ester is 1:0.1:0.08. React at room temperature for 20 h. Dilute the reaction solution twice with deionized water, transfer it to a dialysis bag, and dialyze against deionized water for 36 h, changing the dialysate 6 times during the process. After dialysis, freeze-dry the solution to obtain a polyethylene glycol derivative containing folic acid groups.

[0071] Example 2

[0072] A long-lasting diluted porcine semen powder containing natural antibacterial ingredients, composed of the following ingredients in parts by weight:

[0073] 40 parts glucose, 1.5 parts sodium EDTA, 7 parts sodium citrate, 1.5 parts sodium bicarbonate, 0.8 parts potassium chloride, 0.6 parts compound natural antibacterial ingredients, 0.5 parts novel antibacterial peptides, 0.5 parts polyvinyl alcohol, 0.1 parts cysteine, and 2 parts freeze-dried egg yolk powder;

[0074] The preparation method includes the following steps:

[0075] S1. Accurately weigh each ingredient according to the above weight proportions, and place glucose, sodium EDTA, sodium citrate, sodium bicarbonate, potassium chloride, compound natural antibacterial ingredients, novel antibacterial peptides, polyvinyl alcohol, cysteine, and freeze-dried egg yolk powder in a beaker.

[0076] S2. Add double-distilled water to a beaker, then heat and stir in a 37°C water bath until all ingredients are completely dissolved and equilibrate for more than 1 hour to obtain a uniform diluted solution.

[0077] S3. Spray dry the above diluent to obtain a long-lasting diluent powder for pig semen.

[0078] The compound natural antibacterial components consist of tea polyphenols, allicin, rosemary antibacterial components in a mass ratio of 2:1:1, and chitosan in a total mass of 0.5%.

[0079] The novel antimicrobial peptide is a polyethylene glycol derivative modified with folic acid groups that has sperm-targeting properties.

[0080] The preparation method of the compound natural antibacterial ingredients is as follows:

[0081] High-purity tea polyphenols, refined allicin, and purified rosemary antibacterial components were mixed in a mass ratio of 2:1:1, and 0.8% of the total mass of chitosan was added. The mixture was stirred evenly at a temperature of 45℃ and a stirring speed of 250r / min to obtain a composite natural antibacterial component.

[0082] The preparation method of tea polyphenols includes the following steps:

[0083] 1. Grind the green tea to 50 mesh, place the ground green tea in a supercritical extraction device, and extract for 1.5 hours under the conditions of pressure 25MPa, temperature 40℃ and carbon dioxide flow rate 20L / h.

[0084] 2. Green tea powder that has undergone supercritical treatment was added to a 70% ethanol solution at a material-to-liquid ratio of 1g:12mL. The mixture was ultrasonically extracted for 40min at a temperature of 60℃ and an ultrasonic power of 250W. The mixture was then filtered, the filtrate was collected, and the ethanol was recovered by vacuum distillation to obtain crude tea polyphenol extract.

[0085] 3. The crude extract of tea polyphenols was purified by passing it through a macroporous adsorption resin column. AB-8 type macroporous adsorption resin was selected. The eluent was first eluted with 40% ethanol solution at a flow rate of 1 BV / h, where BV is the resin bed volume. The eluent was collected, concentrated under reduced pressure, and then freeze-dried to obtain high-purity tea polyphenols.

[0086] The preparation method of allicin includes the following steps:

[0087] 1. Peel the garlic and grind it into a homogenate. Add three times the weight of water to the garlic homogenate and enzymatically hydrolyze it at 45℃ for 1.5 hours. Use cellulase and pectinase, with a total enzyme addition of 0.2% and a mass ratio of 1:1. Then, perform vacuum distillation at 65℃ and -0.06MPa, collect the fraction, and obtain crude allicin extract.

[0088] 2. The crude allicin extract was purified by molecular distillation. Molecular distillation was carried out at a temperature of 90℃, a pressure of 0.5Pa, and a scraper rotation speed of 250r / min. The fractions were collected to obtain purified allicin.

[0089] The preparation method of the antibacterial component of rosemary includes the following steps:

[0090] 1. Cut rosemary into 1cm pieces and add 80% acetone solution at a ratio of 1g:12mL. Extract three times at 45℃ and 180r / min for 2.5h each time. Combine the extracts and recover the acetone by vacuum distillation to obtain crude extract of antibacterial components from rosemary.

[0091] 2. The crude extract of antibacterial components of rosemary was purified by silica gel column chromatography with petroleum ether-ethyl acetate (volume ratio 4:1) as the eluent and an elution flow rate of 2 mL / min. The eluent containing antibacterial components was collected, concentrated under reduced pressure, and dried to obtain the purified antibacterial components of rosemary.

[0092] The preparation method of the novel antimicrobial peptide includes the following steps:

[0093] 1. The natural antimicrobial peptide sequence LL-37 with strong antimicrobial activity was screened. The coding gene sequence was modified to the codon preferred by Escherichia coli using codon optimization technology. A fusion expression vector was designed, and a glutathione S-transferase tag gene was linked upstream of the antimicrobial peptide gene. An enterokinase cleavage site sequence was inserted in the middle to construct the recombinant expression plasmid pGEX-AMP. The recombinant plasmid was transformed into Escherichia coli BL21. Through resistance screening and PCR verification, a genetically engineered bacterium that efficiently expresses the antimicrobial peptide fusion protein was obtained.

[0094] 2. Inoculate the engineered bacteria into LB medium containing 100 μg / mL ampicillin and incubate at 37°C with shaking until OD reaches 100%. 600=0.7, add IPTG to a final concentration of 0.3mM, induce expression at 25℃ for 15h, collect bacterial cells, sonicate the bacterial cells, purify the fusion protein by glutathione affinity chromatography column, elute with elution buffer containing 10mM reduced glutathione, add enterokinase (1U / mg fusion protein), digest at 25℃ for 15h, and then separate by ion exchange chromatography to obtain the purified antimicrobial peptide;

[0095] 3. Nanocellulose was selected as the grafting carrier and dispersed in deionized water to prepare a 1% (w / v) suspension. 0.5% by mass of 2,2,6,6-tetramethylpiperidine-1-oxy free radical and 1% sodium bromide were added. Under the condition of pH=10, 5% sodium hypochlorite solution was slowly added dropwise and reacted at room temperature for 1.5 h to obtain a carboxylated nanocellulose suspension.

[0096] 4. The carboxylated nanocellulose suspension and the antimicrobial peptide solution were mixed at a mass ratio of 9:1, the pH was adjusted to 5.5, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added as crosslinking agents at a molar ratio of 1:1 and a final concentration of 5mM for both. The reaction was carried out at room temperature in the dark for 3 hours with continuous stirring. After the reaction was completed, unreacted small molecules were removed by centrifugation and dialysis, and the grafted modified antimicrobial peptide was obtained by freeze drying.

[0097] 5. The grafted modified antimicrobial peptide was mixed with a polyethylene glycol derivative containing folic acid groups at a mass ratio of 4:1. In a borate buffer solution at pH 8.5, 1 mM of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was added and reacted at room temperature for 7 h. The resulting product was obtained by ultrafiltration and lyophilization to obtain a novel antimicrobial peptide with sperm targeting properties.

[0098] The preparation method of polyethylene glycol derivatives containing folic acid groups includes the following steps:

[0099] 1. Folic acid was dissolved in anhydrous N,N-dimethylformamide to prepare a 0.04 g / mL solution. The solution was stirred in an ice bath, and N-hydroxysuccinimide and N,N'-dicyclohexylcarbodiimide were added sequentially. The mass ratio of folic acid, N-hydroxysuccinimide, and N,N'-dicyclohexylcarbodiimide was 1:0.2:0.6. The reaction was carried out at room temperature for 20 h. The dicyclohexylurea precipitate was removed by filtration. The filtrate was concentrated under reduced pressure. The concentrate was slowly added dropwise to ice-cold diethyl ether under vigorous stirring. The precipitate was collected by filtration, washed with cold diethyl ether, and dried under vacuum to obtain folic acid-N-hydroxysuccinimide active ester.

[0100] 2. Dissolve amino-polyethylene glycol in anhydrous dimethyl sulfoxide to prepare a 0.3 g / mL solution. Add triethylamine, and under stirring, slowly add anhydrous dimethyl sulfoxide solution of folic acid-N-hydroxysuccinimide active ester. The mass ratio of amino-polyethylene glycol, triethylamine, and folic acid-N-hydroxysuccinimide active ester is 1:0.12:0.1. React at room temperature for 22 h. Dilute the reaction solution twice with deionized water, transfer it to a dialysis bag, and dialyze against deionized water for 40 h, changing the dialysate 6 times during the process. After dialysis, freeze-dry the solution to obtain a polyethylene glycol derivative containing folic acid groups.

[0101] Example 3

[0102] A long-lasting diluted porcine semen powder containing natural antibacterial ingredients, composed of the following ingredients in parts by weight:

[0103] 50 parts glucose, 2 parts sodium EDTA, 10 parts sodium citrate, 2 parts sodium bicarbonate, 1 part potassium chloride, 2 parts compound natural antibacterial ingredients, 0.8 parts novel antibacterial peptides, 0.8 parts polyvinyl alcohol, 0.2 parts cysteine, and 3 parts freeze-dried egg yolk powder;

[0104] The preparation method includes the following steps:

[0105] S1. Accurately weigh each ingredient according to the above weight proportions, and place glucose, sodium EDTA, sodium citrate, sodium bicarbonate, potassium chloride, compound natural antibacterial ingredients, novel antibacterial peptides, polyvinyl alcohol, cysteine, and freeze-dried egg yolk powder in a beaker.

[0106] S2. Add double-distilled water to a beaker, then heat and stir in a 37°C water bath until all ingredients are completely dissolved and equilibrate for more than 1 hour to obtain a uniform diluted solution.

[0107] S3. Spray dry the above diluent to obtain a long-lasting diluent powder for pig semen.

[0108] The compound natural antibacterial components consist of tea polyphenols, allicin, rosemary antibacterial components in a mass ratio of 2:1:1, and chitosan in a total mass of 0.5%.

[0109] The novel antimicrobial peptide is a polyethylene glycol derivative modified with folic acid groups that has sperm-targeting properties.

[0110] The preparation method of the compound natural antibacterial ingredients is as follows:

[0111] High-purity tea polyphenols, refined allicin, and purified rosemary antibacterial components were mixed in a mass ratio of 2:1:1, and 1% of the total mass of chitosan was added. The mixture was stirred evenly at 50℃ and 300r / min to obtain a composite natural antibacterial component.

[0112] The preparation method of tea polyphenols includes the following steps:

[0113] 1. Grind the green tea to 60 mesh, place the ground green tea in a supercritical extraction device, and extract for 2 hours under the conditions of pressure 30MPa, temperature 45℃ and carbon dioxide flow rate 25L / h.

[0114] 2. Green tea powder that has undergone supercritical treatment was added to an 80% ethanol solution at a material-to-liquid ratio of 1g:15mL. The mixture was ultrasonically extracted for 40min at a temperature of 70℃ and an ultrasonic power of 300W. The mixture was then filtered, the filtrate was collected, and the ethanol was recovered by vacuum distillation to obtain crude tea polyphenol extract.

[0115] 3. The crude extract of tea polyphenols was purified by passing it through a macroporous adsorption resin column. AB-8 type macroporous adsorption resin was selected. The eluent was first eluted with 50% ethanol solution at a flow rate of 2 BV / h, where BV is the resin bed volume. The eluent was collected, concentrated under reduced pressure, and then freeze-dried to obtain high-purity tea polyphenols.

[0116] The preparation method of allicin includes the following steps:

[0117] 1. Peel the garlic and grind it into a homogenate. Add three times the weight of water to the garlic homogenate and enzymatically hydrolyze it at 50°C for 2 hours. Use cellulase and pectinase, with a total enzyme addition of 0.3% and a mass ratio of 1:1. Then, perform vacuum distillation at 70°C and -0.08MPa, collect the fraction, and obtain crude allicin extract.

[0118] 2. The crude allicin extract was purified by molecular distillation. Molecular distillation was carried out at a temperature of 100℃, a pressure of 1Pa, and a scraper rotation speed of 300r / min. The fractions were collected to obtain purified allicin.

[0119] The preparation method of the antibacterial component of rosemary includes the following steps:

[0120] 1. Cut the rosemary into 1cm pieces and add 90% acetone solution at a ratio of 1g:12mL. Extract the rosemary three times at a temperature of 50℃ and a stirring speed of 200r / min, for 3 hours each time. Combine the extracts and recover the acetone by vacuum distillation to obtain the crude extract of antibacterial components from rosemary.

[0121] 2. The crude extract of antibacterial components of rosemary was purified by silica gel column chromatography with petroleum ether-ethyl acetate (v / v) at a flow rate of 2 mL / min. The eluent containing antibacterial components was collected, concentrated under reduced pressure, and dried to obtain the purified antibacterial components of rosemary.

[0122] The preparation method of the novel antimicrobial peptide includes the following steps:

[0123] 1. The natural antimicrobial peptide sequence LL-37 with strong antimicrobial activity was screened. The coding gene sequence was modified to the codon preferred by Escherichia coli using codon optimization technology. A fusion expression vector was designed, and a glutathione S-transferase tag gene was linked upstream of the antimicrobial peptide gene. An enterokinase cleavage site sequence was inserted in the middle to construct the recombinant expression plasmid pGEX-AMP. The recombinant plasmid was transformed into Escherichia coli BL21. Through resistance screening and PCR verification, a genetically engineered bacterium that efficiently expresses the antimicrobial peptide fusion protein was obtained.

[0124] 2. Inoculate the engineered bacteria into LB medium containing 100 μg / mL ampicillin and incubate at 37°C with shaking until OD reaches 100%. 600 =0.8, add IPTG to a final concentration of 0.5 mM, induce expression at 25℃ for 16 h, collect bacterial cells, sonicate the bacterial cells, purify the fusion protein by glutathione affinity chromatography column, elute with elution buffer containing 10 mM reduced glutathione, add enterokinase (1 U / mg fusion protein), digest at 25℃ for 16 h, and then separate by ion exchange chromatography to obtain the purified antimicrobial peptide;

[0125] 3. Nanocellulose was selected as the grafting carrier and dispersed in deionized water to prepare a 1% (w / v) suspension. 0.5% by mass of 2,2,6,6-tetramethylpiperidine-1-oxy free radical and 1% sodium bromide were added. Under the condition of pH=10, 5% sodium hypochlorite solution was slowly added dropwise and reacted at room temperature for 2 hours to obtain a carboxylated nanocellulose suspension.

[0126] 4. The carboxylated nanocellulose suspension and the antimicrobial peptide solution were mixed at a mass ratio of 10:1, the pH was adjusted to 5.5, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added as crosslinking agents at a molar ratio of 1:1 and a final concentration of 5mM for both. The reaction was carried out at room temperature in the dark for 4 hours with continuous stirring. After the reaction was completed, unreacted small molecules were removed by centrifugation and dialysis, and the grafted modified antimicrobial peptide was obtained by freeze drying.

[0127] 5. The grafted modified antimicrobial peptide was mixed with a polyethylene glycol derivative containing folic acid groups at a mass ratio of 5:1. In a borate buffer solution at pH 8.5, 1 mM of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was added and reacted at room temperature for 8 h. The resulting product was obtained by ultrafiltration and lyophilization to obtain a novel antimicrobial peptide with sperm targeting properties.

[0128] The preparation method of polyethylene glycol derivatives containing folic acid groups includes the following steps:

[0129] 1. Folic acid was dissolved in anhydrous N,N-dimethylformamide to prepare a 0.06 g / mL solution. The solution was stirred in an ice bath, and N-hydroxysuccinimide and N,N'-dicyclohexylcarbodiimide were added sequentially. The mass ratio of folic acid, N-hydroxysuccinimide, and N,N'-dicyclohexylcarbodiimide was 1:0.3:0.8. The reaction was carried out at room temperature for 24 h. The dicyclohexylurea precipitate was removed by filtration. The filtrate was concentrated under reduced pressure. Under vigorous stirring, the concentrate was slowly added dropwise to ice-cold diethyl ether, and a precipitate was formed. The precipitate was collected by filtration, washed with cold diethyl ether, and dried under vacuum to obtain folic acid-N-hydroxysuccinimide active ester.

[0130] 2. Dissolve amino polyethylene glycol in anhydrous dimethyl sulfoxide to prepare a 0.4 g / mL solution. Add triethylamine, and under stirring, slowly add anhydrous dimethyl sulfoxide solution of folic acid-N-hydroxysuccinimide active ester. The mass ratio of amino polyethylene glycol, triethylamine, and folic acid-N-hydroxysuccinimide active ester is 1:0.15:0.12. React at room temperature for 24 h. Dilute the reaction solution twice with deionized water, transfer it to a dialysis bag, and dialyze against deionized water for 48 h, changing the dialysate 6 times during the process. After dialysis, freeze-dry the solution to obtain a polyethylene glycol derivative containing folic acid groups.

[0131] Comparative Example 1: Contains no compound natural antibacterial ingredients; the rest of the process is the same as in Example 1.

[0132] Comparative Example 2: Contains no novel antimicrobial peptides; other processes are the same as in Example 1.

[0133] Comparative Example 3: Single antibacterial component tea polyphenols, without the addition of novel antibacterial peptides, and the rest of the process is the same as in Example 1.

[0134] Performance testing:

[0135] 1. Sperm motility testing:

[0136] After collecting bovine semen, it was diluted with various diluents at a ratio of 1:5 and stored at a constant temperature of 17℃. Samples were taken on days 0, 5, 7, and 10, and examined under a microscope using the eosin-aniline black staining method. Live sperm did not stain. Viability (%) was calculated, and the results are shown in Table 1.

[0137] 2. Antibacterial performance test:

[0138] The plate count method was used. 0.1 mL of diluted semen was spread on LB agar plates and incubated at 37°C for 24 h. The total number of colonies (CFU / mL) was then counted. The results are shown in Table 2.

[0139] 3. Sperm plasma membrane integrity test:

[0140] The hypotonic swelling test (HOS) was used. After incubation at 37°C for 30 min, the proportion of swollen sperm in the tail was examined under a microscope, and the plasma membrane integrity rate (%) was calculated. The results are shown in Table 3.

[0141] 4. Acrosome integrity inspection:

[0142] Giemsa staining was used to examine the proportion of intact sperm cells in the acrosome without staining, and the acrosome integrity rate (%) was calculated. The results are shown in Table 4.

[0143] Table 1. Sperm motility test results (%)

[0144] Group 0 days 5 days 7 days 10 days Example 1 85.6 78.3 72.5 65.8 Example 2 85.5 78.1 73.2 66.3 Example 3 85.8 78.6 73.4 66.5 Comparative Example 1 85.2 65.2 52.1 38.7 Comparative Example 2 84.9 70.5 58.6 45.2 Comparative Example 3 85.1 68.7 55.3 42.1

[0145] Table 2. Antibacterial performance test results (CFU / mL)

[0146] Group 0 days 5 days 7 days 10 days Example 1 <![CDATA[<10 2 ]]> <![CDATA[1.2×10 3 ]]> <![CDATA[2.5×10 3 ]]> <![CDATA[3.8×10 3 ]]> Example 2 <![CDATA[<10 2 ]]> <![CDATA[1.2×10 3 ±500]]> <![CDATA[2.5×10 3 ±800]]> <![CDATA[3.8×10 3 ±1000]]> Example 3 <![CDATA[<10 2 ]]> <![CDATA[1.2×10 3 ±500]]> <![CDATA[2.5×10 3 ±800]]> <![CDATA[3.8×10 3 ±1000]]> Comparative Example 1 <![CDATA[<10 2 ]]> <![CDATA[8.5×10 3 ]]> <![CDATA[1.5×10 4 ]]> <![CDATA[2.8×10 4 ]]> Comparative Example 2 <![CDATA[<10 2 ]]> <![CDATA[3.2×10 3 ]]> <![CDATA[6.8×10 3 ]]> <![CDATA[1.2×10 4 ]]> Comparative Example 3 <![CDATA[<10 2 ]]> <![CDATA[5.1×10 3 ]]> <![CDATA[9.3×10 3 ]]> <![CDATA[1.8×10 4 ]]>

[0147] Table 3 Results of sperm plasma membrane integrity testing

[0148] Group 0 days 5 days 7 days 10 days Example 1 88.7 76.5 70.2 63.5 Example 2 88.5 75.2 70.4 62.4 Example 3 88.8 76.3 70.8 63.8 Comparative Example 1 88.2 62.3 49.1 35.8 Comparative Example 2 87.9 68.4 55.3 42.7 Comparative Example 3 88.1 65.7 52.6 39.5

[0149] Table 4. Results of Acrosphere Integrity Testing

[0150] Group 0 days 5 days 7 days 10 days Example 1 90.3 78.9 72.6 65.8 Example 2 90.5 78.2 73.5 65.5 Example 3 90.8 79.5 73.8 66.8 Comparative Example 1 89.8 64.5 51.2 37.9 Comparative Example 2 89.5 70.8 57.6 44.8 Comparative Example 3 89.7 67.3 54.1 41.3

[0151] As shown in the table above, the combination of tea polyphenols, allicin, and rosemary significantly enhances the antibacterial effect compared to tea polyphenols alone, reducing the total bacterial count. This demonstrates that the synergistic effect of multiple components broadens the antibacterial spectrum and inhibits various pathogens. Simultaneously, the novel antimicrobial peptides reduce non-specific damage to sperm from antibacterial components while enhancing local antibacterial concentration, thus improving sperm motility. The diluted powder of this invention allows for a sperm motility rate of ≥65% in boar semen after storage at 17°C for 10 days, significantly superior to traditional formulas, meeting the needs of long-distance transportation and large-scale farming.

[0152] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A long-lasting diluted powder of porcine semen containing natural antibacterial ingredients, characterized in that, Composed of the following raw materials in parts by weight: 30-50 parts glucose, 1-2 parts sodium EDTA, 5-10 parts sodium citrate, 1-2 parts sodium bicarbonate, 0.5-1 part potassium chloride, 0.5-2 parts compound natural antibacterial ingredients, 0.2-0.8 parts novel antibacterial peptides, 0.3-0.8 parts polyvinyl alcohol, 0.05-0.2 parts cysteine, and 1-3 parts freeze-dried egg yolk powder; The compound natural antibacterial components consist of tea polyphenols, allicin, rosemary antibacterial components in a mass ratio of 2:1:1, and chitosan in a total mass of 0.5%. The novel antimicrobial peptide is a polyethylene glycol derivative modified with folic acid groups that has sperm-targeting properties. The method for preparing the polyethylene glycol derivative containing folic acid groups includes the following steps: A1. Folic acid was dissolved in anhydrous N,N-dimethylformamide and stirred under ice bath conditions. N-hydroxysuccinimide and N,N'-dicyclohexylcarbodiimide were added sequentially. The reaction was carried out at room temperature, filtered, and the filtrate was concentrated under reduced pressure. Under vigorous stirring, the concentrate was slowly added dropwise to ice-cold ether, and a precipitate was formed. The precipitate was collected by filtration, washed with cold ether, and dried under vacuum to obtain folic acid-N-hydroxysuccinimide active ester. A2. Dissolve amino polyethylene glycol in anhydrous dimethyl sulfoxide, add triethylamine, and slowly add an anhydrous dimethyl sulfoxide solution of folic acid-N-hydroxysuccinimide active ester under stirring. React at room temperature, dilute the reaction solution with deionized water, transfer it to a dialysis bag for dialyzing, and freeze-dry the solution after dialyzing to obtain a polyethylene glycol derivative containing folic acid groups.

2. The long-lasting diluted porcine semen powder containing natural antibacterial components according to claim 1, characterized in that, The preparation method of tea polyphenols includes the following steps: B1. Grind green tea into 40-60 mesh, place the ground green tea in a supercritical extraction device, and extract for 1-2 hours under the conditions of pressure 20-30 MPa, temperature 35-45℃ and carbon dioxide flow rate 15-25 L / h. B2. Add the supercritical treated green tea powder to a 60-80% ethanol solution at a material-to-liquid ratio of 1g:10-15mL. Under the conditions of 50-70℃ and ultrasonic power of 200-300W, extract by ultrasonication for 30-40min. Filter, collect the filtrate, and recover the ethanol by vacuum distillation to obtain crude tea polyphenol extract. B3. The crude extract of tea polyphenols was purified by passing it through a macroporous adsorption resin column. AB-8 type macroporous adsorption resin was selected. The eluent was first eluted with 30-50% ethanol solution at a flow rate of 1-2 BV / h, where BV is the resin bed volume. The eluent was collected, concentrated under reduced pressure, and then freeze-dried to obtain high-purity tea polyphenols.

3. The long-lasting diluted porcine semen powder containing natural antibacterial components according to claim 1, characterized in that, The preparation method of allicin includes the following steps: C1. Peel the garlic and homogenize it. Add 2 to 3 times its weight of water to the garlic homogenate and enzymatically hydrolyze it at 40 to 50°C for 1 to 2 hours. Use cellulase and pectinase, with a total enzyme addition of 0.1 to 0.3% and a mass ratio of 1:

1. Then, perform vacuum distillation at 60 to 70°C and a vacuum degree of -0.05 to -0.08 MPa, collect the fraction, and obtain crude allicin extract. C2. The crude allicin extract was purified by molecular distillation. Molecular distillation was carried out at a temperature of 80–100℃, a pressure of 0.1–1 Pa, and a scraper rotation speed of 200–300 r / min. The fractions were collected to obtain purified allicin.

4. The long-lasting diluted porcine semen powder containing natural antibacterial components according to claim 1, characterized in that, The preparation method of the antibacterial component of rosemary includes the following steps: D1. Cut rosemary into small pieces of 0.5-1cm, add 70-90% acetone solution at a material-to-liquid ratio of 1g:8-12mL, and extract 2-3 times at a temperature of 40-50℃ and a stirring speed of 150-200r / min, each time for 2-3 hours. Combine the extracts and recover the acetone by vacuum distillation to obtain crude extract of antibacterial components of rosemary. D2. The crude extract of antibacterial components of rosemary was purified by silica gel column chromatography with petroleum ether-ethyl acetate at a volume ratio of 3-5:1 and an elution flow rate of 1-2 mL / min. The eluent containing antibacterial components was collected, concentrated under reduced pressure, and dried to obtain the purified antibacterial components of rosemary.

5. The long-lasting diluted porcine semen powder containing natural antibacterial components according to claim 1, characterized in that, The preparation method of the novel antimicrobial peptide includes the following steps: E1. Screening for the natural antimicrobial peptide sequence LL-37 with potent antimicrobial activity, and modifying its encoding gene sequence to the codons preferred by E. coli using codon optimization technology, designing a fusion expression vector, linking a glutathione S-transferase tag gene upstream of the antimicrobial peptide gene, inserting an enterokinase cleavage site sequence in the middle, constructing the recombinant expression plasmid pGEX-AMP, transforming the recombinant plasmid into E. coli BL21, and obtaining a genetically engineered bacterium that efficiently expresses the antimicrobial peptide fusion protein through resistance screening and PCR verification; E2. Inoculate the engineered bacteria into LB medium containing 100 μg / mL ampicillin and incubate at 37°C with shaking until OD. 600 =0.6-0.8, add IPTG to a final concentration of 0.1-0.5mM, induce expression at 25℃ for 12-16h, collect bacterial cells, sonicate the bacterial cells, purify the fusion protein by glutathione affinity chromatography column, elute with elution buffer containing 10mM reduced glutathione, add enterokinase, digest at 25℃ for 10-16h, and then separate by ion exchange chromatography to obtain the purified antimicrobial peptide; E3. Nanocellulose was selected as the grafting carrier and dispersed in deionized water to prepare a 1% (w / v) suspension. 0.5% by mass of 2,2,6,6-tetramethylpiperidine-1-oxy free radical and 1% sodium bromide were added. Under the condition of pH=10, 5% sodium hypochlorite solution was slowly added dropwise and reacted at room temperature for 1-2 hours to obtain a carboxylated nanocellulose suspension. E4. The carboxylated nanocellulose suspension and the antimicrobial peptide solution were mixed at a mass ratio of 8-10:1, the pH was adjusted to 5.5, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added as crosslinking agents at a molar ratio of 1:1 and a final concentration of 5mM for both. The reaction was carried out at room temperature in the dark for 2-4 hours with continuous stirring. After the reaction was completed, unreacted small molecules were removed by centrifugation and dialysis, and the grafted modified antimicrobial peptide was obtained by freeze drying. E5. The grafted modified antimicrobial peptide was mixed with a polyethylene glycol derivative containing folic acid groups at a mass ratio of 3 to 5:

1. In a borate buffer solution at pH 8.5, 1 mM of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was added and reacted at room temperature for 6 to 8 hours. The mixture was then subjected to ultrafiltration and lyophilization to obtain a novel antimicrobial peptide with sperm targeting properties.

6. A method for preparing a long-lasting diluted porcine semen powder containing natural antibacterial components according to any one of claims 1 to 5, characterized in that, Includes the following steps: S1. Accurately weigh each ingredient according to the above weight proportions, and place glucose, sodium EDTA, sodium citrate, sodium bicarbonate, potassium chloride, compound natural antibacterial ingredients, novel antibacterial peptides, polyvinyl alcohol, cysteine, and freeze-dried egg yolk powder in a beaker. S2. Add double-distilled water to a beaker, then heat and stir in a 37°C water bath until all ingredients are completely dissolved and equilibrate for more than 1 hour to obtain a uniform diluted solution. S3. The above-mentioned diluent is spray-dried to obtain a long-lasting diluent powder for pig semen.